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

Location: Virus and Prion Research

2020 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
Objective 1, Subobjective 1.1: 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. Streptococcus suis (S. suis) is regarded as an emerging human pathogen and a reservoir for antimicrobial resistance (AMR) that can be easily transferred to other bacterial commensals and pathogens of both animals and humans. Most genomic sequence data needed to evaluate potential risks for S. suis isolates to function as zoonotic agents and/or reservoirs for the spread of AMR is derived from non-U.S. isolates. Whole-genome sequence data was obtained for about 100 S. suis isolates collected in the U.S. between 2015-2017. Draft genome assemblies were completed to identify all genomic AMR elements and other genomic elements that could increase the capacity of S. suis to transmit, colonize, and/or cause disease in humans. AMR susceptibility testing was performed on these isolates to determine resistance against specific antibiotics. This analysis demonstrated a high degree of diversity among S. suis isolates in the U.S. Objective 1, Subobjective 1.2: Identify the 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. Based on genetic analysis, previous data demonstrated a clear separation between clinical MRSA ST5 isolates obtained from agricultural and non-agricultural settings. This current research builds on previous studies with analyses of about 150 clinical MRSA ST5 isolates from hospital and community settings from swine production areas in the states of Iowa and Minnesota. Results will determine the extent (if any) to which ST5 MRSA contribute to the burden of human disease in regions of high swine density. Objective 2, Subobjective 2.1: The goal of this subobjective is to determine the role of coinfections on development of disease with bacterial pathogens of swine either via direct mechanisms or alteration of the respiratory microbiota (all the microorganisms that inhabit respiratory sites), which in turn may contribute to carriage and development of disease with respiratory acquired pathogens. Experiments designed to examine the changes in the respiratory microbial community in swine as a result of infection with the common swine respiratory pathogens - porcine reproductive and respiratory syndrome virus, influenza A virus, and Bordetella bronchiseptica, demonstrated these pathogens altered the microbial composition of the respiratory tract. In some cases, increased abundance of potential pathogens such as Actinobacillus and Streptococcus occurred. Additional coinfection studies with Bordetella bronchiseptica and Streptococcus suis were completed to examine 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 Glaesserella (Haemophilus) parasuis and Streptococcus suis that might be cross protective against multiple strains and serotypes. “Immunoproteomic” techniques such as immunoprecipitation and phage display libraries were used to identify proteins that might be candidates for future cross-protective vaccine studies. Objective 2, Subobjective 2.3: Determine the role of biofilms in persistence of pathogens in the respiratory tract of swine. Biofilms are important because they protect the bacteria from a variety of host clearance mechanisms and antimicrobial compounds. The swine bacterial pathogens Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, S. suis, and H. parasuis are capable of forming robust biofilms. Each of these bacterial species typically colonizes the tonsil and nasal cavity of swine causing a variety of symptoms ranging from asymptomatic carriage to lethal systemic disease. Many bacteria use cyclic dimeric GMP (c-di-GMP) to regulate a multitude of virulence functions including biofilm formation. To begin testing the contribution of c-di-GMP to persistent swine respiratory bacterial infections, genes encoding the enzymes responsible for synthesis and degradation of c-di-GMP were constructed in B. bronchiseptica. Biofilm formation, as well as other virulent related functions were evaluated under laboratory conditions. The transcriptional responses of each mutant compared to wild-type bacteria was additionally performed to acquire a comprehensive list of genes that are regulated by c-di-GMP. In addition, the mutants (each harboring a deletion in a gene encoding an enzyme responsible for synthesis or degradation of c-di-GMP) were evaluated in swine infection studies for the ability to colonize, cause disease, result in long-term or persistent colonization, and transmit host-to-host. 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. The majority of vaccines against Glaesserella (Haemophilus) parasuis and Streptococcus suis are based on a killed, whole bacteria platform, for which the immune response tends to be capsule type specific, and the vaccines are thus unable to provide broad cross protection. ARS scientist in Ames, Iowa, tested several G. parasuis proteins for potential cross-protection in vaccine trials in swine. In addition, S. suis proteins newly identified with collaborators were combined with several adjuvants as potential vaccine candidates against Streptococcal disease in swine. ARS scientists in Ames, Iowa, have been working with colleagues in the Animal Plant Health Inspection Service and state veterinary diagnostic laboratory to investigate high mortality outbreaks of Streptococcus equi subsp. zooepidemicus (Strep. zoo) that occurred in swine in the United States and Canada in 2019. Genetic analysis demonstrated these outbreaks appeared to be caused by similar isolates that are closely related to a strain identified in Chinese swine. ARS scientist conducted studies in sows and finishing age swine to examine the severity of disease with these strains, and to compare them to more distantly related Strep. zoo isolates that have been sporadically isolated. The Strep. zoo isolates from the recent outbreaks caused rapid and severe disease compared to other more typically isolated strains.

