Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): Objective 1: Identify the transmission, genetic, and pathogenic mechanisms of the organisms associated with PRDC, concentrating on the bacterial pathogens and their interactions with each other and select swine viruses. Subobjective 1.1: Identify potential virulence factors of H. parasuis through comparative genomics. Subobjective 1.2: Bacterial response to host conditions. Subobjective 1.3: Evaluate ability of PRDC bacterial pathogens to inhibit Influenza A virus vaccine efficacy and/or exacerbate Influenza A virus-associated disease. Objective 2: Identify potential candidates for novel diagnostic assays, vaccines, and biotherapeutics for bacterial pathogens associated with PRDC. Subobjective 2.1. Develop PCR, ELISA, and/or other assays for detection of bacterial pathogens associated with PRDC. Subobjective 2.2. Identify, develop and/or test the efficacy of potential vaccine candidates to control bacterial pathogens associated with PRDC. Subobjective 2.3. Identify potential biotherapeutic candidates to control bacterial pathogens associated with PRDC. Objective 3: Investigate emerging and potential zoonotic bacterial pathogens that could impact the swine industry and design measures to diagnose, prevent, control and eliminate the threat posed to the swine industry. Subobjective 3.1. Evaluate the relationship between highly pathogenic Asian strains of PRRSV and S. suis infection in swine. Subobjective 3.2: Identification of measures that may prevent, control, or eliminate livestock-associated methicillin- resistant Staphylococcus aureus (MRSA) Sequence Type 398 (ST398) in swine. Approach (from AD-416): Use comparative genomic methods, microarray analysis, and co-infection studies to explore pathogenic mechanisms of bacteria associated with the porcine respiratory disease complex and their interactions with each other and swine viruses. Assess the usefulness of selected genes or proteins identified in comparative genomic analyses for DNA-based identification and classification, serological detection of infection, and potentially as vaccine candidates. Strategies for improved heterologous protection will be tested using live attenuated vaccines, as will the use of immunomodulators, such as granulocyte colony stimulating factor (G-CSF), for therapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease and thus reduce antimicrobial usage to treat clinical and subclinical disease. Investigate emerging and potential zoonotic bacteria that could impact the swine industry, including Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus suis for antimicrobial resistance and virulence factors through sequencing and proteomics. Investigations will focus on pathogen strain characteristics and differences, interactions of bacterial and viral pathogens with the swine host and the microbial ecosystems of the pig. Pathology of both zoonotic and endemic bacterial pathogens of swine will be utilized for the purpose of understanding disease pathogenesis and developing effective diagnostic assays. This is the final report for project 5030-32000-109-00D Strategies to Control and Prevent Bacterial Infections in Swine which is terminating October 5, 2016. Progress made over the past year is described below, followed by a summary of the significant progress made over the lifespan of the project the past 5 years. All three objectives and their subobjectives address research components in the Animal Health National Program (NP 103) Action Plan: Component 2: Antimicrobial Resistance, Problem Statement 2B, Alternatives to Antibiotics, and Component 4: Respiratory Diseases, Problem Statement 4B, Porcine Respiratory Diseases. Over the past year progress has been made in all three objectives. For Objective 1, Subobjective 1.1, ten strains of Streptococcus suis were identified with a range of virulence, or ability to cause disease in pigs, and annotated draft genomes have been completed for each. This is providing meaningful data to make genomic comparisons to identify virulence genes. For Subobjective 1.2, transcriptomic and proteomic techniques are being used to examine what genes/proteins are expressed, respectively, by Haemophilus parasuis and Streptococcus suis when they infect a pig. Bacteria have been collected from the respiratory tract and systemic sites such as the brain and joints of infected pigs to determine what genes and proteins are expressed that help the bacteria survive in these in vivo sites. Once the bacteria are isolated, RNA that they produce will be sequenced (RNAseq), which will indicate the quantity of each transcript that the bacteria are producing under the different conditions. Proteins isolated from the bacteria are also being run on two dimensional gels that separate them and will allow us to identify them through mass spectrometry. These are methods of identifying which genes and proteins are important for bacterial survival and causing disease in the pig and will help us identify new vaccine targets. For Subobjective 1.3, a follow up experiment has been conducted to examine whether the bacterial pathogen Bordetella bronchiseptica, inhibits vaccination of pigs with different influenza vaccines. Bordetella bronchiseptica infects the respiratory tract of pigs and can be immunosuppressive and thus may alter the response to other vaccines. Both influenza and Bordetella bronchiseptica contribute to PRDC and coinfection with both pathogens results in more severe disease than with either pathogen alone. In an initial experiment looking at the effects of Bordetella on live attenuated influenza vaccine given intranasally, the immune response to the vaccine was not changed but disease was more severe after infection with influenza virus than in vaccinated pigs that were not infected with Bordetella. An additional experiment was conducted to look at these effects in pigs vaccinated with either live attenuated vaccines or whole inactivated vaccines for influenza and with Bordetella strains that are deficient in one of the genes that cause immunosuppression. For Objective 2, Subobjective 2.2, a study was completed to determine which delivery route was the most effective to administer a Streptococcus suis vaccine that had been developed and it was determined that intramuscular was the most effective route of delivery for protection. This is important for the practicality of administering the vaccine to pigs. Several potential vaccines against Haemophilus parasuis were also tested. A potential subunit vaccine was not protective; the information generated by this study was useful to clarify an earlier report that had indicated one of the proteins in the subunit vaccine was protective in a guinea pig model of infection. Virulence factors for Haemophilus parasuis were identified and an attenuated vaccine made from a deletion mutant in one of these virulence factors, which demonstrated more promise in a protection study. In addition vaccines made from two different strains of the same serotype of Haemophilus parasuis provided contrasting protection results when tested against a heterologous strain of a different serotype, one being protective and the other one not. These results can be used to identify which proteins are cross protective to develop a more universally protective vaccine against Haemophilus parasuis. The role of several Streptococcus suis and Pasteurella multocida genes in virulence were tested by looking for attenuation of deletion mutants of these genes in a pig model of infection and disease. Several of the mutants were attenuated and are potential vaccine candidates to be tested. For Subobjective 2.3., experiments were conducted to examine the ability of interferon alpha to serve as an adjuvant, increasing the immune response to porcine reproductive and respiratory syndrome virus (PRRSV) vaccines and increasing the efficacy of these vaccines. The interferon totally inhibited replication of the vaccine and although it did not have the desired effect in boosting the response to the vaccine, these results provide further evidence that interferon could be used to treat PRRSV infections. For Objective 3, Subobjective 3.2, whole-genome sequences for 156 ST5 strains encompassing both methicillin-sensitive (MSSA) and methicillin- resistant (MRSA) strains from both human and swine related sources were utilized to build phylogenetic or family trees for these strains, identify phages that might be present, and to identify all of the antimicrobial (including zinc) resistance genes carried by these strains. Additionally, the ability of these strains to adhere to skin cells obtained from humans and swine are being tested. Summary - Substantial results were realized over the 5 years of the project in all three objectives. Through in vivo and in vitro testing, strains of Haemophilus parasuis and Streptococcus suis were identified that have varying ability to cause disease, and whole genome sequences on these strains were generated to compare genetic differences associated with fitness for survival of these bacteria in the pig. Through this work bacterial proteins were identified and successfully tested as potential subunit vaccines to protect against diseases associated with Streptococcus suis and Haemophilus parasuis. Further identification of virulence genes in Streptococcus suis, Haemophilus parasuis, Bordetella