Location: Virus and Prion Research
2019 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: To determine how bacterial pathogens respond to the host environment and determine which specific genes enable bacteria to colonize the swine respiratory tract. Haemophilus parasuis is a bacterium that causes Glässer's disease in swine characterized by acute infections and chronic debilitation. 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. Building on our previous work, the gene expression differences for the non-virulent D74 and virulent Nagasaki H. parasuis isolates were evaluated after growth in air supplemented to 5 percent CO2. 284 genes were differentially expressed in strain D74 in response to 5 percent CO2, while only 36 genes were differentially expressed in strain Nagasaki. Our data support the idea that H. parasuis gene expression can be fine-tuned in response to signals specific to different microenvironments within the respiratory tract or deeper tissues within the host. More importantly, these data demonstrate that strain D74 is more transcriptionally responsive to carbon dioxide levels that mimic in vivo conditions within the respiratory tract and suggest that non-virulent H. parasuis strains may be more adaptive to colonization within the respiratory tract than virulent strains. Collectively, the unique genomic and transcriptional features identified in this study provide a foundation for understanding the genomic attributes responsible for the spectrum of virulent phenotypes that exist among H. parasuis isolates. This information is paramount to designing effective vaccines needed by the swine industry to mitigate H. parasuis disease burden.
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 elimination by the host. We previously showed that the capsule of H. parasuis was an important virulence factor. Recent studies indicate that the amount of capsule the bacteria produces not only affects the ability of the bacteria to cause disease but whether or not anti-capsular immunity is an important component of protection. Unlike other strains we have tested, a strain that appears to be highly encapsulated required anti-capsular antibodies to prevent disease in the pig. We believe the capsule is masking other potential protein targets on the surface of the bacteria which is important to know for designing potential non-capsule based cross-protective vaccines.
Subobjective 1.2: To identify the genetic elements that differentiate human and swine methicillin-resistant Staphylococcus aureus (MRSA) isolates. Livestock-associated (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. In contrast, apart from our initial studies, there has been minimal research into the risks of to ST5 LA-MRSA, which are uniquely prevalent to North America. This is particularly problematic, because the ST5 lineage (unlike ST398) is a major cause of human infections globally. Our previous published data enabled us to demonstrate a clear separation between MRSA ST5 isolates obtained from agricultural and clinical settings based on genomic differences identified in clinical isolates from regions where swine exposure was negligible. The overall goal of our current work is to build on our previous published studies by including genomic analyses of clinical MRSA ST5 isolates from the hospital and community settings from areas of swine production. We have received around 150 human clinical MRSA ST5 isolates from the state health laboratory for the states of Iowa and Minnesota. These isolates were obtained from areas of high density swine production. We are currently isolating genomic DNA and preparing these samples for DNA sequencing. The inclusion of these isolates into our established phylogenetic tree will allow us to more fully understand 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. That knowledge will enable us to determine the extent (if any) to which ST5 MRSA contribute to the burden of human disease in regions of high swine density and serve as a blueprint for a complete assessment of the risks associated with occupational exposure to Staphylococcus aureus (S. aureus) and enable assessment of the needs for designing intervention strategies to reduce the risk of S. aureus colonization of workers within the swine industry.
Objective 2, Subobjective 2.1: To establish whether 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 have been analyzing data from 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 (IAV), and Bordetella bronchiseptica. IAV is an important contributing pathogen to the porcine respiratory disease complex (PRDC). Evidence in humans have shown that IAV can disturb the nasal microbiota and increase host susceptibility to secondary infections. Overall, IAV had no marked impact on the swine nasal microbiota during acute infection. However, after the period of acute infection, the nasal microbiota experienced significant changes in microbial composition. Specific genera of bacteria, such as Actinobacillus and Streptococcus that contain pig pathogens, showed significant increases in abundance after IAV infection. Results from this study highlight the need to further study what implications these changes post-infection have on host susceptibility to subsequent infections, such as those caused by PRDC.
Subobjective 2.2: To identify outer membrane proteins of H. parasuis that will be cross protective against multiple strains. 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 is 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 have continued to identify outer membrane proteins with potential broad protective capability from H. parasuis using immunoprecipitation methods with immune serum from pigs that were broadly protected versus pigs that were only protected against one strain of H. parasuis.
Subobjective 2.3: 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. The swine bacterial pathogens Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Streptococcus 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. A key barrier towards the development of improved vaccines or interventions for the infections caused by these bacteria is a gap in our understanding of the mechanisms contributing to persistence in the host, in which colonized pigs continue to shed and transmit these bacteria. To begin addressing the contribution of specific factors produced by one bacterial species to the biofilm capacity of other bacterial species, we have initiated experiments to evaluate the contribution of the capsule of S. suis in biofilm formation for S. suis alone and in combination with other bacterial isolates. Our preliminary data support the idea that capsule production is inhibitory to biofilm formation. While more work is needed, this information is paramount to designing effective intervention strategies to mitigate persistent colonization by bacterial pathogens within the upper respiratory tract of swine.