Accomplishments
1. Genomic regions of Streptococcus suis reveal differences that may contribute to the spectrum of clinical disease. Studies addressing virulence mechanisms used by S. suis have been complicated because different isolates can cause a spectrum of disease. ARS researchers at Ames, Iowa, evaluated the ability of nine isolates of S. suis to cause disease following intranasal challenge in swine followed by comparative genomic analyses to identify genetic differences that could be associated with swine-virulence. Outcomes of intranasal challenge with the isolates ranged from lethal systemic disease to asymptomatic carriage. Whole genome sequencing followed by comparative genomic analyses revealed several notable regions of difference, including regions encoding secreted and membrane-associated factors, which likely contributed to the spectrum of clinical disease observed. In addition, transmissible elements containing antimicrobial resistance genes were identified within the S. suis genomes. Collectively, these results provide a foundation for understanding the genomic attributes responsible for the spectrum of virulence that exist among S. suis isolates. This information is paramount to designing effective vaccines needed by the swine industry to mitigate S. suis disease and decrease public health concerns.

2. Role of the capsule in causing disease with Glaesserella (Haemophilus) parasuis. Glasser’s disease, which can cause high mortality in pigs, is caused by the bacterium Glaesserella (Haemophilus) parasuis. G. parasuis isolates are defined by their capsule type. The capsule of bacteria is composed of surface polysaccharides and is often an important virulence factor enhancing the bacteria’s ability to cause disease. ARS researchers at Ames, Iowa, and their colleagues at the University of Cambridge made a mutant of G. parasuis that is unable to produce capsule to determine what role the capsule plays in the ability of G. parasuis to cause disease. The mutant did not cause disease in pigs and was more susceptible to being killed by the immune system. The mutant was also quickly cleared from the pigs, which may limit its ability to cause disease. Thus, it was shown that the capsule is an important factor for colonization of the nose and protecting G. parasuis from the immune system. This information explains, in part, how G. parasuis causes disease and can be used to explore the importance of capsule in a protective immune response to the bacteria, which will lead to the development of more effective vaccines for use by the swine industry.

3. Route of antibiotic administration impacts the normal respiratory and fecal microbial community in swine and resistance gene abundance in swine feces. Antibiotics are a critical tool for fighting bacterial infections, yet their use can have negative consequences, such as the disturbance of healthy bacterial communities and the dissemination of antibiotic residues in feces. ARS researchers at Ames, Iowa, and their colleagues theorized that the route of antibiotic delivery would influence negative impacts on respiratory and intestinal microbial community and performed a study in pigs to evaluate whether oxytetracycline (oxytet), an antibiotic commonly used in the U.S. swine industry, delivered by injection versus in the feed had a greater impact on microbial diversity and antibiotic resistance genes abundance. Injected oxytet resulted in a higher concentration of the antibiotic in the blood compared to in-feed oxytet delivery, which resulted in higher antibiotic concentrations in feces. Similar changes were observed in microbial community regardless of route of oxytet administration; however, the impact on the microbial community in both the respiratory and intestinal tract was more pronounced with in-feed administration. Fecal antibiotic resistance genes abundance was increased with in-feed administration over injected. The findings are informative for disease management in food animals, but also manure management and antibiotic therapy in human medicine for improved antibiotic stewardship.

