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ARS Home Midwest Area Ames, Iowa National Animal Disease Center Virus and Prion Research Research





- Nicholson, Tracy
- Hau, Samantha
- 2023
- 2022
- Publications
- Animal Health (103)

Research Project: Virulence Mechanisms, Microbiome Changes and Control Strategies for Priority Bacterial Infections in Swine **Location:
                            Virus and Prion Research** 2022 Annual Report **Objectives**
                                

Objective 1: Investigate the role of virulence mechanisms and impact on the swine respiratory microbiota of priority, emerging or re-emerging pathogens such as Glaesserella parasuis and Streptococcus suis. This includes evaluating the presence and mechanisms of transfer of antimicrobial resistance (AMR) genes harbored by these bacterial pathogens. 
Subobjective 1.1: Identify genetic determinants contributing to the virulence of G. parasuis and S. suis through the use of comparative genomics, functional genomics, and proteomics. 
Subobjective 1.2: Evaluate the presence of AMR genes, determine the genomic location of identified AMR genes, and determine which specific type of MGEs most abundantly contain AMR genes harbored by priority, emerging or re-emerging swine bacterial pathogens. 
Subobjective 1.3: Determine the impact of infection with priority swine pathogens on the respiratory microbiota and development of secondary bacterial infections. 
Objective 2: Develop and evaluate novel non-antibiotic intervention and management strategies to control priority bacterial diseases in swine, including vaccine platforms and therapeutics. 
Subobjective 2.1: Develop novel vaccines and therapeutics to prevent clinical disease or decrease colonization caused by priority, emerging or re-emerging swine bacterial pathogens. 
Subobjective 2.2: Evaluate the host response to vaccination or infection with bacterial pathogens such as G. parasuis or S. suis to identify mechanisms of cross protective immunity. **Approach**
                                

The first goal for this research plan is to investigate the role of virulence mechanisms and impact on the swine respiratory microbiota of priority, emerging or re-emerging pathogens such as Glaesserella parasuis and Streptococcus suis. This includes evaluating the presence and mechanisms of transfer of antimicrobial resistance (AMR) genes harbored by these bacterial pathogens. First, we will use genome sequence data to identify genes encoding virulence factors and compare population structure of isolates, determine whether and how bacterial gene and protein expression responds to mammalian host signals, and determine which genes enable bacteria to colonize the swine respiratory tract and cause invasive disease. Next, we will compare the whole genome sequences of swine bacterial pathogens to identify AMR genes and determine whether or not identified AMR genes are located within mobile genetic elements (MGEs) such as plasmids, prophages, integrative and conjugative elements (ICEs), insertion sequences, and transposons. Finally, we will determine whether and how the microbiota changes following infection with swine respiratory pathogens and whether changes contribute to enhanced disease and evaluate the impact of pathogen-pathogen interactions occurring during infections with G. parasuis or S. suis. The second goal for this research plan is to develop and evaluate novel non-antibiotic intervention and management strategies to control priority bacterial diseases in swine, including vaccine platforms and therapeutics. First, we will develop novel vaccines that can prevent clinical disease with priority bacterial pathogens of swine, such as G. parasuis and S. suis. Examples of these novel vaccines include a G. parasuis capsule mutant bacterin, protein subunit vaccines for S. suis and G. parasuis, a S. suis capsule mutant, a conjugated capsule vaccine for G. parasuis, and a SEZ bacterin vaccine. We will additionally isolate bacteriophages active against swine LA-MRSA ST398 isolates and evaluate the use of bacteriophage treatment to reduce colonization. Finally, we will identify immunogenic, protective, and conserved outer membrane proteins of G. parasuis through immunoproteomics that will be cross protective against multiple serotypes. **Progress Report**
                                