Objective 3, Subobjective 3.1: 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 H. parasuis and S. 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. We created a strain of H. parasuis that does not produce capsule and previously found that using the unencapsulated strain as a vaccine protected against the parent strain that it was created from and another strain from a different capsule type. We further tested the unencapsulated strain against several other common capsule types found in pigs and found it to be protective against a broad range of common capsular types. We also tested several H. parasuis proteins we identified as potentially important in cross protection as a vaccine and although the pigs developed good antibody responses against the proteins, they were not protective by themselves. We are currently identifying other proteins that we can try as vaccine candidates. We are also currently testing newly identified proteins of S. suis with several adjuvants as potential vaccine candidates.
Accomplishments
1. Subinhibitory concentrations of antibiotics commonly used to treat swine increase Streptococcus suis (S. suis) biofilm formation. S. suis is the leading bacterial swine pathogen worldwide causing a wide variety of clinical presentations in pigs ranging from asymptomatic carriage to lethal systemic disease. S. suis is also a zoonotic pathogen capable of causing invasive disease in humans. A barrier towards the development of improved vaccines or interventions for S. suis infections is a gap in our understanding of the factors contributing to persistence in the host, in which colonized pigs continue to shed and transmit S. suis. Biofilms are adherent communities of bacteria that are protected from clearance mechanisms and are considered a key factor contributing to chronic or persistent bacterial infections. Routine management practices involve treating all pigs in the same pen or barn with appropriate antibiotics upon observing any pig exhibiting clinical signs associated with a bacterial infection. ARS researchers at Ames, Iowa, studied the effects of sub-minimal inhibitory concentrations of antibiotics commonly used by the swine industry on the development of S. suis biofilms and found amoxicillin, lincomycin, and oxytetracycline increase biofilm formation while bacitracin, carbadox, chlortetracycline, enrofloxacin, gentamicin, neomycin, sulfadimethoxine, tiamulin, and tylosin did not. Collectively, our data demonstrate that exposure to some commonly used antibiotics contributes to increased biofilm formation of S. suis, thereby potentially increasing survival and persistence within the respiratory tract of swine and increasing transmission dynamics among animals. This data is critical for proper selection of antibiotics for successful treatment of swine bacterial diseases while minimizing potential collateral consequences.
2. Antimicrobial resistance distribution differs among methicillin resistant Staphylococcus aureus (MRSA) sequence type (ST) 5 isolates from health care and agricultural sources. Antimicrobial resistance is an expanding public health concern and MRSA is a notable example. Since the discovery of livestock associated MRSA (LA-MRSA), public health concerns have arisen surrounding the potential of LA-MRSA isolates to serve as a reservoir for antimicrobial resistance determinants. ARS researchers at Ames, Iowa, compared swine associated LA-MRSA ST5 and human clinical MRSA ST5 isolates for antimicrobial susceptibilities and genes associated with antimicrobial resistance. Swine associated LA-MRSA ST5 isolates exhibited resistance to fewer antibiotics than clinical MRSA ST5 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 associated LA-MRSA ST5 and clinical MRSA ST5 isolates. Our results demonstrate that antimicrobial susceptibilities and genes that encode antimicrobial resistance among swine associated LA-MRSA ST5 and clinical MRSA ST5 isolates are separate and distinct suggesting that swine do not play a major role in maintaining a MRSA ST5 reservoir for humans.
3. Genomic analysis indicates distinct populations of methicillin resistant Staphylococcus aureus (MRSA) isolates from swine compared to human clinical isolates. Livestock-associated MRSA (LA-MRSA) are lineages adapted to livestock species. LA-MRSA can be transmitted to humans and public health concerns exist because livestock may be the largest MRSA reservoir outside of hospital settings. Although the predominant European (ST398) and Asian (ST9) lineages of LA-MRSA are considered livestock adapted, North American swine also harbor ST5, a globally disseminated and highly pathogenic lineage. ARS researchers at Ames, Iowa, applied whole genome sequencing and single nucleotide polymorphism (SNP) typing to compare the population structure and genetic relatedness between swine-associated and human clinical MRSA ST5 isolates. The results revealed that swine-associated LA-MRSA and human clinical MRSA ST5 are genetically distinct. LA-MRSA isolates were found to be more similar to each other within farms, while greater genome diversity was observed among sampled human clinical MRSA ST5. Further analysis demonstrated that LA-MRSA ST5 isolates and clinical MRSA ST5 isolates harbor different virulence factors, consistent with the SNP analysis. Collectively, our data indicate LA-MRSA and clinical MRSA ST5 isolates are distinct and the swine reservoir is likely of minimal significance as a source for transmission of MRSA ST5 to humans.