bronchiseptica, and Pasteurella multocida was accomplished through genomic comparison and testing of deletion mutants of potential virulence genes to determine if attenuation of the bacteria occurred. Several mutants that were attenuated were successfully tested as potential vaccine candidates. The most promising of these vaccine candidates are in the process of further development. Two biotherapeutic cytokine candidates were tested for their ability to control pathogens associated with the porcine respiratory disease complex. Granulocyte-colony stimulating factor (G-CSF) and interferon-alpha both showed promise in treating bacterial and viral pathogens, respectively, associated with disease in swine. The impact of viruses such as porcine reproductive and respiratory disease virus (PRRSV) and influenza virus on causing secondary bacterial infections in swine was evaluated and mechanisms of immunosuppression or impact on vaccination were identified. This information will help us develop mechanisms to mitigate these responses through different vaccination strategies or immunotherapy. Whole genome sequences were generated for 156 strains encompassing both methicillin- susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus from both human and swine related sources and screened for the presence of human-specific virulence factors and antibiotic resistance markers. Combining these genetic comparisons with the results of in vitro assays will provide the information needed to deliver a comprehensive understanding of the origin, evolution, and zoonotic potential of livestock associated MRSA strains associated with swine to both the public health and veterinary communities. Accomplishments 01 A potential new Haemophilus parasuis vaccine for swine. Haemophilus parasuis is a bacterium that causes Gl�sser's disease in swine, a disease characterized by chronic debilitation and often death that costs the swine industry millions in losses annually. H. parasuis isolates display a wide range of virulence, or ability to cause disease, but the virulence factors are largely unknown. Strain variability and lack of cross-reactivity make current vaccines an ineffective means of protection. ARS scientists in Ames, Iowa with collaborators identified a virulence factor by demonstrating that a deletion mutant, that does not produce the factor, was attenuated when given to pigs. A vaccine made of this mutant was able to protect against multiple strains of H. parasuis. The creation of a cross-protective vaccine would minimize disease loss and greatly reduce the economic impact to the swine industry. 02 Determining genes responsible for disease development with Streptococcus suis infection. Streptococcus suis is a bacterium that causes meningitis and arthritis in swine and 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 S. suis strains and the genetic factors that contribute to its ability to cause disease. ARS scientists in Ames, Iowa identified 10 strains of S. suis with varying ability to cause disease and then determined the DNA genomic sequence of those 10 strains. Now comparison of the sequence from these strains will identify genes in certain strains that are likely responsible for their ability to cause disease. These genes can be targeted to produce vaccines that will protect against disease with this costly pathogen. 03 Feeding zinc does not play a role in the prevalence and persistence of LA-MRSA in US swine. Livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA) draws concern from the public health community because in some countries these organisms may represent the largest reservoir of MRSA outside hospital settings. The emergence and prevalence of LA-MRSA isolates in Europe has been attributed, in part, to the in-feed use of zinc as an antidiarrheal agent. Since the gene encoding zinc resistance and the gene encoding methicillin resistance are collocated on the same segment of DNA, it has been suggested that the use of in feed zinc as an antimicrobial has the potential to contribute to the emergence and spread of MRSA in swine by increasing the selective pressure to maintain this DNA segment in isolates from pigs. ARS scientists in Ames, Iowa determined the prevalence of the zinc resistance gene and phenotypic zinc resistance in US swine associated LA-MRSA isolates and MRSA isolates from humans with no swine contact. The prevalence of zinc resistance in US swine associated LA- MRSA isolates was significantly lower than the prevalence of zinc resistance in MRSA isolates from humans with no swine contact, and previous reports describing zinc resistance in other LA-MRSA isolates. Collectively this data suggests the application of zinc in feed is not playing a role in the prevalence and persistence of LA-MRSA in the US swine population, which is important for swine producers in making informed decisions on whether or not it is safe to include zinc in swine diets. 04 Treatment option for porcine reproductive and respiratory syndrome (PRRSV) in pigs. PRRSV is the most costly viral disease for pig producers. Interferon-alpha (IFN-a) is an antiviral agent produced by cells in the body in response to viral infection. Little IFN-a is produced during PRRSV infection in pigs, which is potentially the reason for viral persistence and delay in an adaptive immune response to the virus. ARS scientists in Ames, Iowa completed a study giving an adenovirus vector which produces IFN-a to pigs to determine if it would boost the immune response to PRRSV vaccine and determine the effectiveness of IFN-a used metaphylactically, meaning in the face of a PRRSV outbreak in a herd to both treat infected pigs and prevent further spread to pigs that have not been infected. Use of IFN-a was shown to reduce the severity of disease and reduce transmission of the virus showing promise as a metaphylactic treatment in herds that break with PRRSV. The IFN-a totally inhibited replication of the vaccine and although it did not have the desired effect in boosting the response to the vaccine, these results provide further evidence that interferon could be used to treat PRRSV infections. 05 Determining the best route of administration for a new vaccine against Streptococcus suis for 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 scientists in Ames, Iowa along with collaborators previously tested a subunit vaccine of Streptococcus suis that was found to be protective against disease. In the initial experiment the vaccine was given intramuscularly and intranasally to the pigs. Additional vaccine trials have examined which route, intramuscular or intranasal, is the most effective and found that intramuscular was the most effective route of delivery for protection. This is important for the practicality of administering the vaccine to pigs in the field. Partnership with a private company for further vaccine development is under way to provide swine producers an efficacious vaccine against this devastating pathogen. 06 Sequence comparison of a Bordetella bronchiseptica isolate that causes disease in pigs. The well-characterized Bordetella bronchiseptica strain KM22, originally isolated from a pig with atrophic rhinitis, has been used to develop a reproducible swine respiratory disease model. To gain a broader perspective of the genetic relationship of KM22 among other B. bronchiseptica strains, ARS scientists in Ames, Iowa compared selected genes of KM22 to five other B. bronchiseptica strains isolated from different hosts. Overall, the KM22 genome sequence is more similar to the genome sequences of the strains isolated from animals than the strains isolated from humans. The annotation of the KM22 genome sequence, coupled with the comparative genomic analyses reported in this study, can be used to facilitate the development of vaccines with improved efficacy towards B. bronchiseptica in swine to decrease the prevalence and disease burden caused by this pathogen.
Impacts
(N/A)
Publications
- Nicholson, T.L., Shore, S., Register, K.B., Bayles, D.O., Kingsley, R.A., Brunelle, B.W. 2016. Comparative genomic analysis of the swine pathogen Bordetella bronchiseptica strain KM22. Veterinary Microbiology. 182:87-94.
- Trainor, E., Nicholson, T.L., Merkel, T.J. 2015. Bordetella pertussis transmission. Pathogens and Disease. 10.1093/femspd/ftv068.
- Hau, S.J., Sun, J., Davies, P.R., Frana, T.S., Nicholson, T.L. 2015. Comparative prevalence of immune evasion complex genes associated with beta-hemolysin converting bacteriophages in MRSA ST5 isolates from swine, swine facilities, humans with swine contact, and humans with no swine contact. PLoS One. 10(11):e0142832.
- Hau, S.J., Bayles, D.O., Alt, D.P., Brockmeier, S.L., Frana, T.S., Nicholson, T.L. 2015. Draft genome sequences of nine Streptococcus suis strains isolated in the United States. Genome Announcements. 3(6):e01301- 15.
- McCaig, W.D., Loving, C.L., Hughes, H.R., Brockmeier, S. 2016. Characterization and vaccine potential of outer membrane vesicles produced by Haemophilus parasuis. PLoS One. 11(3):e0149132.
- Hughes, H.R., Vincent, A.L., Brockmeier, S.L., Gauger, P.C., Pena, L., Santos, J., Braucher, D.R., Perez, D.R., Loving, C.L. 2015. Oral fluids as a live-animal sample source for evaluating cross-reactivity and cross- protection following intranasal influenza A virus vaccination in pigs. Clinical and Vaccine Immunology. 22(10):1109-1120.
