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

Location: Virus and Prion Research

2021 Annual Report

Objectives
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.

Approach
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.

Progress Report
This is the final report for project 5030-32000-119-00D terminating September 30, 2021. 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. We performed genomic studies on S. suis isolates that exhibit different pathogenic capacities and identified several differences in regions encoding secreted and membrane-associated factors, providing diagnostic and vaccine targets. Most data needed to evaluate risks for S. suis isolates to function as zoonotic agents and/or AMR reservoirs are derived from non-U.S. isolates. To fill this gap, we performed genomic studies on recently collected U.S. S. suis isolates and identified AMR elements and factors that could enable S. suis to transmit, and/or cause disease in humans. Our results demonstrated a high degree of diversity among S. suis isolates in the U.S. and helps elucidate any risk associated with S. suis. H. parasuis 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 can cause disease. To identify genetic factors contributing to the ability of H. parasuis to cause disease, we compared the genome sequence of a virulent strain to a non-virulent strain and successfully identified factors likely contributing to virulence. We isolated and sequenced RNA obtained from H. parasuis grown in the laboratory and from animals that have developed disease. Data is being analyzed to determine which genes are expressed in each condition. We performed swine studies using pools of the transposon mutants of both H. parasuis and S. suis, collected samples, extracted DNA, and sent the DNA to collaborators for sequencing and analysis to identify genes required for survival at various anatomical sites in the pig. We collected samples from both respiratory (lung) and systemic (serosa, joint, CSF, and serum) sites from S. suis infected pigs and utilized both RNA sequencing and proteomic techniques to measure gene expression and protein production in these collected samples. Combined, this work has identified H. parasuis and S. suis factors expressed during respiratory tract colonization and systemic disease that can be used to develop vaccine, diagnostic, and non-antibiotic intervention strategies. Objective 1, Subobjective 1.2: The goal of this subobjective is to identify genetic elements that differentiate human and swine MRSA isolates. Livestock-associated methicillin-resistant S. aureus (LA-MRSA) is at the center of debates about antibiotic use in the swine industry. It is well demonstrated that the original ST398 LA-MRSA in Europe poses a low risk to public health. Apart from initial studies conducted by ARS scientist in Ames, Iowa, there has been minimal research into the risks of ST5 LA-MRSA, which are uniquely prevalent to North America. The ST5 lineage (unlike ST398) is a major cause of human infections globally. Building on our previous results, we obtained and completed whole genome sequencing for 150 human clinical MRSA ST5 isolates from the states of Iowa and Minnesota, representing areas of high-density swine production. 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 AMR. Results indicate distinct genetic lineages for swine versus human isolates, suggesting swine isolates are not likely to contribute to human disease. Objective 2, Subobjective 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. We completed swine studies examining the effect of the antibiotic oxytetracycline delivered in feed or by injection on the respiratory microbial community. Results indicate that short-term antibiotic exposure from a broad-spectrum antibiotic like oxytetracycline influences the upper respiratory microbiota. We performed swine studies to evaluate changes in the respiratory microbial community due to infection from common swine respiratory pathogens, porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus (IAV), and B. bronchiseptica. Specific genera of bacteria, such as Actinobacillus and Streptococcus that contain pig pathogens, showed significant increases in abundance after IAV infection. Additional coinfection studies with B. bronchiseptica and S. suis were completed to test whether Bordetella directly increased colonization and development of disease with Streptococcus. Objective 2, Subobjective 2.2: The purpose of this subobjective is to identify conserved outer membrane proteins of H. parasuis that might be cross protective against multiple strains and serotypes. We vaccinated pigs with bacterins made from two H. parasuis serovar 5 strains and evaluated 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. We used an outer membrane protein (OMP) enrichment method and identified OMPs that reacted with antiserum from either protected or nonprotected pigs, which revealed 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. Combined, we identified potential protective factors for future vaccine studies. Objective 2, Subobjective 2.3: The goal of this subobjective is to determine the role of biofilms in persistence of pathogens in the respiratory tract of swine. The swine bacterial pathogens B. bronchiseptica, A. pleuropneumoniae, S. suis and H. parasuis can form robust biofilms, which protects them from antimicrobials and enables them to avoid host clearance mechanisms. We conducted multispecies biofilm studies employing a variety of B. bronchiseptica mutants harboring an in-frame deletion of specific genes. We additionally tested the effects of sub-minimal inhibitory concentrations of antibiotics commonly used by the swine industry on the capacity of S. suis to form biofilms. We demonstrated 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 biofilm formation, likely increasing survival and persistence of S. suis within the respiratory tract. Many bacteria use cyclic dimeric GMP (c-di-GMP) to regulate aspects of virulence, including biofilm formation. We generated mutants harboring deletions in genes encoding the enzymes responsible for synthesis and degradation of c-di-GMP and tested these mutants for the ability to colonize, cause disease, persist within the respiratory tract, and transmit host-to-host. While further work is needed, our preliminary findings suggests that maximum regulation of the c-di-GMP system is required for maximal persistence and disease severity. These results provide a promising target for future vaccine and biotherapeutic development. Objective 3, Subobjective 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 conducted swine studies to evaluate a capsule deficient P. multocida mutant for the ability to colonize and cause disease and demonstrated that the capsule mutant failed to survive host immune mechanisms and failed to colonize and cause disease in the pig, providing potential vaccine development. We tested the ability of an unencapsulated H. parasuis strain to function as a vaccine and demonstrated that the unencapsulated strain protected against the parent H. parasuis strain and a strain from a different capsule type, suggesting heterologous protection. We then demonstrated that the unencapsulated strain was protective against a broad range of common capsular types. Most vaccines used against H. parasuis and S. suis consist of inactivated, whole bacteria, which tends to generate a capsule or strain specific response, limiting the efficacy of the vaccine against other strains and serovars. To avoid this, we identified H. parasuis proteins and conducted swine studies to determine if these proteins can provide cross protection against other strains and serovars. We additionally tested five individual S. suis proteins with several adjuvants for their ability to induce a protective immune response against S. suis infection and demonstrated that when given together, the proteins are protective. We demonstrated that Streptococcus equi subspecies zooepidemicus (SEZ) isolates from the recent 2019 outbreaks cause rapid and severe disease in pigs compared to other more typically isolated strains. We also tested the use of an inactivated vaccine against the newly identified highly virulent strain of SEZ in pigs. Objective 3, Subobjective 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 successfully demonstrated that pigs given the vectored G-CSF had an improved outcome when infected with S. suis.