4. Blood or serum exposure induce changes in gene expression, altered protein expression, and increased cell death by the Bordetella bacterial species. The classical bordetellae bacteria (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica) sense and respond to a variety of environments outside and within their mammalian hosts. By causing inflammation and tissue damage, the bordetellae are likely to encounter components of blood and/or serum during the course of a respiratory infection, and theoretically the ability of the bacteria to respond to these should be advantageous. ARS researchers at Ames, Iowa, with collaborators found that exposure to blood or serum resulted in substantial gene expression changes in B. bronchiseptica, including enhanced expression of many virulence-associated genes. Exposure to blood or serum additionally elicited production of multiple proteins not otherwise detectable, and led to increased bacterial killing of macrophages. Similar gene expression changes in response to blood/serum were observed for the other classical bordetellae, B. pertussis and B. parapertussis. These data suggest the classical bordetellae respond to signals present in blood and serum by changing their behavior in ways that likely contribute to their remarkable success, via effects on pathogenesis, persistence and/or transmission between hosts.
5. Comparative genomic and methylome analysis of non-virulent D74 and virulent Nagasaki Haemophilus parasuis (H. parasuis) isolates reveals genomic attributes underlying virulence differences. H. parasuis is a respiratory pathogen of swine and the etiological agent of Glässer's disease. H. parasuis isolates can exhibit different virulence capabilities ranging from lethal systemic disease to subclinical carriage. To identify genomic differences between disease-causing and non-disease-causing strains, ARS researchers at Ames, Iowa, obtained the closed whole-genome sequence and genome-wide methylation patterns for the highly virulent Nagasaki strain and for the non-virulent D74 strain. Evaluation of the virulence-associated genes contained within the genomes of D74 and Nagasaki showed genes unique to each strain. DNA methylation is a process by which a chemical methyl group is added to the DNA molecule and this can change the activity of a DNA segment without changing the sequence and can enhance or repress gene expression. Multiple methylation sequence motifs were identified in both D74 and in Nagasaki, but only one of the methylation sequence motifs was observed in both strains indicative of the diversity between D74 and Nagasaki. The collective information reported in this study will aid in the understanding of how some strains of H. parasuis cause disease in the pig while others exist as normal respiratory resident bacteria, as well as identifying potential targets for vaccine development to decrease the prevalence and disease burden caused by H. parasuis.
6. Blocking the ability of bacteria to “sort” proteins prevents disease due to Streptococcus suis (S. suis). S. suis colonizes the upper respiratory tracts of pigs potentially causing septicemia, meningitis and death, placing a severe burden on the agricultural industry worldwide. It is also a zoonotic pathogen known to cause systemic infections and meningitis in humans. Understanding how S. suis interacts with and colonizes its host is important for future strategies of drug and vaccine development. Some bacteria can utilize enzymes known as sortases to “sort” or direct proteins to certain areas such as the surface of the bacteria where they can play a role in host-pathogen interactions. ARS researchers at Ames, Iowa, with collaborators found that the sortases, and thus their associated sorted proteins, are essential to the outcome of disease in pigs. This both adds to our knowledge of S. suis pathogenesis and suggests that generating an immune response to the putative sorted proteins may be useful in developing a vaccine that protects against S. suis infection in swine.
7. Pre-existing respiratory infections can affect the outcome of subsequent influenza infection even after intranasal vaccination. Influenza virus can predispose pigs to pneumonia with bacteria such as Bordetella bronchiseptica. Live-attenuated influenza virus (LAIV) vaccines given intranasally provide significant protection against a range of influenza virus exposures in swine. B. bronchiseptica commonly colonizes the nasal cavity of swine and thus could be expected to be present when LAIV vaccines are given. ARS researchers at Ames, Iowa, evaluated the effects of B. bronchiseptica colonization on the performance of a LAIV vaccine in swine and found the immune response to LAIV was not impacted, and although LAIV vaccination provided significant protection against a different IAV challenge, pneumonia was more severe in pigs colonized with B. bronchiseptica and challenged with IAV, regardless of LAIV vaccination status. This study demonstrated that LAIV vaccine did reduce IAV challenge disease, but interaction between IAV challenge and B. bronchiseptica produced more disease than either respiratory pathogen by itself which demonstrates the importance of controlling both viral and bacterial pathogens.