- Register, K.B., Nicholson, T.L., Brunelle, B.W. 2016. Comparison of Ribotyping and sequence-based typing for discriminating among isolates of Bordetella bronchiseptica. Journal of Microbiological Methods. 129:117-126. doi: 10.1016/j.mimet.2016.08.008.
- Schlink, S.N., Lager, K.M., Brockmeier, S.L., Loving, C.L., Miller, L.C., Vorwald, A.C., Yang, H., Kehrli, Jr., M.E., Faaberg, K.S. 2016. Enhancement of innate immunity with granulocyte colony-stimulating factor did not mitigate disease in pigs infected with a highly pathogenic Chinese PRRSV strain. Veterinary Immunology and Immunopathology. 179:70-76.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): Objective 1: Identify the transmission, genetic, and pathogenic mechanisms of the organisms associated with PRDC, concentrating on the bacterial pathogens and their interactions with each other and select swine viruses. Subobjective 1.1: Identify potential virulence factors of H. parasuis through comparative genomics. Subobjective 1.2: Bacterial response to host conditions. Subobjective 1.3: Evaluate ability of PRDC bacterial pathogens to inhibit Influenza A virus vaccine efficacy and/or exacerbate Influenza A virus-associated disease. Objective 2: Identify potential candidates for novel diagnostic assays, vaccines, and biotherapeutics for bacterial pathogens associated with PRDC. Subobjective 2.1. Develop PCR, ELISA, and/or other assays for detection of bacterial pathogens associated with PRDC. Subobjective 2.2. Identify, develop and/or test the efficacy of potential vaccine candidates to control bacterial pathogens associated with PRDC. Subobjective 2.3. Identify potential biotherapeutic candidates to control bacterial pathogens associated with PRDC. Objective 3: Investigate emerging and potential zoonotic bacterial pathogens that could impact the swine industry and design measures to diagnose, prevent, control and eliminate the threat posed to the swine industry. Subobjective 3.1. Evaluate the relationship between highly pathogenic Asian strains of PRRSV and S. suis infection in swine. Subobjective 3.2: Identification of measures that may prevent, control, or eliminate livestock-associated methicillin- resistant Staphylococcus aureus (MRSA) Sequence Type 398 (ST398) in swine. Approach (from AD-416): Use comparative genomic methods, microarray analysis, and co-infection studies to explore pathogenic mechanisms of bacteria associated with the porcine respiratory disease complex and their interactions with each other and swine viruses. Assess the usefulness of selected genes or proteins identified in comparative genomic analyses for DNA-based identification and classification, serological detection of infection, and potentially as vaccine candidates. Strategies for improved heterologous protection will be tested using live attenuated vaccines, as will the use of immunomodulators, such as granulocyte colony stimulating factor (G-CSF), for therapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease and thus reduce antimicrobial usage to treat clinical and subclinical disease. Investigate emerging and potential zoonotic bacteria that could impact the swine industry, including Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus suis for antimicrobial resistance and virulence factors through sequencing and proteomics. Investigations will focus on pathogen strain characteristics and differences, interactions of bacterial and viral pathogens with the swine host and the microbial ecosystems of the pig. Pathology of both zoonotic and endemic bacterial pathogens of swine will be utilized for the purpose of understanding disease pathogenesis and developing effective diagnostic assays. We are testing what role the capsule of H. parasuis has in establishing infection and causing disease. Bacterial capsules often protect the bacteria from the host immune response. With collaborators we created a mutant of H. parasuis that does not make a capsule and have found that the mutant was unable to cause disease, and are now testing whether that mutant could be used as an attenuated vaccine. In support of Objective 1, Subobjective 1.2, we are using a technique known as RNAseq to examine what genes are expressed by Haemophilus parasuis when it encounters environments similar to those encountered when infecting a pig. For example, bacteria that infect the lungs of pigs and cause pneumonia are exposed to higher levels of carbon dioxide, thus we have exposed the bacteria to physiologic levels of carbon dioxide in the laboratory to see how they respond. Once we collect the bacteria, we sequence the RNA (RNAseq) that they produce, which indicates the quantity of each gene that the bacteria are producing under the different conditions. This is an alternative way of identifying which genes are important for survival and causing disease in the pig and will help us identify new vaccine targets. In support of Objective 1, Subobjective 1.3, we have conducted experiments to examine whether the bacterial pathogen Bordetella bronchiseptica, inhibits vaccination of pigs with different influenza vaccines. B. bronchisptica infects the respiratory tract of pigs and can be immunosuppressive and thus may alter the response to other vaccines. Both influenza and B. bronchiseptica contribute to PRDC and coinfection with both pathogens results in more severe disease than with either pathogen alone. In an initial experiment looking at the effects of Bordetella on live attenuated influenza vaccine given intranasally, the immune response to the vaccine was not changed but disease was more severe after infection with influenza virus than in vaccinated pigs that were not infected with Bordetella. We are continuing to look at these effects in pigs vaccinated with either live attenuated vaccines or whole inactivated vaccines for influenza and with Bordetella strains that are deficient in one of the genes that cause immunosuppression. In support of Objective 2, Subobjective 2.1, we have adapted the standard gel-based PCR for a selected gene of Haemophilus parasuis to a real-time PCR assay to increase the sensitivity of the assay. We have completed construction and validation of the plasmid in which the gene was cloned for generating a standard curve for real-time PCR. We have also optimized the extraction protocol for isolation of bacterial DNA from swabs (nasal or systemic) for use in the diagnostic PCR assay. DNA was extracted from swabs collected during a H. parasuis study and was used in the real-time PCR for validation. Collectively, this will lead to the development of a sensitive and specific assay for the detection of H. parasuis DNA from swab samples. This approach is ideal because it is often difficult to recover viable bacteria through culture and instead, testing for isolated genomic DNA will be the primary sample type. An additional 80 isolates of H. parasuis from collaborators in Canada, Australia, and the United States have been sequenced using MiSeq. Data analysis is ongoing and is focused on the specific locus to further validate a molecular capsule typing scheme for H. parasuis. The capsule of H. parasuis is considered an important component for virulence of H. parasuis and we are testing whether using the capsule would be an effective vaccine. In addition we are testing whether a capsule mutant can be used as an attenuated vaccine for H. parasuis. Finally, outer membrane vesicles (OMVs) are spherical particles naturally released from the membrane of bacteria. These structures have been shown to be enriched in certain bacterial components. Examination of these structures in numerous bacteria has led to identification of virulence factors and subunit vaccines comprised of OMVs have been successfully used to protect against bacterial pathogens. We successfully isolated OMVs from H. parasuis and tested their use as a vaccine and found them to be protective. In support of Objective 2, Subobjective 2.3, a series of neutrophil functional assays are being developed and validated to confirm that neutrophils elicited