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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Virus and Prion Research » Research » Research Project #432023

Research Project: Non-Antibiotic Strategies to Control Priority Bacterial Infections in Swine

Location: Virus and Prion Research

2017 Annual Report


1a. Objectives (from AD-416):
Objective 1: Determine molecular mechanisms for virulence of bacterial diseases of swine, including the genetic determinants of bacterial virulence of important swine bacterial pathogens such as Haemophilus parasuis and Streptococcus suis through the use of functional genomics and proteomics, and identify the genetic determinants that differentiate human and swine methicillin-resistant Staphylococcus aureus (MRSA) strains. Subobjective 1.1: Identify genetic determinants contributing to the virulence of H. parasuis and S. suis through the use of functional genomics and proteomics. Subobjective 1.2: Identify the genetic determinants that differentiate human and swine MRSA strains. Objective 2: Determine mechanisms of host susceptibility/resistance to bacterial diseases of swine, including the role of coinfections, physiological, and/or environmental factors on development of disease with bacterial pathogens of swine, identify mechanisms of cross protective immunity to important swine bacterial pathogens such as Haemophilus parasuis and Streptococcus suis, and determine the role of biofilms in persistence of pathogens in the respiratory tract of swine. Subobjective 2.1: Determine the role of coinfections, physiological, and/or environmental factors on development of disease with bacterial pathogens of swine. Subobjective 2.2: Identify mechanisms of cross protective immunity to important swine bacterial pathogens such as H. parasuis and S. suis. Subobjective 2.3: Determine the role of biofilms in persistence of pathogens in the respiratory tract of swine. Objective 3: Develop novel non-antibiotic intervention strategies to control bacterial diseases in swine, including the discovery of effective vaccine platforms to prevent the pathogenesis and clinical disease caused by important swine bacterial pathogens such as Haemophilus parasuis and Streptococcus suis, and determine the feasibility of using biotherapeutics to treat or prevent infectious disease in swine. Subobjective 3.1: Discover vaccine platforms to prevent clinical disease caused by important swine bacterial pathogens such as H. parasuis and S. suis. Subobjective 3.2: Determine the feasibility of using biotherapeutics to treat or prevent infectious disease in swine.


1b. Approach (from AD-416):
The first goal for this research plan is to determine molecular mechanisms for virulence of bacterial pathogens of swine. By combining the genomic work we accomplished during the previous project plan with functional genomic and proteomic studies we expect to identify genes and proteins that are expressed by respiratory pathogens during infection of swine. Combining these techniques will help to refine and confirm prospective virulence targets. We will then be able to test whether these potential targets are involved in pathogenesis through virulence testing in our swine models. Results from these studies will lead to an improved understanding of pathogenic mechanisms of infection, as well as provide novel targets for vaccine strategies. The second goal of this research plan is to determine mechanisms of host susceptibility and resistance to bacterial diseases of swine. There are three areas we have chosen to focus on for this objective. First, we plan to examine how environmental and physiologic factors affect the composition of the upper respiratory microbiome and the establishment and maintenance of pathogens at these sites. For this plan we will examine how in feed and parenteral antibiotics that weaned pigs are exposed to affect the respiratory microbiome. Eventually this will lead to future experiments that will examine the effects of physiologic and environmental stressors and coinfections on carriage of respiratory acquired pathogens. Secondly, we will use immunoproteomics to identify potential cross protective immunogens of bacterial pathogens, such as H. parasuis, that have many serotypes. Finally, we will examine the role of biofilms in persistence of pathogens in the respiratory tract of swine. The third goal of this plan is to develop novel non-antibiotic intervention strategies to control bacterial diseases in swine. One obvious method to reduce antibiotic usage is prevention of disease through the development of efficacious vaccines. We will ultimately use results obtained from the first two objectives to help develop broadly efficacious vaccines. We will be focusing largely on developing improved vaccines against H. parasuis and S. suis, two bacteria in which the current vaccines are limited in their efficacy due to a large number of serotypes that are present in the swine population. In addition to vaccines, we will examine the use of immunomodulators as a promising area of therapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease during periods of peak disease incidence.