4. Importance of using a relevant model for testing vaccine candidates for swine diseases. The bacterium Glaesserella parasuis causes severe disease in pigs. It is difficult to prevent because vaccines made from inactivated whole cells do not prevent disease with all strains. There is now focus on developing vaccines from G. parasuis proteins that can provide protection against many strains. ARS researchers at Ames, Iowa, and their colleagues at the University of Cambridge identified and evaluated the use of two G. parasuis proteins as vaccine candidates (RlpB and VacJ). The proteins induced high antibody levels; however, these antibodies were not able to protect pigs from challenge with G. parasuis. A previous publication by another laboratory indicated that VacJ was protective against G. parasuis in a mouse model. Although these proteins were not effective as a vaccine in swine, this work demonstrates the importance of confirming results from other animal models in pigs, so that non-efficacious vaccines are not disseminated to the swine industry.

Review Publications
Brockmeier, S., Hau, S.J., Mou, K.T., Bayles, D.O. 2019. Transcriptomic differences noted in Glasserella parasuis between growth in broth and on agar. PLoS One. 14(8):e0220365. https://doi.org/10.1371/journal.pone.0220365.
Hau, S.J., Luan, S., Loving, C.L., Nicholson, T.L., Wang, J., Peters, S.E., Seilly, D., Weinert, L., Langford, P.R., Rycroft, A., Wren, B.W., Maskell, D.J., Tucker, A.W., Brockmeier, S. 2020. Evaluation of the recombinant proteins RlpB and VacJ as a vaccine for protection against Glaesserella parasuis in pigs. BMC Veterinary Research. 16:167. https://doi.org/10.1186/s12917-020-02377-5.
Segura, M., Aragon, V., Brockmeier, S., Gebhart, C., De Greeff, ., Kerdsin, ., O’Dea, M.A., Okura, M., Saléry, M., Schults, C., Valentin-Weigand, P., Weinert, L.A., Wells, J.M., Gottschalk, M. 2020. Update on Streptococcus suis research and prevention in the era of antimicrobial restriction: 4th International Workshop on S. suis. Pathogens. 9(5). https://doi.org/10.3390/pathogens9050374.
Eberle, K.C., Hau, S.J., Luan, S., Weinert, L., Stasko, J.A., Wang, J., Peters, S.E., Langford, P.R., Rycroft, A., Wren, B.W., Maskell, D.J., Tucker, A.W., Brockmeier, S. 2020. Generation and evaluation of a Glaesserella parasuis capsular mutant. Infection and Immunity. 88(5):e00879-19. https://doi.org/10.1128/IAI.00879-19.
Mou, K.T., Allen, H.K., Alt, D.P., Trachsel, J.M., Hau, S.J., Coetzee, J.F., Holman, D.B., Kellner, S.G., Loving, C.L., Brockmeier, S. 2019. Shifts in the swine nasal microbiota of swine in response to different dosing regimens of oxytetracycline administration. Veterinary Microbiology. 237(1084020). https://doi.org/10.1016/j.vetmic.2019.108386.
Ricker, N., Trachsel, J.M., Colgan, P., Jones, J., Choi, J., Lee, J., Coetzee, J., Howe, A., Brockmeier, S., Loving, C.L., Allen, H.K. 2020. Toward antibiotic stewardship: Route of antibiotic administration impacts the microbiota and resistance gene diversity in swine feces. Frontiers in Veterinary Science. 7:255. https://doi.org/10.3389/fvets.2020.00255.
Stuart, K.L., Shore, S., Nicholson, T.L. 2019. Complete genome sequence of Escherichia coli antibiotic resistance (AR) isolate bank #0349. Microbiology Resource Announcements. 8(48). https://doi.org/10.1128/MRA.01078-19.
Nicholson, T.L., Bayles, D.O., Shore, S. 2020. Complete genome sequence of Bordetella bronchiseptica strain KM22. Microbiology Resource Announcements. 9(4). https://doi.org/10.1128/MRA.01207-19.