following granulocyte colony stimulating factor (G- CSF) administration are functioning. In addition, the assays are being used to determine the mechanism of protection for ongoing Streptococcus suis (S. suis) and Haemophilus parasuis vaccine studies. The protocol for isolation of functional, viable neutrophils from porcine blood has been optimized (updated from older protocols) and functional assays, including bacterial killing, formation of neutrophil extracellular traps, and oxidative burst, are being performed using different isolates of S. suis, including mutants generated by collaborators. We are conducting an experiment to examine the ability of IFNalpha to serve as an adjuvant, increasing the immune response to PRRSV vaccines and increasing the efficacy of these vaccines. We have also conducted an experiment to determine the effectiveness of IFNalpha used metaphylactically, meaning in the face of a PRRSV outbreak in a herd to both treat infected pigs and prevent further spread to pigs that have not been infected. Use of IFNapha was shown to reduce the severity of disease and reduce transmission of the virus showing promise as a metaphylactic treatment in herds that break with PRRSV. In support of Objective 3, Subobjective 3.2, we examined ST5 MRSA isolates collected from swine, swine facilities, and humans with short- and long-term swine contact and screened for the presence of certain human-specific virulence factors. All LA-MRSA ST5 isolates screened lacked the virulence factors, which is consistent with the hypothesis that LA-MRSA strains that are adapted to swine have a reduced capacity to cause significant disease in immunocompetent humans. Accomplishments 01 A new vaccine against Streptococcus suis for 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 scientists in Ames, Iowa along with collaborators identified bacterial genes related to fitness for survival of the bacterium and tested the proteins that these genes encode as a potential subunit vaccine. The proteins were delivered to pigs with adjuvants that help stimulate an immune response and found to be protective against S. suis. Partnership with a private company for further vaccine development is under way to provide growers alternatives to this devastating pathogen. 02 Absence of human virulence factors in livestock-associated (LA), methicillin-resistant Staphylococcus aureus (MRSA). Staphylococcus aureus is a common yet devastating human pathogen that has the ability to acquire resistance to antibiotics resulting in the development of lineages with resistance to multiple antimicrobial drugs (including methicillin), resulting in MRSA. Swine can carry specific strains of MRSA that do not appear to cause disease in swine, but it is unclear whether these swine LA-MRSA can cause disease in humans. To test the possible role of MRSA in human diseases, ARS scientists in Ames, Iowa along with collaborators collected MRSA isolates from swine, swine facilities, and humans with short- and long-term swine contact, and screened the bacterial isolates for the presence of human-specific virulence factors (specific bacterial-associated proteins that contribute to disease in people). All LA-MRSA isolates screened lacked the human-specific virulence factors, indicating the LA-MRSA bacteria found in pigs have adapted to swine and they have a reduced capacity to cause significant disease in healthy humans. These results indicate that the risk of humans developing MRSA-associated disease from contact with swine is low. 03 A potential new Haemophilus parasuis vaccine for swine. Haemophilus parasuis is a bacterium that causes Gl�sser's disease in swine, a disease characterized by chronic debilitation and often death that costs the swine industry millions in losses annually. H. parasuis isolates display a wide range of virulence, or ability to cause disease, but the virulence factors are largely unknown. Strain variability and lack of cross-reactivity make current vaccines an ineffective means of protection. Outer membrane vesicles (OMVs) are spherical particles naturally released from the membrane of bacteria that have been shown to be enriched in bacterial virulence factors. Examination of these structures in numerous bacteria has led to identification of virulence factors and subunit vaccines comprised of OMVs have been successfully used to protect against bacterial pathogens. ARS scientists in Ames, Iowa successfully isolated OMVs from H. parasuis, determined the protein content of these vesicles, and determined that they have potential for use as a subunit vaccine. Protecting against multiple strains of H. parasuis through the creation of a cross-reactive subunit vaccine would minimize loss of life and greatly reduce the economic impact to the swine industry. 04 Use of attenuated vaccines to vaccinate against swine influenza. It has been repeatedly demonstrated experimentally that live-attenuated influenza virus (LAIV) vaccines provide significant protection against infection with divergent strains of influenza virus. These studies are often performed in specific-pathogen free pigs, which may not represent conditions in commercial settings. Previous work has shown that influenza A virus infection can exacerbate Bordetella bronchiseptica (B. bronchiseptica) a respiratory disease of swine. Given that LAIV vaccines are delivered by the intranasal route, and the ubiquitous nature of B. bronchiseptica nasal colonization in commercial swine, we investigated the effect of B. bronchiseptica colonization on the immune response to LAIV and its efficacy. B. bronchiseptica had some impact on LAIV efficacy, but not the immune response to the vaccine, suggesting that the effect is related to the interaction of influenza virus and B. bronchiseptica, not that B. bronchiseptica inhibits LAIV immunity. Protection was still significant when LAIV was delivered to pigs colonized with B. bronchiseptica. Our results reiterate the superior protective efficacy provided by LAIV vaccines and the need to have these vaccines available for use in the swine industry in addition to the currently available inactivated vaccines.
Impacts
(N/A)
Publications
- Brockmeier, S.L., Register, K.B., Kuehn, J.S., Nicholson, T.L., Loving, C. L., Shore, S.M., Phillips, G.J. 2014. Virulence and draft genome sequence overview of multiple strains of the swine pathogen Haemophilus parasuis. PLoS ONEe. 9(8):e103787.
- Nicholson, T.L., Shore, S., Bayles, D.O., Register, K.B., Kingsley, R.A. 2014. Draft genome sequence of the Bordetella bronchiseptica swine isolate KM22. Genome Announcements. 2(4). e00670-14. DOI: 10.1128/genomeA.00670-14.
- Loving, C.L., Brockmeier, S.L., Vincent, A.L., Gauger, P.C., Zanella, E.L., Lager, K.M., Kehrli, Jr., M.E. 2014. Cross-fostering to prevent maternal cell transfer did not prevent vaccine-associated enhanced respiratory disease that occurred following heterologous influenza challenge of pigs vaccinated in presence of maternal immunity. Viral Immunology. 27(7):334- 342.
- Butler, J.E., Lager, K.M., Golde, W., Faaberg, K.S., Sinkora, M., Loving, C., Zhang, Y.I. 2014. Porcine reproductive and respiratory syndrome (PRRS) : an immune dysregulatory pandemic. Immunologic Research. 59(1-3):81-108.
- Raj�o, D.S., Loving, C.L., Gauger, P.C., Kitikoon, P., Vincent, A.L. 2014. Influenza A virus hemagglutinin protein subunit vaccine elicits vaccine- associated enhanced respiratory disease. Vaccine. 32(40):5170-5176.
- Lorusso, A., Ciacci-Zanella, J.R., Zanella, E.L., Pena, L., Perez, D.R., Lager, K.M., Rajao, D.S., Loving, C.L., Kitikoon, P., Vincent, A.L. 2014. Polymorphisms in the hemagglutinin gene influenced the viral shedding of pandemic 2009 influenza virus in swine. Journal of General Virology. 95(Pt 12):2618-2626.
- Lager, K.M., Schlink, S.N., Brockmeier, S.L., Miller, L.C., Henningson, J. N., Kappes, M.A., Kehrli, M.E., Loving, C.L., Guo, B., Swenson, S.L., Yang, H., Faaberg, K.S. 2014. Efficacy of type 2 PRRSV vaccine against Chinese and Vietnamese HP-PRRSV challenge in pigs. Vaccine. 32(48):6457�6462.