Objective 1, Subobjective 1.1: The goal of this subobjective is to use genome sequence data to identify genes encoding virulence factors and compare population structure of isolates, determine whether and how bacterial gene and protein expression responds to mammalian host signals, and determine which genes enable bacteria to colonize the swine respiratory tract and cause invasive disease. Swine experiments have been completed in which samples were collected from respiratory tract and systemic site from pigs challenged with Glaesserella parasuis. Samples are currently being further processed for the purpose of RNA sequencing to determine the transcriptional response of G. parasuis during respiratory tract colonization and systemic disease. Whole-genome sequencing, assembly, and annotation for Streptococcus suis isolates obtained from swine production facilities within the U.S. have been completed. These genome assemblies have been screened for genes encoding any putative factors that could increase the capacity of these isolates to cause disease using both publicly available databases as well as published literature detailing experimentally verified virulence factors. These genome assemblies have also been used to construct phylogenetic trees for the purpose of determining the population structure and genetic relatedness of S. suis isolates obtained from within the U.S.
Objective 1, Subobjective 1.2: The goal of this subobjective is to compare the whole genome sequences of swine bacterial pathogens to identify antimicrobial resistance (AMR) genes and determine whether or not identified AMR genes are located within mobile genetic elements (MGEs) such as plasmids, prophages, integrative and conjugative elements (ICEs), insertion sequences, and transposons. Phenotypic antimicrobial susceptibility testing has been completed for Streptococcus suis isolates obtained from within the U.S. Over ninety percent of the isolates tested exhibited resistance to two or more antimicrobial classes, with the highest frequencies of resistance observed for tetracycline and macrolide/lincosamide/streptogramin (MLSb) classes. Numerous genes conferring antimicrobial resistance were found among the S. suis isolates, many were located within MGEs. To build on previous published studies demonstrating a clear separation between livestock associated methicillin resistant Staphylococcus aureus (LA-MRSA) sequence type five (ST5)  and clinical human MRSA ST5 isolates we completed AMR analysis, detection of Mobile Genetic Elements, virulence factor analysis, and phylogenetic analysis for clinical human MRSA ST5 isolates obtained from swine dense regions. The inclusion of the data from these isolates into our established phylogenetic tree provides a more complete understanding of the population structure and genetic relatedness between swine-associated and clinical MRSA ST5 isolates to fully evaluate origin, evolution, and zoonotic potential of LA-MRSA ST5 isolates.
Objective 1, Subobjective 1.3: The goal of this subobjective is to determine whether and how the microbiota changes following infection with swine respiratory pathogens and whether changes contribute to enhanced disease and evaluate the impact of pathogen-pathogen interactions occurring during infections with G. parasuis or S. suis. Swine experiments have been completed for the purpose of determining the changes in the respiratory microbial community in swine as a result of infection with common swine respiratory pathogens, such as porcine reproductive and respiratory syndrome virus, influenza A virus, and Bordetella bronchiseptica. Following infection with some pathogens we found increased abundance of potential pathogens such as Actinobacillus and Streptococcus occurred. However, following infection with influenza A virus, minimal changes in the upper respiratory microbiome was observed.
Objective 2, Subobjective 2.1: The goal of this subobjective is to develop novel vaccines that can prevent clinical disease with priority bacterial pathogens of swine, such as G. parasuis and S. suis. Swine experiments have been completed in which a G. parasuis mutant was evaluated for the ability to prevent disease with heterologous G. parasuis strains, including a serovar 1 isolate and a serovar 4 isolate. Autogenous vaccines are vaccines made from inactivated bacteria isolated from an affected farm and are commonly used by the swine industry to prevent bacterial infections. Swine experiments have been completed in which an inactivated Streptococcus zooepidemicus bacterin was evaluated for the ability to prevent disease. 
Objective 2, Subobjective 2.2: The goal of this subobjective is to identify immunogenic, protective, and conserved outer membrane proteins of G. parasuis through immunoproteomics that will be cross protective against multiple serotypes. Experiments have been completed in which sera from pigs immune to G. parasuis was used to identify proteins produced by G. parasuis for the purpose of identifying proteins that can serve as protective antigens and then be used in a vaccine to prevent diseases caused by G. parasuis. Additionally, a G. parasuis phage display library is currently being constructed for the purpose of identifying proteins that can serve as protective antigens, which can be used in a vaccine to prevent diseases caused by G. parasuis. **Accomplishments**
                                