Accomplishments
1. Streptococcus equi subspecies zooepidemicus causes disease in healthy, conventionally raised pigs. Streptococcus equi subspecies zooepidemicus (SEZ) was isolated from pigs that died during high mortality events in 2019. These events reported severe disease and death of 30-50% of exposed animals. Before 2019, SEZ was not considered an important cause of disease in pigs. ARS researchers in Ames, Iowa, in collaboration with Iowa State University and the National Veterinary Services Laboratories have determined SEZ causes disease in healthy, non-stressed sows and 4-month-old pigs. When scientists exposed pigs to a 2019 SEZ field isolate, the pigs developed severe disease. This research confirmed that healthy, conventionally raised pigs can develop SEZ disease without stress or coinfection. Thus, the general swine population is susceptible to SEZ infection. These findings are important in evaluating the risk of SEZ to the swine industry. The disease model developed can also be used to evaluate treatment and prevention strategies for SEZ.

Review Publications
Nicholson, T.L., Waack, U., Anderson, T.K., Bayles, D.O., Zaia, S.R., Goertz, I., Eppinger, M., Hau, S.J., Brockmeier, S., Shore, S. 2021. Comparative virulence and genomic analysis of streptococcus suis isolates. Frontiers in Microbiology. 11. Article 620843. https://doi.org/10.3389/fmicb.2020.620843.
Hau, S.J., Eberle, K.C., Brockmeier, S. 2021. Importance of strain selection in the generation of heterologous immunity to Glaesserella (Haemophilus) parasuis. Veterinary Immunology and Immunopathology. 234. Article 110205. https://doi.org/10.1016/j.vetimm.2021.110205.
Stuart, K.L., Bayles, D.O., Shore, S., Nicholson, T.L. 2021. Complete genome sequence of Escherichia coli Antibiotic-Resistance Isolate AR Bank #0346. Microbiology Resource Announcements. 10(23). https://doi.org/10.1128/MRA.00305-21.