- Gauger, P.C., Loving, C.L., Khurana, S., Lorusso, A., Perez, D.R., Kehrli, Jr., M.E., Roth, J.A., Golding, H., Vincent, A.L. 2014. Live attenuated influenza A virus vaccine protects against A(H1N1)pdm09 heterologous challenge without vaccine associated enhanced respiratory disease. Virology. 471-473:93-104.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): Objective 1: Identify the transmission, genetic, and pathogenic mechanisms of the organisms associated with PRDC, concentrating on the bacterial pathogens and their interactions with each other and select swine viruses. Subobjective 1.1: Identify potential virulence factors of H. parasuis through comparative genomics. Subobjective 1.2: Bacterial response to host conditions. Subobjective 1.3: Evaluate ability of PRDC bacterial pathogens to inhibit Influenza A virus vaccine efficacy and/or exacerbate Influenza A virus-associated disease. Objective 2: Identify potential candidates for novel diagnostic assays, vaccines, and biotherapeutics for bacterial pathogens associated with PRDC. Subobjective 2.1. Develop PCR, ELISA, and/or other assays for detection of bacterial pathogens associated with PRDC. Subobjective 2.2. Identify, develop and/or test the efficacy of potential vaccine candidates to control bacterial pathogens associated with PRDC. Subobjective 2.3. Identify potential biotherapeutic candidates to control bacterial pathogens associated with PRDC. Objective 3: Investigate emerging and potential zoonotic bacterial pathogens that could impact the swine industry and design measures to diagnose, prevent, control and eliminate the threat posed to the swine industry. Subobjective 3.1. Evaluate the relationship between highly pathogenic Asian strains of PRRSV and S. suis infection in swine. Subobjective 3.2: Identification of measures that may prevent, control, or eliminate livestock-associated methicillin- resistant Staphylococcus aureus (MRSA) Sequence Type 398 (ST398) in swine. Approach (from AD-416): Use comparative genomic methods, microarray analysis, and co-infection studies to explore pathogenic mechanisms of bacteria associated with the porcine respiratory disease complex and their interactions with each other and swine viruses. Assess the usefulness of selected genes or proteins identified in comparative genomic analyses for DNA-based identification and classification, serological detection of infection, and potentially as vaccine candidates. Strategies for improved heterologous protection will be tested using live attenuated vaccines, as will the use of immunomodulators, such as granulocyte colony stimulating factor (G-CSF), for therapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease and thus reduce antimicrobial usage to treat clinical and subclinical disease. Investigate emerging and potential zoonotic bacteria that could impact the swine industry, including Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus suis for antimicrobial resistance and virulence factors through sequencing and proteomics. Investigations will focus on pathogen strain characteristics and differences, interactions of bacterial and viral pathogens with the swine host and the microbial ecosystems of the pig. Pathology of both zoonotic and endemic bacterial pathogens of swine will be utilized for the purpose of understanding disease pathogenesis and developing effective diagnostic assays. In support of Objective 1, Subobjective 1.1 to identify potential virulence factors through comparative genomics, we have continued to compare the genomic sequence of the 10 Haemophilus parasuis strains for which we have draft genome sequences and have identified several differences in coding regions. These coding regions include predicted outer membrane, metabolism, and pilin or adhesin related genes, some of which likely contributed to the differences in virulence and systemic disease observed following challenge. We have also performed another sequencing run on one of the avirulent strains of H. parasuis in order to close the genome. In addition, we have completed the annotation of the whole genome sequence for the swine pathogen Bordetella bronchiseptica strain KM22. We then used the genome sequence to perform comparative genomics analyses to identify genes and genomic sequences unique to KM22 compared to the other six previously sequenced B. bronchiseptica strains to identify possible virulence factors that contribute to disease. Finally we have completed preliminary studies using transposon-directed insertion site sequencing to identify bacterial genes related to fitness for survival of H. parasuis and Streptococcus suis in the pig. In support of Objective 1, Subobjective 1.2, to examine the bacterial response to host conditions, we developed assays to test the effect of complement in serum on various H. parasuis isolates and have identified strains that are both sensitive and resistant to serum compliment mediated killing. We will now use this to isolate RNA from these strains after exposure to serum to determine what genes are expressed that allows the bacteria to resist complement mediated killing. In support of Objective 1, Subobjective 1.3 to evaluate ability of porcine respiratory disease complex (PRDC) bacterial pathogens to inhibit Influenza A virus vaccine efficacy, we have completed a study in which pigs were colonized with B. bronchiseptica during vaccination with live- attenuated influenza virus (LAIV) vaccine and whole-inactivated virus (WIV) to determine if active B. bronchiseptica infection decreases the efficacy of LAIV vaccination. This is important given the prevalence of B. bronchiseptica in the field and its ability to alter adaptive immune responses. In support of Objective 2, Subobjective 2.1, to develop PCR assays for detection of bacterial pathogens associated with porcine respiratory disease complex (PRDC), we have adapted the standard gel-based PCR for the wza gene of H. parasuis, a gene responsible for capsule expression, to a real-time PCR assay to increase the sensitivity of the assay. We have completed construction and validation of the plasmid in which the wza gene was cloned for generating a standard curve for real-time PCR. We have focused on developing the diagnostic assay for use with swab samples (nasal or systemic) and are working on identifying the best kit for extraction of bacterial genomic DNA from swab samples. Collectively, this will lead to the development of a sensitive and specific assay for the detection of H. parasuis DNA from swab samples. This approach is ideal because it is often difficult to recover viable bacteria through culture and instead, testing for isolated genomic DNA will be the primary sample type. In addition, we collected nearly 70 isolates of H. parasuis from collaborators in Canada, Australia, and the United States for genomic DNA sequencing using MiSeq. Data analysis will focus on the capsule locus to determine if a molecular typing scheme is possible. In support of Objective 2, Subobjective 2.2 to develop potential vaccine candidates to control bacterial pathogens associated with PRDC, we completed the construction of 5 suicide vectors for the purpose of generating markerless mutations in H. parasuis strains (these include a red fluorescent protein, a green fluorescent protein, a yellow fluorescent protein, a kanamycin resistance gene, and an aroA deletion). The aroA mutant will be tested for attenuation and the potential to be used as a vaccine. In addition, we examined the efficacy of a bacterin (killed vaccine) of the H. parasuis strain Nagasaki (a highly virulent strain) and found it was protective against disease with this strain, we can now test whether it is protective against other strains of H. parasuis and use this as a comparison for other vaccine candidates. We are also developing methods to isolate outer membrane vesicles secreted by H. parasuis to evaluate the role these have in virulence and to test whether they would be good vaccine candidates. In support of Objective 2, Subobjective 2.3, to identify potential biotherapeutic candidates to control bacterial pathogens associated with PRDC, we have tested the immunomodulator G-CSF, which increased the production of neutrophils (one of the white blood cells that clear bacterial infections), with infection models of Salmonella and Streptococcus suis in pigs with some promising results. We have also completed and have ongoing studies comparing the ability of type I vs. type III interferons (antiviral compounds) to inhibit the replication of swine viruses in vitro and in vivo. In support of Objective 3, Subobjective 3.1 to investigate potential zoonotic bacterial pathogens we tested several different Streptococcus suis strains for virulence in cesarean-derived colostrum-deprived pigs (CDCD) pigs and identified strains of varying virulence that can be used in further studies examining the role of coinfection with porcine reproductive and respiratory syndrome virus (PRRSV), vaccine studies, and studies on virulence mechanisms of this potentially zoonotic agent. In support of Objective 3, Subobjective 3.2 to prevent, control, or eliminate livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in swine, we have constructed an LA-MRSA strain producing a fluorescent marker (Green Fluorescence Protein) in collaboration with researchers at the Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland in order to facilitate planned in vitro and in vivo studies. Accomplishments 01 Genes responsible for disease development with Haemophilus parasuis infection. Haemophilus parasuis is a bacterium that causes Gl�sser's disease in swine, a disease characterized by chronic debilitation and often death 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. ARS scientists at the National Animal Disease Center in Ames, Iowa had previously identified 10 strains of H. parasuis with varying ability to cause disease and then determined the DNA genomic sequence of those 10 strains. Now comparison of the sequence from these strains has identified genes in certain strains that are likely responsible for their ability to cause disease. These genes can be targeted to produce vaccines that will protect against disease with this costly pathogen. 02 An increase in pig innate defenses decreases Salmonella typhimurium shedding from pigs. Neutrophils are cells of the innate immune system that play an important role in combatting infections, particularly bacterial infections. Granulocyte-colony stimulating factor (G-CSF) is an immune protein produced normally by the body to keep neutrophils in the blood and also induce production of neutrophils in times of need, such as infection. The use of compounds like G-CSF can be used to modulate the immune system and could be given to animals to prevent and/ or combat infectious diseases. ARS scientists at the National Animal Disease Center in Ames, Iowa completed a study in which a replication- defective adenovirus (non-infectious) that contained the genetic sequence for porcine G-CSF was given to pigs to increase the number of neutrophils and determine the effects on infection with Salmonella typhimurium. Administration of G-CSF prior to S. typhimurium challenge led to a significant reduction in shedding of bacteria in the feces and a decrease in the ability to isolate the bacteria from the tonsils of G- CSF treated pigs. These findings will benefit producers and pharmaceutical companies working towards non-antibiotic methods to reduce shedding and colonization of S. typhimurium in pigs. 03 A secretion system plays a role in the persistence, disease and immunosuppression caused by Bordetella bronchiseptica. Bordetella bronchiseptica is a bacterium that is widespread in swine herds and plays multiple roles in respiratory disease, causing both upper- respiratory illness and pneumonia, and predisposes pigs to infection with other bacteria. The type III secretion system (T3SS) is a needle- like structure that the bacterium uses to inject proteins directly into host cells to aid in the infection process. ARS scientists at the National Animal Disease Center in Ames, Iowa constructed a mutant of B. bronchiseptica that does not make the T3SS to determine what role this system plays in colonizing the respiratory tract, causing disease, and transmitting pig-to-pig. Infection with the Bordetella T3SS mutant was cleared more rapidly, indicating the T3SS is required for the bacteria to persist in the pig�s respiratory tract. Additionally, disease was milder in pigs infected with the Bordetella T3SS mutant. The presence of the T3SS was found to hinder the pig�s immune response to Bordetella, which likely plays a role in the inability of swine to clear the bacteria and is why Bordetella can normally persist in the swine respiratory tract for long periods of time. However, the T3SS mutant was still capable of transmitting from one pig to another, demonstrating that even with minimal disease and lower colonization transmission can occur. Targeting proteins secreted by the T3SS for future vaccines will decrease disease caused by Bordetella and secondary bacterial infections, resulting in healthier pigs and fewer losses due to respiratory disease.
