2012 Annual Report
1a.Objectives (from AD-416):
Objective 1: Identify the transmission, genetic, and pathogenic mechanisms of the organisms associated with PRDC, concentrating on the bacterial pathogens and their interactions with each other and select swine viruses. Subobjective 1.1: Identify potential virulence factors of H. parasuis through comparative genomics. Subobjective 1.2: Bacterial response to host conditions. Subobjective 1.3: Evaluate ability of PRDC bacterial pathogens to inhibit Influenza A virus vaccine efficacy and/or exacerbate Influenza A virus-associated disease.
Objective 2: Identify potential candidates for novel diagnostic assays, vaccines, and biotherapeutics for bacterial pathogens associated with PRDC. Subobjective 2.1. Develop PCR, ELISA, and/or other assays for detection of bacterial pathogens associated with PRDC. Subobjective 2.2. Identify, develop and/or test the efficacy of potential vaccine candidates to control bacterial pathogens associated with PRDC. Subobjective 2.3. Identify potential biotherapeutic candidates to control bacterial pathogens associated with PRDC.
Objective 3: Investigate emerging and potential zoonotic bacterial pathogens that could impact the swine industry and design measures to diagnose, prevent, control and eliminate the threat posed to the swine industry. Subobjective 3.1. Evaluate the relationship between highly pathogenic Asian strains of PRRSV and S.
suis infection in swine. Subobjective 3.2: Identification of measures that may prevent, control, or eliminate livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type 398 (ST398) in swine.
1b.Approach (from AD-416):
Use comparative genomic methods, microarray analysis, and co-infection studies to explore pathogenic mechanisms of bacteria associated with the porcine respiratory disease complex and their interactions with each other and swine viruses. Assess the usefulness of selected genes or proteins identified in comparative genomic analyses for DNA-based identification and classification, serological detection of infection, and potentially as vaccine candidates. Strategies for improved heterologous protection will be tested using live attenuated vaccines, as will the use of immunomodulators, such as granulocyte colony stimulating factor (G-CSF), for therapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease and thus reduce antimicrobial usage to treat clinical and subclinical disease. Investigate emerging and potential zoonotic bacteria that could impact the swine industry. Investigations will focus on pathogen strain characteristics and differences, interactions of bacterial and viral pathogens with the swine host and the microbial ecosystems of the pig. Pathology of both zoonotic and endemic bacterial pathogens of swine will be utilized for the purpose of understanding disease pathogenesis and developing effective diagnostic assays and strategies to control these pathogens and diseases in swine and potentially in humans.
We continued genomic sequencing efforts for Haemophilus parasuis (HPS). We now have annotated draft genomes for 10 strains of HPS, and are in the process of comparing the relative virulence of all these isolates in both animal model systems and in vitro assays. This will provide meaningful data on which to base genomic comparisons. These efforts support subobjective 1.1. We constructed a HPS DNA microarray using genome sequence information from isolate SH0165 and other isolates that we have sequenced and confirmed its functionality. Working with a collaborator at the University of California, Santa Barbara Westmont College, we used our Bordetella-specific microarray to identify the genes regulated by a newly identified two-component transactional regulatory system shared between both B. bronchiseptica and B. pertussis. These efforts support subobjective 1.2. Viral infection is thought to predispose the host to secondary infection with bacteria. We have continued to investigate when and how swine influenza virus by itself and as a component of vaccine associated enhanced respiratory disease predisposes pigs to secondary bacterial pneumonia. These efforts support subobjective 1.3.
We cloned and expressed recombinant P2 and P5 proteins from a virulent and avirulent isolate of HPS. The respective P2 and P5 alleles are highly divergent and represent different major clades in a phylogeny based on the DNA sequence. We began evaluating the immunogenicity of different HPS outer membrane proteins that may be of value in diagnostic assays and/or as vaccines. We initiated construction of a novel suicide vector for the purpose of generating markerless mutations in HPS. We completed cloning the bsp22 gene from B. bronchiseptica that encodes a Type 3 secretion needle protein that was shown to be immunogenic in mice, and a recombinant replication-defective adenovirus vaccine vector expressing the protein has been generated for testing in pigs. These efforts support subobjectives 2.1.and 2.2. We also completed initial experiments evaluating protection provided by the administration of Granulocyte-Colony Stimulating Factor, a compound that enhances immunity against bacterial infections by increasing the number of circulating neutrophils. These efforts support subobjective 2.3.
We completed a swine experiment designed to compare 2 Asian isolates of porcine reproductive and respiratory syndrome virus obtained from the porcine high fever disease outbreaks to high and low virulence isolates obtained from the United States for their alteration of innate immune functions and their ability to predispose to secondary bacterial infections in swine. This experiment supports subobjective 3.1. We completed studies investigating the ability of ST398 methicillin-resistant S. aureus (MRSA) swine isolates to form biofilms and mechanisms to disperse or eliminate biofilms that could be used or evaluated in vivo in the future. In collaboration with scientists at the New Jersey Dental School, we tested the ability of an exopolysaccharide prepared from a nonpathogenic bacterium to inhibit biofilm formation by ST398 MRSA swine isolates. These efforts support subobjective 3.2.
