2008 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 a. Genomic sequencing of two important bacteria that cause respiratory disease in pigs, Haemophilus parasuis and Bordetella bronchiseptica, has been performed. The sequence of two outer membrane proteins of 30 isolates of H. parasuis has been compared. Variability has been found in the sequence of the proteins that correspond to areas exposed on the surface of the bacteria, which could be a reason that vaccines to one strain of H. parasuis often don’t protect against other strains. Experiments comparing the ability of the H. parasuis isolates to cause disease in pigs have been conducted as well, in hopes of identifying genomic differences that correlate to differences in their ability to cause disease. The sequence data has also been used to identify the capsule genes of H. parasuis, which could be used to generate a more relevant strain identification scheme than the one currently used. b. B. bronchiseptica strains with both decreased and increased abilities to cause disease have been analyzed. Using a Bordetella specific microarray, which can determine what genes are being expressed by the bacteria, it was determined that that the strain with decreased ability to cause disease lacked the genes that are required for the production of a well characterized toxin, the adenylate cyclase toxin (ACT). The strain with increased ability to cause disease was found to produce more of the Type Three Secretion System, which allows the bacteria to secrete toxins into host cells. c. Experiments were conducted investigating the role that Bvg (a system which turns on and off a set of genes in B. bronchiseptica) has in disease, survival, and transmission in the pig. The studies indicate that the Bvg system needs to be functioning for the bacteria to survive and cause disease in the pig and transmit from pig to pig. d. Experiments were conducted to investigate the ability of B. bronchiseptica to sense and respond to the presence of CO2, which is elevated in the respiratory tract. These initial experiments indicate that the production of many disease causing factors are upregulated in the presence of CO2. e. Studies were conducted to investigate how previous swine influenza virus (SIV) infection alters the lungs ability to resist secondary bacterial infection. The studies showed that alveolar macrophages (a protective cell in the lung) collected from pigs that had previously been infected with SIV had an altered immune response to bacteria. f. The induction of immune mediators by different strains of H. parasuis in alveolar macrophages was examined and it was found that these cells produce elevated levels of proteins that cause inflammation regardless of the strain they were exposed to. These results provide information that different strains of H. parasuis incite a similar immune response in alveolar macrophages and may not be related to strains ability to cause disease. This accomplishment addresses Animal Health National Program Component 4: Countermeasures to prevent and control respiratory diseases and Problem Statement 4B: Porcine respiratory diseases. 4.Accomplishments 1. Contribution of Bordetella bronchiseptica Filamentous Hemagglutinin and Pertactin to Respiratory Disease in Swine. Respiratory disease in pigs is the most important health concern for swine producers today, and Bordetella bronchiseptica is a bacterium pervasive in swine populations that has multiple roles in respiratory disease. To investigate the role that two proteins of B. bronchiseptica, filamentous hemagglutinin (FHA) and pertactin (PRN), have in the ability of this pathogen to cause disease, mutants containing a deletion of the FHA or the PRN structural gene in a B. bronchiseptica swine isolate were constructed. A comparison of these mutants to the wild-type swine isolate for their ability to colonize and cause disease revealed that FHA was important in overall colonization of B. bronchiseptica, while PRN was important for colonization during early and late infection. The decreased colonization observed early suggests that PRN plays a role in attachment, while the decreased colonization levels observed late implies a role for PRN in persistence in the swine respiratory tract. Higher anti-Bordetella antibodies were also detected in pigs infected with the PRN mutant. These results provide a crucial understanding of the role of PRN and FHA in swine respiratory disease, which will expedite the discovery of new vaccine therapy. This accomplishment addresses Animal Health National Program 103, Component 4: Countermeasures to Prevent and Control Respiratory Diseases and Problem Statement 4B: Porcine Respiratory Diseases. 2. Synergistic Effect of Swine Influenza and Bordetella bronchiseptica coinfection. Respiratory disease is one of the most important health issues for the swine industry, and coinfection with two or more pathogens is a common occurrence. Bordetella bronchiseptica and swine influenza virus (SIV) are important and common respiratory pathogens of pigs. A study examining the effect of coinfection of SIV and B. bronchiseptica in pigs found that pneumonia was seen very early, one day after infection, only in the coinfected pigs as opposed to pigs infected with either pathogen alone. Increased levels of immune mediators that cause inflammation were found in the respiratory tracts of these coinfected pigs. This data suggests that there is a synergistic effect during coinfection with SIV and B. bronchiseptica leading to the early and more pronounce production of these immune mediators, contributing to the early pneumonia seen in coinfected pigs. Understanding the mechanism by which multiple agents cause disease will help in the development of more effective therapies. This accomplishment addresses Animal Health National Program 103, Component 4: Countermeasures to Prevent and Control Respiratory Diseases and Problem Statement 4B: Porcine Respiratory Diseases. 3. Specificity of DNA-based tests for identification of Bordetella. Although primarily a pathogen in mammals other than humans, B. bronchiseptica is increasingly implicated in human respiratory disease which can be especially severe in very young, elderly, or immunocomprised patients. It is important for clinicians to distinguish between these species since they require different treatment regimens. Genetic characterization of B. bronchiseptica virulence genes related to disease in swine has fortuitously revealed that methods of identification once considered specific for B. pertussis may give false positive results with some strains of B. bronchiseptica. These observations have a significant impact on the ongoing debate related to diagnostic standards for B. pertussis and also highlight the role of B. bronchiseptica as an occasional, but serious, human pathogen. This accomplishment addresses Animal Health National Program 103, 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 Commercial Licenses Executed 1 Number of Other Technology Transfer 1 Review Publications Brockmeier, S., Register, K.B. 2007. Expression of the dermonecrotic toxin by Bordetella bronchiseptica is not necessary for predisposing to infection with toxigenic Pasteurella multocida. Veterinary Microbiology. 125(3-4):284-289. Register, K.B., Nicholson, T.L. 2007. Misidentification of Bordetella bronchiseptica as Bordetella pertussis using a newly described RT-PCR targeting the pertactin gene. Journal of Medical Microbiology. 56:1608-1610. Brockmeier, S., Loving, C.L., Nicholson, T.L., Palmer, M.V. 2008. Coinfection of pigs with Porcine Respiratory Coronavirus and Bordetella bronchisphica. Veterinary Microbiology. 128(1-2):36-47. Rath, B.A., Register, K.B., Wall, J., Sokol, D.M., Van Dyke, R.B. 2008. Bordetella bronchiseptica Pneumonia in an Infant and Genetic Comparison of Clinical Isolates with Veterinary Kennel Cough Vaccines. Clinical Infectious Diseases. 46(6):905-908.