8. Antibiotics alter the normal respiratory microbial community in swine. Antibiotic stewardship is of the utmost importance to improve animal health outcomes and prevent selection of antimicrobial resistance. There is increasing evidence of the important role the normal respiratory microbial community (microbiota) plays in shaping immune and respiratory health. However, there is little knowledge on the effects of antibiotics on the swine respiratory microbiota. Oxytetracycline is a broad-spectrum antibiotic that is used for treatment of bacterial respiratory disease in swine. ARS researchers at Ames, Iowa, showed the respiratory microbiota diversity decreased in response to oxytetracycline administration. In addition, giving the antibiotic in the feed had a greater and longer lasting impact on the nasal microbiota than the injectable route. There were also increased abundances of some pathogenic bacteria and decreased abundances of normal respiratory resident bacteria after antibiotic treatment. These results highlight the need to further assess how these changes can ultimately affect the animal’s respiratory health and risk to disease.
9. The use of immunomodulators as an alternative to antibiotic use 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 that giving an immunomodulatory protein to pigs 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 protein had an improved outcome when infected with Streptococcus suis, the leading cause of meningitis in weaned pigs. Thus, the use of this immunomodulatory protein 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.
10. Optimizing growth conditions for Haemophilus parasuis (H 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. ARS researchers at Ames, Iowa, compared the genetic expression of H. parasuis grown under different laboratory growth conditions. This information will be used to determine which conditions are best for growth of bacteria to produce vaccines and mimic animal disease in the laboratory.
Review Publications
Abente, E.J., Rajao, D.S., Santos, J., Kaplan, B.S., Nicholson, T.L., Brockmeier, S.L., Gauger, P.C., Perez, D.R., Vincent, A.L. 2018. Comparison of adjuvanted-whole inactivated virus and live-attenuated virus vaccines against challenge with contemporary, antigenically distinct H3N2 influenza A viruses. Journal of Virology. 92(22):e01328-18. https://doi.org/10.1128/JVI.01323-18.
Gestal, M.C., Rivera, I., Howard, L.K., Dewan, K.K., Soumana, I.H., Dedloff, M., Nicholson, T.L., Linz, B., Harvill, E.T. 2018. Blood or serum exposure induce global transcriptional changes, altered antigenic profile, and increased cytotoxicity by classical Bordetellae. Frontiers in Microbiology. 9:1969. https://doi.org/10.3389/fmicb.2018.01969.
Hau, S.J., Haan, J.S., Davies, P.R., Frana, T., Nicholson, T.L. 2018. Antimicrobial resistance distribution differs among methicillin resistant Staphylococcus aureus sequence type (ST) 5 isolates from health care and agricultural sources. Frontiers in Microbiology. 9:2102. https://doi.org/10.3389/fmicb.2018.02102.
Hau, S.J., Allué-Guardia, A., Rusconi, B., Haan, J.S., Davies, P.R., Frana, T.S., Eppinger, M., Nicholson, T.L. 2018. Single nucleotide polymorphism analysis indicates genetic distinction and reduced diversity of swine-associated methicillin resistant Staphylococcus aureus (MRSA) ST5 isolates compared to clinical MRSA ST5 isolates. Frontiers in Microbiology. 9:2078. https://doi.org/10.3389/fmicb.2018.02078.
Nicholson, T.L., Brunelle, B.W., Bayles, D.O., Alt, D.P., Shore, S. 2018. Comparative genomic and methylome analysis of non-virulent D74 and virulent Nagasaki Haemophilus parasuis isolates. PLoS One. 13(11):e0205700. https://doi.org/10.1371/journal.pone.0205700.
Waack, U., Nicholson, T.L. 2018. Subinhibitory concentrations of amoxicillin, lincomycin, and oxytetracycline commonly used to treat swine increase Streptococcus suis biofilm formation. Frontiers in Microbiology. 9:2707. https://doi.org/10.3389/fmicb.2018.02707.
Brockmeier, S., Loving, C.L., Eberle, K.C., Hau, S.J., Mou, K.T., Kehrli Jr., M.E. 2019. Administration of granulocyte-colony stimulating factor (G-CSF) to pigs results in a longer mean survival time after exposure to Streptococcus suis. Veterinary Microbiology. 231:116-119. https://doi.org/10.1016/j.vetmic.2019.03.010.
Hughes, H.R., Brockmeier, S., Loving, C.L. 2018. Bordetella bronchiseptica colonization limits efficacy, but not immunogenicity, of live-attenuated influenza virus vaccine and enhances pathogenesis after influenza challenge. Frontiers in Immunology. 9:2255. https://doi.org/10.3389/fimmu.2018.02255.
Faulds-Pain, A., Shaw, H.A., Terra, V.S., Kellner, S., Brockmeier, S L., Wren, B.W. 2018. The Streptococcos suis sortases SrtB and SrtF are essential for disease in pigs. Microbiology. 165:163-173. https://doi.org/10.1099/mic.0.000752.