Determination of the genetic basis by which Haemophilus parasuis (H. parasuis) causes disease. H. parasuis is a bacterium that causes Glasser's disease in swine, a disease characterized by chronic debilitation and often death that costs the swine industry millions in losses annually. However, not all strains of the bacterium cause disease. To date, little is known about genetic differences among H. parasuis strains and the genetic factors that contribute to its ability to cause disease. ARS scientists at the National Animal Disease Center in Ames, Iowa determined the DNA genomic sequence of 10 strains of H. parasuis with varying capability of causing disease. The DNA sequence can now be used to compare the different isolates for identification of genes contributing to disease development. These data comprise the first publicly available genome sequence for this bacterium.
Virulence determinants of porcine reproductive and respiratory syndrome virus (PRRSV) are complex. Demonstrated the correlation of secondary bacterial infections in pigs infected with genetically varied isolates of PRRSV. PRRSV is one of the most devastating and costly diseases to the swine industry world-wide. PRRSV is ubiquitous and mutates rapidly making it difficult to develop vaccines that protect pigs from disease. ARS scientists at the National Animal Disease Center in Ames, Iowa determined the whole genomic sequence of 7 PRRSV isolates and found that the isolates varied considerably in their genomic sequence as well as their capacity to replicate in pigs and predispose to secondary bacterial pneumonia. No specific mutations correlated with disease, indicating specific genomic determinants of disease are complex. These experiments are elucidating mechanisms by which the virus causes disease and alters the pig's immune response to cause increased susceptibility to secondary bacterial infections.
Determined a mechanism by which porcine reproductive and respiratory syndrome virus (PRRSV) causes immunosuppression. PRRSV alters the pig's immune response to cause increased susceptibility to secondary bacterial infections. Neutrophils are cells of the immune system that play an important role in combating bacterial infection. Granulocyte-Colony Stimulating Factor (G-CSF), a compound normally produced by the body, increases the number of circulating neutrophils. The use of immunomodulators like G-CSF is a promising area for therapeutic and prophylactic use to prevent and combat infectious disease while eliminating or reducing the use of antibiotics. ARS scientists at the National Animal Disease Center in Ames, Iowa, completed a study in which pigs were given G-CSF to determine if it would reduce the incidence of secondary bacterial infections subsequent to PRRSV infection. Although G-CSF did not decrease the prevalence of secondary bacterial infections in PRRSV infected pigs, a paradoxical outcome of the viral infection was an abolition of the surge in circulating neutrophils created by G-CSF, which represents a previously unrecognized effect of PRRSV on the host immune system and may explain how PRRSV contributes to development of secondary bacterial infections.
Developed strategies to eliminate antibiotic resistant Staphylococcus bacteria in swine. Staphylococcus aureus (S. aureus) is an extremely common yet devastating human pathogen. S. aureus has an outstanding ability to acquire resistance to antibiotics, and in 2003, a new methicillin-resistant S. aureus (MRSA) lineage emerged associated with livestock species including pigs. Therefore the identification of factors or strategies to eliminate the presence of MRSA from swine herds is a top research priority for U.S. swine producers. Bacterial persistence is often linked to the development of microbial communities known as biofilms that make the bacteria resistant to immune responses, disinfectants, and antibiotics. ARS scientists at the National Animal Disease Center in Ames, Iowa determined that MRSA isolated from swine are capable of forming biofilms and found two compounds able to inhibit or disperse biofilm formation of these isolates. The rationale behind this approach is that compounds that can prevent or disperse biofilm formation will cause the bacteria to either revert to or be held in a free-living state where they are sensitive to host immune responses, disinfectants, available antimicrobial therapies, and other environmental pressures that lead to the clearance of the organism. Collectively, these findings provide a critical first step in designing strategies to control or eliminate MRSA in swine herds.
Brockmeier, S.L., Loving, C.L., Nelson, E.A., Miller, L.C., Nicholson, T.L., Register, K.B., Grubman, M.J., Brough, D.E., Kehrli, Jr., M.E. 2012. The presence of alpha interferon at the time of infection alters the innate and adaptive immune responses to porcine reproductive and respiratory syndrome virus. Clinical and Vaccine Immunology. 19(4):508-514.
Mullins, M.A., Register, K.B., Bayles, D.O., Dyer, D.W., Kuehn, J.S., Phillips, G.J. 2011. Genome sequence of Haemophilus parasuis strain 29755. Standards in Genomic Sciences. 5(1):61-68.
Nicholson, T.L., Brockmeier, S.L., Loving, C.L., Register, K.B., Kehrli, Jr., M.E., Stibitz, S.E., Shore, S.M. 2012. Phenotypic modulation of the virulent Bvg phase is not required for pathogenesis and transmission of Bordetella bronchiseptica in swine. Infection and Immunity. 80(3):1025-1036.
Register, K.B., Sukumar, N., Palavecino, E.L., Rubin, B.K., Deora, R. 2012. Bordetella bronchiseptica in a paediatric cystic fibrosis patient: possible transmission from a household cat. Zoonoses and Public Health. 59(4):246-250.
Loving, C.L., Vincent, A.L., Pena, L., Perez, D.R. 2012. Heightened adaptive immune responses following vaccination with a temperature-sensitive, live-attenuated influenza virus compared to adjuvanted, whole-inactivated virus in pigs. Vaccine. 30(40):5830-5838.