Impacts
(N/A)
Publications
- Nicholson, T.L., Brockmeier, S.L., Loving, C.L., Register, K.B., Kehrli, Jr., M.E., Shore, S.M. 2014. The Bordetella bronchiseptica type III secretion system is required for persistence and disease severity but not transmission in swine. Infection and Immunity. 82(3):1092-1103.
- Nicholson, T.L., Shore, S.M., Smith, T.C., Fraena, T.S. 2013. Livestock- associated methicillin-resistant Staphylococcus aureus (LA-MRSA) isolates of swine origin form robust biofilms. PLoS One. 8(8):e73376.
- Sukumar, N., Nicholson, T.L., Conover, M.S., Ganguly, T., Deora, R. 2014. Comparative analyses of a cystic fibrosis isolate of Bordetella bronchiseptica reveal differences in important pathogenic phenotypes. Infection and Immunity. 82(4):1627-1637.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): Objective 1: Identify the transmission, genetic, and pathogenic mechanisms of the organisms associated with PRDC, concentrating on the bacterial pathogens and their interactions with each other and select swine viruses. Subobjective 1.1: Identify potential virulence factors of H. parasuis through comparative genomics. Subobjective 1.2: Bacterial response to host conditions. Subobjective 1.3: Evaluate ability of PRDC bacterial pathogens to inhibit Influenza A virus vaccine efficacy and/or exacerbate Influenza A virus-associated disease. Objective 2: Identify potential candidates for novel diagnostic assays, vaccines, and biotherapeutics for bacterial pathogens associated with PRDC. Subobjective 2.1. Develop PCR, ELISA, and/or other assays for detection of bacterial pathogens associated with PRDC. Subobjective 2.2. Identify, develop and/or test the efficacy of potential vaccine candidates to control bacterial pathogens associated with PRDC. Subobjective 2.3. Identify potential biotherapeutic candidates to control bacterial pathogens associated with PRDC. Objective 3: Investigate emerging and potential zoonotic bacterial pathogens that could impact the swine industry and design measures to diagnose, prevent, control and eliminate the threat posed to the swine industry. Subobjective 3.1. Evaluate the relationship between highly pathogenic Asian strains of PRRSV and S. suis infection in swine. Subobjective 3.2: Identification of measures that may prevent, control, or eliminate livestock-associated methicillin- resistant Staphylococcus aureus (MRSA) Sequence Type 398 (ST398) in swine. Approach (from AD-416): Use comparative genomic methods, microarray analysis, and co-infection studies to explore pathogenic mechanisms of bacteria associated with the porcine respiratory disease complex and their interactions with each other and swine viruses. Assess the usefulness of selected genes or proteins identified in comparative genomic analyses for DNA-based identification and classification, serological detection of infection, and potentially as vaccine candidates. Strategies for improved heterologous protection will be tested using live attenuated vaccines, as will the use of immunomodulators, such as granulocyte colony stimulating factor (G-CSF), for therapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease and thus reduce antimicrobial usage to treat clinical and subclinical disease. Investigate emerging and potential zoonotic bacteria that could impact the swine industry. Investigations will focus on pathogen strain characteristics and differences, interactions of bacterial and viral pathogens with the swine host and the microbial ecosystems of the pig. Pathology of both zoonotic and endemic bacterial pathogens of swine will be utilized for the purpose of understanding disease pathogenesis and developing effective diagnostic assays and strategies to control these pathogens and diseases in swine and potentially in humans. We completed virulence testing for the 10 Haemophilus parasuis (HPS) isolates we have sequenced. This will provide meaningful data on which to base genomic comparisons. These efforts support subobjective 1.1. We developed assays to examine the transcriptional response of virulent and avirulent isolates HPS after exposure to serum and complement. These efforts support Subobjective 1.2. We identified a conserved gene in HPS, called wza, that produces a protein that is part of the capsule. A PCR assay has been developed and shown to be specific to HPS, and we have begun evaluating the assay in a blinded fashion and have initiated the cloning of the gene for development of a quantitative assay. We have screened convalescent and naive sera for reactivity to HPS P2 and P5 proteins and concluded that these are not optimal diagnostic targets given the reactivity of naive sera to these proteins and numerous differences between P2 and P5 proteins in different HPS isolates. These efforts support subobjective 2. 1. We completed the construction of a novel suicide vector that will allow us to make markerless mutations in H. parasuis, including an AroA mutant to test as a vaccine candidate. The genome sequence data files of an identified virulent (Nagasaki) and avirulent (D74) strain of HPS have been annotated and pseudogene candidates identified in order to introduce a gene encoding fluorescent protein for the purpose of marking a strain. A recombinant Ad5 vector expressing the Bsp22 protein has been generated and pigs were immunized with the vector to test for immunogenicity. These efforts support Subobjective 2.2. We completed in vitro assays evaluating the function of neutrophils isolated from pigs that had been given granulocyte-colony stimulating factor (G-CSF), a compound that enhances immunity against bacterial infections by increasing the number of circulating neutrophils, and found the administration of the compound does not alter cell function. We continued experiments evaluating protection provided by the administration of G-CSF. These efforts support subobjective 2.3. We examined a number of Streptococcus suis strains for virulence and effects of coinfection with other bacteria and viruses. These efforts support subobjective 3.1. To begin experimentally addressing mechanisms contributing to colonization, carriage, and competitive exclusion of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) strains, we have begun constructing mutants in our MRSA isolates that contain a gene encoding fluorescent protein. We have also performed qPCR to evaluate expression of genes that potentially lead to biofilm formation and tested a collection of strains for production of secreted proteases and nucleases when grown as a biofilm. We found certain strains to be the most sensitive to both inhibition of biofilm formation and dispersal of biofilms by DNaseI, and Proteinase K to both inhibit biofilm formation and disperse mature biofilms in all LA-MRSA strains. These experiments demonstrate the sensitivity is not due to differences in gene expression, secreted proteases, or extracellular nucleases. These efforts support subobjective 3.2. Accomplishments 01 Developed an assay to detect newly emerging strains of porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV is a virus that infects pigs and causes reproductive losses and respiratory disease. It is the top disease problem for pig producers in the United