1. Streptococcus suis isolates obtained from within the U.S. harbor antimicrobial resistance genes in mobile genetic elements, but do not harbor genes encoding the capacity to cause disease in humans. Streptococcus suis is a swine pathogen that infects multiple species including humans and causes extensive health and economic burdens to the pork industry worldwide. Most genomic sequence data needed to evaluate potential risks for S. suis to infect humans or harbor antimicrobial resistance (AMR) is derived from isolates obtained outside the U.S. ARS researchers at Ames, Iowa, determined the antimicrobial resistance profiles and performed comparative genomic analyses among S. suis obtained from within the U.S. While genes conferring AMR were found within the genomes of the U.S. S. suis isolates, no genes encoding the increased capacity of S. suis to cause disease in humans were detected. Collectively, these results provide a detailed assessment of the genetic diversity, including virulence-related factors and AMR genes, of S. suis within the U.S. swine production environment and serve as a blueprint for determining any potential risks associated with occupational exposure to these bacteria, while also providing important data to address public concerns. 2. Antibiotic treatment but not autogenous vaccination effective against Streptococcus equi subspecies zooepidemicus. In 2019, a novel Streptococcus equi subspecies zooepidemicus (SEZ) strain was identified in pigs in North America. SEZ causes severe disease in pigs resulting in death losses up to 50% under field conditions. No research has investigated intervention strategies for the novel North American SEZ isolates. Two common ways the swine industry prevents losses due to bacterial infections are antibiotic treatment and autogenous vaccines. Autogenous vaccines are vaccines made from inactivated bacteria isolated from the affected farm. ARS researchers in Ames, Iowa, evaluated antibiotic treatment and autogenous vaccination as intervention strategies against SEZ. Researchers demonstrated the efficacy of ceftiofur treatment for SEZ disease in pigs. Survival rate for clinically ill pigs treated with ceftiofur was 94% while survival rate for untreated pigs was 6%. Researchers also showed vaccination with an autogenous vaccine was unable to prevent the development of SEZ disease. These results are important to inform producers on management of SEZ outbreaks including treatment and vaccine strategy. This will reduce losses following SEZ exposure and prevent the implementation of costly, ineffective preventive measures against SEZ. 3. Minimal changes in the upper respiratory microbiome of pigs does not explain secondary infections of porcine respiratory disease complex (PRDC). Influenza A virus (IAV) is an important cause of respiratory disease in pigs. Often, IAV infections are complicated by secondary bacterial infections leading to more severe disease. These complicated respiratory infections, termed the porcine respiratory disease complex (PRDC), are one of the most costly diseases and most frequent causes for antibiotic use in the swine industry. The mechanism leading to these secondary bacterial infections is not well understood. ARS researchers in Ames, Iowa, investigated the impact of IAV infection on the upper respiratory tract microbiome to help understand the development of secondary bacterial infections. Following IAV infection, there was no change to the microbial diversity but some minor changes in the abundance of bacterial groups containing respiratory pathogens. The data showed that secondary infections following IAV are not associated with changes in the prevalence of specific bacteria in the upper respiratory tract. This work contributes to the general understanding of respiratory infections in pigs and will help direct future research investigating the development of and intervention strategies for PRDC. **Review Publications**
                            *Hau, S.J., Devries, A.C., Brockmeier, S. 2022. Bacterin vaccination provides insufficient protection against Streptococcus equi subspecies zooepidemicus infection in pigs. Frontiers in Veterinary Science. 9. Article 827082. https://doi.org/10.3389/fvets.2022.827082.*
                                *Hau, S.J., Brockmeier, S., Lantz, K., Stuart, K.L., Sitthicharoenchai, P., Macedo, N., Derscheid, R.J., Burrough, E.R., Robbe-Austerman, S. 2022. Replication of Streptococcus equi subspecies zooepidemicus disease in swine. Veterinary Microbiology. 264. https://doi.org/10.1016/j.vetmic.2021.109271.*


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