3. Progress Report:
Objective 1, Subobjective 1.1: The goal of this subobjective is to determine how bacterial pathogens respond to the host environment and determine which specific genes enable bacteria to colonize the swine respiratory tract and cause invasive acute disease. Transposon-directed insertion-site sequencing (TraDIS) and transcript sequencing (RNAseq) are recently developed methodologies that allow a quantitative assessment of the genes required for the survival of microorganism in certain environments. We have conducted swine infection experiments with pools of the transposon mutants of both Haemophilus parasuis and Streptococcus suis, collected samples, extracted DNA, and sent the DNA to U.K. collaborators for sequencing and analysis to determine which genes are important for survival at various anatomical sites in the pig. We have also performed experiments exposing H. parasuis to carbon dioxide levels that are experienced in the respiratory tract and have isolated the RNA for sequencing (RNAseq) for transcriptomic analysis to determine genes that are highly expressed under these conditions. We have performed in vitro assays and animal studies to examine the role of capsule in the virulence of H. parasuis, S. suis, and Pasteurella multocida and the role of sortases in S. suis virulence. 1.2: The goal of this subobjective is to compare the genome sequences, along with phenotypic adherence assays, to elucidate the evolution and zoonotic potential of livestock-associated (LA)-methicillin-resistant Staphylococcus aureus (MRSA) strains associated with swine. In support of Objective 1.2, whole genome sequencing has been completed on 175 MRSA isolates obtained from swine related and clinical human sources. Draft genome assemblies were used to ascertain the genetic similarity between swine and human isolates and have been analyzed for the presence of mobile genetic elements and antimicrobial resistance genes. Findings indicate distinct genetic lineages for swine versus human isolates, suggesting swine isolates are not likely to contribute to human disease. Objective 2, 2.1 The goal of this subobjective is to establish that environmental factors such as antibiotic usage, physiologic factors around farrowing and weaning, and infections with viral and bacterial pathogens that alter the respiratory microbiome, which in turn plays a role in carriage and development of disease with respiratory acquired pathogens. A swine experiment examining the effect of the antibiotic oxytetracycline delivered in the feed or by injection on the respiratory microbial community was completed and samples from the nasal cavity and tonsil have been processed and are being sequenced for analysis of microbiome changes. In addition we have examined the effectiveness of oxytetracycline given by these routes in clearing Bordetella bronchiseptica and Pasteurella multocida from the respiratory tract. We are currently implementing a swine experiment examining the changes in the respiratory microbial community as a result of infection with the common swine respiratory pathogens, porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus, and B. bronchiseptica. 2.2: The purpose of this subobjective is to identify immunogenic, protective, and conserved outer membrane proteins of Haemophilus parasuis through immunoproteomics that will be cross protective against multiple serotypes. We vaccinated pigs with bacterins made from two H. parasuis serovar 5 strains and examined both homologous and heterologous protective antibodies produced during infection. Both bacterins protected against challenge with the homologous strain, but a difference in protection was observed against a heterologous challenge with a serovar 1 strain. We used an outer membrane protein (OMP) enrichment method and identified OMPs that reacted with antiserum from either protected or nonprotected pigs which revealed stark differences in the possible protective antibodies produced during vaccination. Through an immunoproteomic approach using 2-D gel electrophoresis, we further separated proteins of interest and identified them by mass spectrometry. These bacterial OMPs are potential protective factors for future vaccine studies. 2.3: The goal of this subobjective is to demonstrate whether factors produced by one bacterial species impact the biofilm development and persistence of other bacterial species. Biofilms are important because they protect the bacteria from a variety of host clearance mechanisms and antimicrobial compounds. We conducted microtiter plate assays to examine multispecies biofilm formation employing a Bordetella bronchiseptica mutant harboring an in-frame deletion of the gene encoding FHA along with several isolates of S. suis. Objective 3, 3.1: The goal of this subobjective is to develop efficacious vaccines that are cross protective and prevent disease associated with respiratory bacterial pathogens of swine without the use of antibiotics. We are currently testing five individual Streptococcus suis proteins with several adjuvants for their ability to induce a protective immune response against S. suis infection. We have shown that the five proteins when given together are protective and this experiment will determine whether certain of the proteins or certain adjuvants are better at inducing protection for an improved vaccine formulation. We have tested four Haemophilus parasuis proteins for their ability to induce a protective immune response against H. parasuis infection and found one protein that provides partial protection and one protein that although others have reported protection in a mouse model, did not provide protection in swine. We have also tested an H. parasuis capsule mutant as a vaccine which was protective against both homologous and heterologous strains of H. parasuis demonstrating that an immune response to the capsule is not necessary for protection and may be a more efficacious vaccine against strains of multiple capsule types. 3.2.1. The goal of this subobjective is to determine if the administration of granulocyte-colony stimulating factor (G-CSF) will prevent the development of disease in nursery age pigs challenged with pathogenic S. suis. We conducted studies that demonstrated that pigs given the vectored G-CSF had an improved outcome when infected with S. suis.