States and many other parts of the world. This virus mutates very rapidly and new strains of PRRSV are constantly emerging. Current differentiation of PRRSV strains involves a few selected U.S. veterinary diagnostic laboratories performing techniques that typically take two weeks or more for results. ARS scientists at the National Animal Disease Center in Ames, Iowa, have developed a new assay called a microarray specific for PRRSV that can function as a diagnostic tool that will rapidly identify and differentiate viral strains. Our novel PRRSV microarray recognizes multiple regions throughout the PRRSV genome and as a result, our PRRSV microarray has a greater sensitivity than currently used methods while simultaneously detecting genetic diversity. This test is a faster and more reliable test that can detect newly emerging domestic and foreign strains of PRRSV that threaten the national swine herd. 02 Determination of influenza vaccine efficacy for limiting transmission between pigs and people. Influenza virus spillover from pigs to people is concern for both public health officials as well as swine producers. In 2012 there were more than 300 cases reported in which a particular strain of swine influenza virus was isolated from people, the majority occurring at agricultural fairs in the summer. One method to limit transmission of the virus from pigs to people is to control the virus in pigs through vaccination. ARS scientists at the National Animal Disease Center in Ames, Iowa, evaluated the ability of currently available commercial swine influenza vaccines and experimental vaccines to limit replication and spread of a strain of swine influenza associated with spillover at agricultural fairs in 2012. One commercial vaccine provided significant protection, but it did not prevent transmission of the virus to non-vaccinated pigs in a neighboring pen. One experimental vaccine that is a live-attenuated virus given to pigs in the nose was able to prevent infection. The results from this work will provide pig owners and agricultural personnel experimental data for determining the value of commercial vaccines at limiting infection with this particular strain of swine influenza virus, which should contribute to decreasing the frequency of spillover from pigs to people. It also highlights the need for future work to develop assays that are predictive of a vaccine's effectiveness. 03 Determined that enhanced immune response may limit the need for antibiotic use. Overuse of antibiotics has been blamed for the growing resistance of bacteria to their effects. Neutrophils are white blood cells that circulate in the blood and play an important role in combating infections. Granulocyte-Colony Stimulating Factor (G-CSF) is a substance produced by the body in response to infection that increases the number of circulating neutrophils. The use of immunomodulators like G-CSF is a promising area that enhances the body's own ability to prevent and combat infectious disease while eliminating or reducing the use of antibiotics. ARS scientists at the National Animal Disease Center in Ames, Iowa, completed a series of studies in which pigs were given a G-CSF in a vector which allows it to have longer lasting effects. The vector worked better than predicted resulting in enhanced neutrophil levels in the blood of pigs for up to 3 weeks after a single injection. This novel delivery of G-CSF precludes the need for manufacturing G-CSF and elicits a longer duration of effect in the body than current modalities provide today. These findings will benefit veterinarians and pharmaceutical companies working towards novel methods to reduce antibiotic usage in pigs that develop bacterial infections. 04 Determined a mechanism by which porcine reproductive and respiratory syndrome virus (PRRSV) causes immunosuppression. PRRSV is a virus that infects pigs and causes reproductive losses and respiratory disease. It is the top disease problem for pig producers in the United States and many other parts of the world. The immune response to PRRSV following infection has been characterized as weak and delayed; thus, the infection is not controlled and becomes chronic and vaccines tend to be ineffective. ARS scientists at the National Animal Disease Center in Ames, Iowa completed a study in which pigs were given one of four different PRRSV isolates, each with varying ability to cause disease. Within 2 days of infection all of the pigs had a significant decrease in the number of circulating lymphocytes, a type of white blood cell involved in immunity. While the number of cells in circulation steadily increased over time, by day 10 following infection, the number of circulating lymphocytes was still below normal. In addition, there were significant decreases in the cellularity of the thymus, an organ required for the development of a population of circulating lymphocytes. The decrease in thymus size correlated with the ability of the virus to cause disease and predispose to secondary infections. These results indicate that PRRSV causes significant changes in the population of lymphocytes which may explain the inadequate adaptive immune response elicited following infection. If we can find ways to counteract these effects we can improve the protective effects of PRRSV vaccines.
Impacts
(N/A)
Publications
- Brockmeier, S.L., Loving, C.L., Vorwald, A.C., Kehrli, Jr., M.E., Baker, R. B., Nicholson, T.L., Lager, K.M., Miller, L.C., Faaberg, K.S. 2012. Genomic sequence and virulence comparison of four Type 2 porcine reproductive and respiratory syndrome virus strains. Virus Research. 169(1) :212-221.
- Braucher, D.R., Henningson, J.N., Loving, C.L., Vincent, A.L., Kim, E., Steitz, J., Gambotto, A.A., Kehrli, Jr., M.E. 2012. Intranasal vaccination with replication-defective adenovirus type 5 encoding influenza virus hemagglutinin elicits protective immunity to homologous challenge and partial protection to heterologous challenge in pigs. Clinical and Vaccine Immunology. 19(11):1722-1729.
- Nicholson, T.L., Conover, M.S., Deora, R. 2012. Transcriptome profiling reveals stage-specific production and requirement of flagella during biofilm development in Bordetella bronchiseptica. PLoS ONE. 7(11):e49166.
- Hester, S.E., Lui, M., Nicholson, T., Nowacki, D., Harvil, E.T. 2012. Identification of a CO2 responsive regulon in Bordetella. PLoS One. 7(10) :e47635.
- Loving, C.L., Kehrli, M.E., Brockmeier, S.L., Bayles, D.O., Michael, D.D., Schlink, S.N., Lager, K.M. 2013. Porcine granulocyte-colony stimulating factor (G-CSF) delivered via replication-defective adenovirus induces a sustained increase in circulating peripheral blood neutrophils. Biologicals. Available:
- Brockmeier, S.L., Register, K.B., Nicholson, T.L., Loving, C.L. 2012. Bordetellosis. In: Zimmerman, J., Karriker, L., Ramirez, A., Schwartz, K., Stevenson, G., editors.Diseases of Swine. 10th edition. Ames, IA: Iowa State University Press. pp. 670-679.
- Brockmeier, S.L., Loving, C.L., Mullins, M., Register, K.B., Nicholson, T. L., Wiseman, B.S., Baker, R.B., Kehrli, Jr., M.E. 2013. Virulence, transmission, and heterologous protection of four isolates of Haemophilus parasuis. Clinical and Vaccine Immunology. 20(9):1466-1472.
