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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

2018 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. Haemophilus parasuis (H. parasuis) is a bacterium that causes Glässer's disease in swine, a disease characterized by acute infections and chronic debilitation 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. To identify genetic factors that contribute to the ability of H. parasuis to cause disease, we obtained the closed whole-genome sequence for the virulent Nagasaki strain and for the commensal D74 strain. Evaluation of the genomes is revealing differences in the presence of virulence-associated genes between the two strains. Different methylation patterns and diversity in the restriction-modification systems have been found between D74 and Nagasaki. These systems provide a bacterial defense mechanism that effectively destroys foreign DNA such as that introduced by a bacteriophage, a virus that infects bacteria. In addition, we have isolated RNA from H. parasuis grown in the laboratory and from animals that have developed disease with this bacteria. The RNA has been sequenced and the data is being analyzed to determine which genes are being expressed in these different environmental conditions. This information will aid in the development of diagnostics, vaccines and intervention strategies to decrease the prevalence and disease burden caused by H. parasuis. Streptococcus suis (S. suis) is a significant swine pathogen and a zoonotic agent causing severe infections in people through consumption of raw pork products or occupational exposure. We performed comparative genomic analyses of S. suis isolates that exhibit different pathogenic capacities to identify genomic attributes associated with virulent phenotypes. Nine genetically diverse strains isolated within the U.S. were chosen for whole genome sequence analysis and virulence assessment by evaluating disease following intranasal challenge. S. suis strains were identified that were highly and moderately virulent as well as avirulent. Whole genome sequencing followed by comparative genomic analyses is revealing several notable differences in regions encoding secreted and membrane-associated factors that have been previously proposed to serve a role in virulence. These genomic regions of difference may contribute to the spectrum of clinical disease observed following challenge with these isolates. These results are providing a foundation for understanding the genomic attributes responsible for the spectrum of virulent phenotypes that exist among S. suis isolates. This information is paramount to designing effective vaccines needed by the swine industry to mitigate S. suis disease. Objective 2, 2.1: The goal of this subobjective is to establish how factors such as antibiotic usage and infections with viral and bacterial pathogens alter the respiratory microbiome (all the microorganisms that inhabit respiratory sites), which in turn plays a role in carriage and development of disease with respiratory acquired pathogens. We analyzed the data generated from a study that characterized how administration of the antibiotic oxytetracycline, given either parenterally or in medicated feed, affected the composition and diversity of the nasal and tonsil microbiotas of post-weaned pigs over a two-week period. Analyses of the data is showing that the tonsil microbiota was unaffected by either form of oxytetracycline treatment and remained relatively stable. The nasal microbiota diversity decreased as a result of both oral and parenteral oxytetracycline treatments. The nasal microbiota also showed greater shifts in microbial composition that persisted from day 4 until the end of the study trial. Numerous genera were differentially abundant in response to either of the two oxytetracycline treatments. This included high abundances of genera that contain pathogens such as Actinobacillus, Streptococcus, Pseudomonas, as well as decreased abundances of multiple commensal genera. Though both forms of oxytetracycline administration resulted in numerous changes in the microbiota composition, the magnitude of changes from oral oxytetracycline treatment were greater. The findings from this study reveal that short-term antibiotic exposure from a broad-spectrum antibiotic like oxytetracycline has an effect on the upper respiratory microbiota. In addition, results from this study acknowledge the need for further assessment on how such changes in the population can ultimately affect the animal's disease risk and respiratory health. We also completed a swine experiment designed to examine 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 Bordetella bronchiseptica. We have isolated and sequenced the DNA from the nasal and tonsil samples and are currently analyzing the data from this experiment. 2.2: The purpose of this subobjective is to identify outer membrane proteins of Haemophilus parasuis (H. parasuis) that will be cross protective against multiple strains. H. parasuis, the causative agent of Glasser's disease in pigs, is widespread and results in significant financial losses to the swine industry annually. The majority of vaccines are based on a killed, whole bacteria platform, in which the immune response tends to be serotype or strain specific and are unable to provide broad cross protection against H. parasuis. This has stimulated interest in the development of subunit vaccines, where the immune response can be directed to highly conserved, surface exposed proteins. We identified outer membrane proteins with potential broad protective capability from H. parasuis by determining differences in the immune response to H. parasuis proteins from pigs that were broadly protected versus pigs that were only protected against one strain of H. parasuis. 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. Streptococcus suis (S. suis) is a bacterial swine pathogen with a significant economic burden. It typically colonizes the tonsil and nasal cavity of swine causing a variety of symptoms ranging from asymptomatic carriage to lethal systemic disease. A key barrier towards the development of improved vaccines or interventions for S. suis infections is a gap in our understanding of the mechanisms contributing to persistence in the host, in which colonized pigs continue to shed and transmit S. suis. We hypothesized that exposure to sub-minimal inhibitory concentrations of antibiotics commonly used by the swine industry would increase the biofilm capacity of S. suis strains. Using biofilm assays, we have found that sub-minimal inhibitory concentrations of bacitracin, carbadox, chlortetracycline, enrofloxacin, gentamicin, neomycin, sulfadimethoxine, tiamulin, and tylosin did not increase S. suis biofilms. In contrast, we found that sub-minimal inhibitory concentrations of amoxicillin, lincomycin, and oxytetracycline increased overall biofilm formation, which may increase survival and persistence within the respiratory tract of swine. 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. Streptococcus suis (S. suis) is a bacterium that is an important and common cause of meningitis and arthritis in pigs that costs the swine industry millions in losses annually. Unfortunately there are few efficacious vaccines available for this disease. We identified five proteins of S. suis that were selected for inclusion in an experimental vaccine that was found to be effective at preventing disease. We further examined whether a subset of these proteins, a solitary protein, or different adjuvants used to stimulate the immune response would be effective at preventing disease. So far no combination has been as efficacious as the five original proteins together with the original adjuvant used. Haemophilus parasuis (H.parasuis), the causative agent of Glasser's disease in pigs, is widespread and results in significant financial losses to the swine industry annually. The majority of vaccines are based on a killed, whole bacteria platform, in which the immune response tends to be capsule type specific, and the vaccines are thus unable to provide broad cross against the many capsule types of H. parasuis. We created a strain of H. parasuis that does not produce capsule and are testing it as a potential vaccine candidate. We found that the unencapsulated vaccine protected against the parent strain that it was created from and another strain from a different capsule type. We are currently testing the unencapsulated vaccine against several other common capsule types found in pigs. In addition we are testing several of the H. parasuis proteins we identified as potentially important in cross protection as a vaccine.