4. Accomplishments
1. Biofilm plays a role in persistence of Bordetella bronchiseptica in the lung. B. bronchiseptica is a bacterial respiratory swine pathogen that routinely infects pigs for long periods of time. This holds true despite the use of vaccines, where B. bronchiseptica is frequently isolated from the nose of vaccinated animals. Like many bacteria, B. bronchiseptica can form biofilms, which protects the bacteria from a variety of host clearance mechanisms and antimicrobial compounds. ARS researchers at Ames, Iowa tested a known biofilm factor produced by bacteria termed Bps for its role in biofilm formation by swine isolates of B. bronchiseptica and its role in swine respiratory disease. Results indicated that Bps was required for biofilm formation and for infecting the lungs or lower respiratory tract of swine. These findings provide critical information needed to design improved vaccines and intervention strategies to control or eliminate chronic carriage of B. bronchiseptica and other bacterial pathogens in swine.

2. Antimicrobial resistance in swine isolates of MRSA is lower than in human isolates. Staphylococcus aureus is a common and sometimes devastating human pathogen that has the ability to acquire resistance to antibiotics resulting in methicillin-resistant Staphylococcus aureus (MRSA). Swine can carry strains of MRSA that do not appear to cause disease in swine, but it is unclear whether these swine livestock associated (LA)-MRSA are a risk for humans. ARS researchers at Ames, Iowa determined the antimicrobial resistance profiles and genetic mechanisms of antimicrobial resistance among swine LA-MRSA and human clinical MRSA isolates. Swine LA-MRSA isolates exhibited resistance to fewer antibiotics than MRSA isolates from humans with no swine contact. Distinct genomic antimicrobial resistance elements were harbored by each subgroup, with little overlap in shared antimicrobial resistance genes between swine LA-MRSA and human clinical MRSA isolates. These results indicate there are distinct populations of MRSA in swine and humans, antibiotic resistance is more prevalent in human strains, swine to human transmission is infrequent, and that LA-MRSA may not be a common zoonotic threat.

3. Capsule plays a key role in virulence of the swine pathogen Haemophilus parasuis. Bacterial capsules, largely made of polysaccharide material that surrounds the bacteria, have long been associated with virulence. They can function in adherence, prevent phagocytosis by white blood cells, and protect the bacteria from antimicrobial agents produced by the host. ARS researchers at Ames, Iowa with collaborators examined the role of capsule in enhancing the ability of H. parasuis to cause disease by creating a mutant of H. parasuis unable to produce capsule and testing whether this mutant was attenuated. Results confirmed the capsule mutant was easily killed by immune mechanisms of the host and deficient in its ability to colonize and cause disease in the pig, furthering our understanding of how this bacteria causes disease in pigs. The next steps are to determine whether the attenuated capsule mutant can be used as a successful vaccine against Glässer's disease, an important cause of arthritis and meningitis in swine that is caused by H. parasuis.

