2007 Annual Report 1a.Objectives (from AD-416) 1. Characterize mechanisms of respiratory disease infections by defining determinants of virulence and respiratory pathogen interactions that lead to polymicrobial infections. 2. Characterize changes in host-pathogen gene expression that are associated with the PRDC. 3. Design novel vaccine approaches for swine respiratory pathogens of interest, using bacterial ghost technology, and assess their safety and efficacy in validated PRDC disease models. 1b.Approach (from AD-416) Objective 1: Identifying the contribution of the different pathogens involved in porcine respiratory disease is important in understanding the complex. We will characterize and compare the ability of the bacterial and viral pathogens involved in PRDC to act alone and in concert to exacerbate disease. These studies will involve developing in vivo porcine animal models of single and mixed infections involving PRDC pathogens and determining important interactions among these agents. Subobjective 1.2 describes plans to investigate the role of putative virulence factors of PRDC bacterial pathogens through the generation of mutants and in vitro and in vivo functional testing of these mutants. Objective 2: Infected hosts recognize the presence of pathogens and mobilize specific immune defense mechanisms. Pathogens in turn can actively modulate host-signaling and immune-pathways to enhance their persistence and survival. Global networks for gene regulation in bacteria allow these organisms to adapt to different environmental niches and host microenvironments; such adaptation underlies the capacity of infectious agents to persist in and damage host tissues. We plan to perform comprehensive profiling of the transcriptional response of both the host, porcine respiratory tract, and the pathogen, Bordetella bronchiseptica, by exploiting two approaches. First, using a B. bronchiseptica microarray, we will analyze the global expression profile of B. bronchiseptica during various in vitro growth conditions and during respiratory tract infections. Secondly, utilizing tissue samples from the previously mentioned infection models and a porcine immune microarray, we will thoroughly investigate the immune response pathways that underlie respiratory infections. Objective 3: Improved vaccines for swine respiratory disease agents are needed. Commercially available products sometimes fail to provide reliable protection against disease, are costly, and cumbersome to administer, and retain undesirable side-effects. We will determine the efficacy of bacterial ghosts (BG) as vaccines against respiratory pathogens of swine in our swine respiratory infection models. 3.Progress Report (1) Conducted a pig experiment in collaboration with the swine virology group at NADC examining whether an Haemophilus parasuis and/or bovine viral diarrhea virus (BVDV) isolate was responsible for a high mortality outbreak in swine. Findings from this study indicated it was most likely the H. parasuis isolate that was responsible for the mortality and that this strain is a highly virulent isolate that we can continue to work with. (2) Initiated experiments to characterize an in-frame sigma E deletion mutant of B. bronchiseptica using the newly constructed B. bronchiseptica DNA microarrays. Sigma E is an alternative sigma factor responsible for stress survival in other bacteria. The information gained from this study should provide insight as to how B. bronchiseptica survives the stress encountered during an infection in swine. (3) Conducted experiments investigating the ability of two in-frame deletion mutants, of B. bronchiseptica, to colonize and cause disease using an in vivo swine respiratory infection model. These deletions were constructed in the swine isolate KM22 and are known virulence factors, FHA and pertactin, of the B. bronchiseptica RB50 (rabbit isolate), which is the strain most routinely used in mouse infection models and previous literature reports. (4)Initiated the in vitro characterization of three newly constructed Bvg-phase locked mutants, in the B. bronchiseptica KM22 (swine isolate) strain. These mutants will be used in swine studies elucidate the role of Bvg regulation in swine respiratory disease. (5) Purified a BrpL, B. bronchiseptica transcriptional regulator responsible for regulating type three secretion virulence factors. This protein will be used in in vitro transcription assays to identify all genes encompassing the BrpL regulon. (6) Conducting purification of BvgA, the DNA-binding partner of the B. bronchiseptica two-component sensory transduction system, BvgAS. This protein will be used in in vitro transcription assays to identify all genes encompassing the BvgAS regulon. (7) Conducting purification of RpoD and core RNA polymerase (RNAP) from B. bronchiseptica. These proteins together form the haloRNAP that is needed to perform all in vitro transcription assays, which will be used to identify and understand the regulatory mechanisms underlying Bordetella pathogenesis. (8) Conducted experiments investigating the transcriptional response of B. bronchiseptica during biofilm formation. (9) Conducting studies examining the transcriptional response of immune genes in different tissues from pigs infected with swine influenza virus (SIV), Bordetella bronchiseptica, or both agents to better understand the responses involved in protection and pathogenesis of respiratory disease in pigs. (10) Studies of porcine macrophage regulatory pathways have been hampered by a lack of appropriate species-specific reagents. It is known that dsRNA can signal through the toll-like receptor (TLR)-3. Reagents are not presently available that specifically detect porine TLR-3 from lung lavage cells. 4.Accomplishments Construction and validation of a first-generation Bordetella bronchiseptica long-oligonucleotide microarray by transcriptional profiling the Bvg regulon. Bordetella bronchiseptica is a bacterial respiratory pathogen that infects a broad range of mammals. Gene expression in Bordetella is controlled by a set of proteins, called BvgAS, which control the expression of a spectrum of phases transitioning between an infectious (Bvg+) phase and a non-infectious (Bvg-) phase. This report describes the design and construction of a method, termed DNA microarray, which allows people to tell whether or not a gene is expressed. The special aspect of this method is that gene activity, whether or not a gene is expressed, can be determined for the entire Bordetella genome in a single experiment. This method was tested and validated by comparing the gene activity between two different strains of Bordetella, one normal strain and the other altered so that the set of proteins called BvgAS were inactivated or no longer functioning. Data from this comparison revealed 1,667 genes that are regulated by BvgAS. The results provide a comprehensive, genome-wide portrait of genes regulated by BvgAS, while also providing data validating the use of this method for studying gene expression in Bordetella. These results will expedite the discovery of new potential targets for drug and vaccine therapy, which will aid the swine industry in controlling respiratory disease. This accomplishment addresses 103 Animal Health National Program Component 4: Countermeasures to prevent and control respiratory diseases and Problem Statement 4B: Porcine respiratory diseases. Coinfection studies with agents of PRDC. Porcine respiratory disease complex (PRDC) is a common pneumonia in pigs caused by infection with multiple pathogens. We conducted studies looking into interactions among common respiratory pathogens in pigs to determine whether interactions exist between swine influenza virus (SIV) and B. bronchiseptica. Results of these studies indicate that coinfection with SIV and B. bronchiseptica leads to earlier and more persistent pneumonia in pigs. These results stress the need to properly diagnose all pathogens involved in respiratory disease in order to most effectively address the problem. Disease models of mixed infections can be used to further investigate host-pathogen interactions and determine the effectiveness of vaccines to prevent respiratory disease in pigs. This accomplishment addresses 103 Animal Health National Program Component 4: Countermeasures to prevent and control respiratory diseases and Problem Statement 4B: Porcine respiratory diseases. Construction of swine immune microarray. There is currently no method to comprehensively evaluate the host response to infection in pigs. Given that the porcine genome sequence is incomplete and unavailable, we constructed a swine immune-specific DNA microarray that will be used as a tool for evaluating transcriptional responses of swine during a variety of bacterial and viral infections. Understanding how a host responds to invasion by a pathogen will lead to development of a whole new series of methods to prevent primary infection and abrogate the development of respiratory disease, helping the swine industry to raise disease free pigs. This accomplishment addresses 103 Animal Health National Program Component 4: Countermeasures to prevent and control respiratory diseases and Problem Statement 4B: Porcine respiratory diseases. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of new CRADAs and MTAs 2