4. Accomplishments
1. A subunit vaccine against Streptococcus suis (S. suis) in swine. Streptococcus suis is a bacterium that is an important and common cause of meningitis and arthritis in pigs that costs the swine industry millions in losses annually. Unfortunately there are few efficacious vaccines available for this disease. ARS researchers at Ames, Iowa with collaborators from the University of Cambridge identified five candidate proteins of S. suis that were selected for inclusion in an experimental vaccine by identifying bacterial genes required for survival of the bacterium in the pig. The proteins were delivered to pigs with different adjuvants that help stimulate an immune response. The vaccine was found to be effective at preventing disease caused by S. suis. In addition, antiserum from the vaccinated pigs was reactive against whole S. suis bacteria of differing serotypes indicating a potential for cross-protection. These proteins are now being developed into a vaccine by a biologics company that can be used by swine producers to protect against this devastating and costly swine disease.

2. Ability of methicillin resistant Staphylococcus aureus (S. aureus) to adhere to human and swine skin. Methicillin resistant Staphylococcus aureus (MRSA) is a bacterium that causes severe disease in humans and can be obtained from health care settings (HA-MRSA), community settings (CA-MRSA), or contact with livestock including swine (LA-MRSA). While LA-MRSA infrequently causes disease in pigs, there have been concerns in the public health community that livestock may act as a reservoir for MRSA isolates affecting people. To understand the implications of the LA-MRSA isolates for swine producers and the risk these isolates may pose outside of the agricultural setting, ARS researchers at Ames, Iowa compared MRSA isolates from health care settings and from swine associated sources for the ability to attach to human and swine skin. The HA-MRSA and LA-MRSA isolates tested were found to adhere equivalently to human skin and a comparison of genes involved in initial attachment revealed a similar genetic potential for adherence. However, the genes that contribute to persistent colonization and disease with S. aureus were absent in the LA-MRSA isolates. This suggests that humans in contact with swine have the potential to become transiently colonized with these LA-MRSA isolates but that these isolates are probably less likely to cause disease in humans compared to human isolates acquired from health care settings.