4. Granulocyte-colony stimulating factor (G-CSF) helps control Streptococcus suis disease in swine. The use of immunomodulators is a promising alternative to the use of antibiotics to prevent and combat infectious disease. Previously ARS researchers at Ames, Iowa demonstrated a replication-defective adenovirus vector that expresses G-CSF elicited a sustained increase in circulating neutrophils, a type of white blood cell that is beneficial in preventing bacterial diseases. In new studies, pigs given the vectored G-CSF had an improved outcome when infected with Streptococcus suis, the leading cause of meningitis in weaned pigs. Thus, the use of G-CSF in pigs to induce an increase in circulating neutrophil numbers may be a useful alternative to antibiotics for prevention of Streptococcal and other bacterial diseases, especially during times of stress and pathogen exposure such as post-weaning. Finding alternatives to antibiotics is important to reduce the use of antibiotics in livestock species and reduce the development of antibiotic resistance.


Review Publications
Brockmeier, S.L., Loving, C.L., Palmer, M.V., Spear, A., Nicholson, T.L., Faaberg, K.S., Lager, K.M. 2017. Comparison of Asian porcine high fever disease isolates of porcine reproductive and respiratory syndrome virus to United States isolates for their ability to cause disease and secondary bacterial infection in swine. Veterinary Microbiology. 203:6-17.
Gutierrez, A.H., Loving, C., Moise, L., Terry, F.E., Brockmeier, S.L., Hughes, H.R., Martin, W.D., De Groot, A.S. 2016. In vivo validation of predicted and conserved T cell epitopes in a swine influenza model. PLoS One. 11(7):e0159237.
Hau, S.J., Bayles, D.O., Alt, D.P., Nicholson, T.L. 2017. Complete genome sequences of two Staphylococcus aureus sequence type 5 isolates from California, USA. Genome Announcements. 5:e00099-17. doi.org/10.1128/genomeA.00099-17.
Hau, S.J., Bayles, D.O., Alt, D.P., Nicholson, T.L. 2017. Draft genome sequences of 14 Staphylococcus aureus sequence type 5 isolates from California, USA. Genome Announcements. 5(13):e00098-17. doi.org/10.1128/genomeA.00098-17.
Kumar, S., Bass, B., Bandrick, M., Loving, C.L., Brockmeier, S., Looft, T.P., Trachsel, J., Madson, D.M., Thomas, M., Casey, T., Frank, J.W., Stanton, T., Allen, H.K. 2017. Fermentation products as feed additives mitigate some ill-effects of heat stress in pigs. Journal of Animal Science. 95:279-290. doi: 10.2527/jas.2016.0662.
Linz, B., Ivanov, Y., Preston, A., Brinkac, L., Parkhill, J., Kim, M., Harris, S.R., Goodfield, L.L., Fry, N.K., Gorring, A.R., Nicholson, T.L., Register, K.B., Losada, L., Harvill, E.T. 2016. Acquisition and loss of virulence-associated factors during genome evolution and speciation in three clades of Bordetella species. Biomed Central (BMC) Genomics. 17:767. doi: 10.1186/s12864-016-3112-5.
Hau, S.J., Frana, T.S., Sun, J., Davies, P.R., Nicholson, T.L. 2017. Zinc resistance within swine associated methicillin resistant Staphylococcus aureus (MRSA) isolates in the USA is associated with MLST lineage. Applied and Environmental Microbiology. pii: AEM.00756-17. doi: 10.1128/AEM.00756-17.
Nicholson, T.L., Brockmeier, S.L., Sukumar, N., Paharik, A.E., Lister, J.L., Horswill, A.R., Kehrli, M.E. Jr., Loving, C.L., Shore, S.M., Deora, R. 2017. The Bordetella Bps polysaccharide is required for biofilm formation and enhances survival in the lower respiratory tract of swine. Infection and Immunity. 00261-17. doi:10.1128/IAI.00261-17.