3. Antiviral treatment for porcine reproductive and respiratory syndrome virus (PRRSV) infection in pigs. Interferon is produced in response to virus infections to help the host inhibit viral replication and develop a stronger immune response to the pathogen. Porcine reproductive and respiratory syndrome virus is one of the most devastating and costly viruses to the swine industry world-wide and has been shown to induce a meager interferon response in pigs. ARS researchers at Ames, Iowa explored the use of interferon as an adjuvant given with live-attenuated PRRSV vaccine to determine if it would result in an enhanced immune response to the vaccine. Giving interferon totally abolished replication of the vaccine virus and as a result there was no detectible adaptive immune response after vaccination. Although interferon did not have the desired adjuvant effect, the results are promising for the use of interferon as a treatment for PRRSV infection.


Review Publications
Brockmeier, S.L., Loving, C.L., Eberle, K.C., Hau, S.J., Buckley, A., Van Geelen, A., Montiel, N.A., Nicholson, T., Lager, K.M. 2017. Interferon alpha inhibits replication of a live-attenuated porcine reproductive and respiratory syndrome virus vaccine preventing development of an adaptive immune response in swine. Veterinary Microbiology. 212:48-51.
Hau, S.J., Bayles, D.O., Alt, D.P., Davies, P.R., Haan, J.S., Nicholson, T.L. 2017. Draft genome sequences of nine livestock-associated methicillin-resistant Staphylococcus aureus sequence type 5 isolates from humans with long-term swine contact. Genome Announcements. 5(41):e01079-17.
Hau, S.J., Bayles, D.O., Alt, D.P., Frana, T.S., Nicholson, T.L. 2017. Draft genome sequences of nine livestock-associated methicillin-resistant Staphylococcus aureus sequence type 5 isolates obtained from humans after short-term swine contact. Genome Announcements. 5(41):e01080-17.
Hau, S.J., Bayles, D.O., Alt, D.P., Frana, T.S., Nicholson, T.L. 2017. Draft genome sequences of 63 swine-associated methicillin-resistant Staphylococcus aureus sequence type 5 isolates from the United States. Genome Announcements. 5(44):e01081-17.
Hau, S.J., Bayles, D.O., Alt, D.P., Frana, T.S., Nicholson, T.L. 2017. Draft genome sequences of 14 livestock-associated methicillin-resistant Staphylococcus aureus sequence type 398 isolates from swine farms in the United States. Genome Announcements. 5(44):e01082-17.
Hau, S.J., Bayles, D.O., Alt, D.P., Nicholson, T.L. 2017. Draft genome sequences of 50 methicillin-resistant Staphylococcus aureus sequence type 5 isolates obtained from a U.S. hospital. Genome Announcements. 5(44):e01083-17.
Hau, S.J., Bayles, D.O., Alt, D.P., Nicholson, T.L. 2017. Draft genome sequences of one methicillin-sensitive and seven methicillin-resistant Staphylococcus aureus sequence type 5 Isolates obtained in California. Genome Announcements. 5(42):e01084-17.
Hau, S.J., Bayles, D.O., Alt, D.P., Frana, T.S., Nicholson, T.L. 2017. Complete genome sequence of livestock-associated methicillin-resistant Staphylococcus aureus sequence type 398 isolated from swine in the United States. Genome Announcements. 5(32):pii: e00790-17. https://doi.org/10.1128/genomeA.00790-17.
Hau, S.J., Bayles, D.O., Alt, D.P., Frana, T.S., Nicholson, T.L. 2017. Complete genome sequence of a livestock-associated methicillin-resistant Staphylococcus aureus sequence type 5 isolate from the United States. Genome Announcements. 5(32):e00791-17. https://doi.org/10.1128/genomeA.00791-17.
Hau, S.J., Kellner, S., Eberle, K.C., Waack, U., Brockmeier, S.L., Haan, J.S., Davies, P.R., Frana, T., Nicholson, T.L. 2018. Methicillin-resistant Staphylococcus aureus sequence type (ST) 5 isolates from health care and agricultural sources adhere equivalently to human keratinocytes. Applied Environmental Microbiology. 84(2):e02073-17. https://doi.org/10.1128/AEM.02073-17.
Brockmeier, S.L., Loving, C.L., Nicholson, T.L., Wang, J., Peters, S.E., Weinert, L., Chaudhuri, R., Seilly, D.J., Langford, P.R., Rycroft, A., Wren, B.W., Maskell, D.J., Tucker, A.W. 2018. Use of proteins identified through a functional genomic screen to develop a protein subunit vaccine that provides significant protection against virulent Streptococcus suis in pigs. Infection and Immunity 86(3):e00559-17. https://doi.org/10.1128/IAI.00559-17.