- Loving, C.L., Lager, K.M., Vincent, A.L., Brockmeier, S.L., Gauger, P.C., Anderson, T.K., Kitikoon, P., Perez, D.R., Kehrli, Jr., M.E. 2013. Efficacy in pigs of inactivated and live attenuated influenza virus vaccines against infection and transmission of an emerging H3N2 similar to the 2011-2012 H3N2v. Journal of Virology. 87(17):9895-903.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): Objective 1: Identify the transmission, genetic, and pathogenic mechanisms of the organisms associated with PRDC, concentrating on the bacterial pathogens and their interactions with each other and select swine viruses. Subobjective 1.1: Identify potential virulence factors of H. parasuis through comparative genomics. Subobjective 1.2: Bacterial response to host conditions. Subobjective 1.3: Evaluate ability of PRDC bacterial pathogens to inhibit Influenza A virus vaccine efficacy and/or exacerbate Influenza A virus-associated disease. Objective 2: Identify potential candidates for novel diagnostic assays, vaccines, and biotherapeutics for bacterial pathogens associated with PRDC. Subobjective 2.1. Develop PCR, ELISA, and/or other assays for detection of bacterial pathogens associated with PRDC. Subobjective 2.2. Identify, develop and/or test the efficacy of potential vaccine candidates to control bacterial pathogens associated with PRDC. Subobjective 2.3. Identify potential biotherapeutic candidates to control bacterial pathogens associated with PRDC. Objective 3: Investigate emerging and potential zoonotic bacterial pathogens that could impact the swine industry and design measures to diagnose, prevent, control and eliminate the threat posed to the swine industry. Subobjective 3.1. Evaluate the relationship between highly pathogenic Asian strains of PRRSV and S. suis infection in swine. Subobjective 3.2: Identification of measures that may prevent, control, or eliminate livestock-associated methicillin- resistant Staphylococcus aureus (MRSA) Sequence Type 398 (ST398) in swine. Approach (from AD-416): Use comparative genomic methods, microarray analysis, and co-infection studies to explore pathogenic mechanisms of bacteria associated with the porcine respiratory disease complex and their interactions with each other and swine viruses. Assess the usefulness of selected genes or proteins identified in comparative genomic analyses for DNA-based identification and classification, serological detection of infection, and potentially as vaccine candidates. Strategies for improved heterologous protection will be tested using live attenuated vaccines, as will the use of immunomodulators, such as granulocyte colony stimulating factor (G-CSF), for therapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease and thus reduce antimicrobial usage to treat clinical and subclinical disease. Investigate emerging and potential zoonotic bacteria that could impact the swine industry. Investigations will focus on pathogen strain characteristics and differences, interactions of bacterial and viral pathogens with the swine host and the microbial ecosystems of the pig. Pathology of both zoonotic and endemic bacterial pathogens of swine will be utilized for the purpose of understanding disease pathogenesis and developing effective diagnostic assays and strategies to control these pathogens and diseases in swine and potentially in humans. We continued genomic sequencing efforts for Haemophilus parasuis (HPS). We now have annotated draft genomes for 10 strains of HPS, and are in the process of comparing the relative virulence of all these isolates in both animal model systems and in vitro assays. This will provide meaningful data on which to base genomic comparisons. These efforts support subobjective 1.1. We constructed a HPS DNA microarray using genome sequence information from isolate SH0165 and other isolates that we have sequenced and confirmed its functionality. Working with a collaborator at the University of California, Santa Barbara Westmont College, we used our Bordetella-specific microarray to identify the genes regulated by a newly identified two-component transactional regulatory system shared between both B. bronchiseptica and B. pertussis. These efforts support subobjective 1.2. Viral infection is thought to predispose the host to secondary infection with bacteria. We have continued to investigate when and how swine influenza virus by itself and as a component of vaccine associated enhanced respiratory disease predisposes pigs to secondary bacterial pneumonia. These efforts support subobjective 1.3. We cloned and expressed recombinant P2 and P5 proteins from a virulent and avirulent isolate of HPS. The respective P2 and P5 alleles are highly divergent and represent different major clades in a phylogeny based on the DNA sequence. We began evaluating the immunogenicity of different HPS outer membrane proteins that may be of value in diagnostic assays and/or as vaccines. We initiated construction of a novel suicide vector for the purpose of generating markerless mutations in HPS. We completed cloning the bsp22 gene from B. bronchiseptica that encodes a Type 3 secretion needle protein that was shown to be immunogenic in mice, and a recombinant replication-defective adenovirus vaccine vector expressing the protein has been generated for testing in pigs. These efforts support subobjectives 2.1.and 2.2. We also completed initial experiments evaluating protection provided by the administration of Granulocyte- Colony Stimulating Factor, a compound that enhances immunity against bacterial infections by increasing the number of circulating neutrophils. These efforts support subobjective 2.3. We completed a swine experiment designed to compare 2 Asian isolates of porcine reproductive and respiratory syndrome virus obtained from the porcine high fever disease outbreaks to high and low virulence isolates obtained from the United States for their alteration of innate immune functions and their ability to predispose to secondary bacterial infections in swine. This experiment supports subobjective 3.1. We completed studies investigating the ability of ST398 methicillin- resistant S. aureus (MRSA) swine isolates to form biofilms and mechanisms to disperse or eliminate biofilms that could be used or evaluated in vivo in the future. In collaboration with scientists at the New Jersey Dental School, we tested the ability of an exopolysaccharide prepared from a nonpathogenic bacterium to inhibit biofilm formation by ST398 MRSA swine isolates. These efforts support subobjective 3.2. Accomplishments 01 Developed strategies to eliminate antibiotic resistant Staphylococcus bacteria in swine. Staphylococcus aureus (S. aureus) is an extremely common yet devastating human pathogen. S. aureus has an outstanding ability to acquire resistance to antibiotics, and in 2003, a new methicillin-resistant S. aureus (MRSA) lineage emerged associated with livestock species including pigs. Therefore the identification of factor or strategies to eliminate the presence of MRSA from swine herds is a to research priority for U.S. swine producers. Bacterial persistence is oft linked to the development of microbial communities known as biofilms tha make the bacteria resistant to immune responses, disinfectants, and antibiotics. ARS scientists at the National Animal Disease Center in Ame Iowa determined that MRSA isolated from swine are capable of forming biofilms and found two compounds able to inhibit or disperse biofilm formation of these isolates. The rationale behind this approach is that compounds that can prevent or disperse biofilm formation will cause the bacteria to either revert to or be held in a free-living state where the are sensitive to host immune responses, disinfectants, available antimicrobial therapies, and other environmental pressures that lead to the clearance of the organism. Collectively, these findings provide a critical first step in designing strategies to control or eliminate MRSA in swine herds. 02 Determination of the genetic basis by which Haemophilus parasuis (H. parasuis) causes disease. H. parasuis is a bacterium that causes Glasser disease in swine, a disease characterized by chronic debilitation and often death 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. ARS scientists at the National Animal Disease Center in Ames, Iowa determine the DNA genomic sequence of 10 strains of H. parasuis with varying capability of causing disease. The DNA sequence can now be used to compa the different isolates for identification of genes contributing to disea development. These data comprise the first publicly available genome sequence for this bacterium. 03 Virulence determinants of porcine reproductive and respiratory syndrome virus (PRRSV) are complex. Demonstrated the correlation of secondary bacterial infections in pigs infected with genetically varied isolates o PRRSV. PRRSV is one of the most devastating and costly diseases to the swine industry world-wide. PRRSV is ubiquitous and mutates rapidly makin it difficult to develop vaccines that protect pigs from disease. ARS scientists at the National Animal Disease Center in Ames, Iowa determine the whole genomic sequence of 7 PRRSV isolates and found that the isolat varied considerably in their genomic sequence as well as their capacity replicate in pigs and predispose to secondary bacterial pneumonia. No specific mutations correlated with disease, indicating specific genomic determinants of disease are complex. These experiments are elucidating mechanisms by which the virus causes disease and alters the pig's immune response to cause increased susceptibility to secondary bacterial infections. 04 Determined a mechanism by which porcine reproductive and respiratory syndrome virus (PRRSV) causes immunosuppression. PRRSV alters the pig's immune response to cause increased susceptibility to secondary bacterial infections. Neutrophils are cells of the immune system that play an important role in combating bacterial infection. Granulocyte-Colony Stimulating Factor (G-CSF), a compound normally produced by the body, increases the number of circulating neutrophils. The use of immunomodulators like G-CSF is a promising area for therapeutic and prophylactic use to prevent and combat infectious disease while eliminating or reducing the use of antibiotics. ARS scientists at the National Animal Disease Center in Ames, Iowa, completed a study in which pigs were given G-CSF to determine if it would reduce the incidence of secondary bacterial infections subsequent to PRRSV infection. Although G CSF did not decrease the prevalence of secondary bacterial infections in PRRSV infected pigs, a paradoxical outcome of the viral infection was an abolition of the surge in circulating neutrophils created by G-CSF, whic represents a previously unrecognized effect of PRRSV on the host immune system and may explain how PRRSV contributes to development of secondary bacterial infections.
Impacts
(N/A)
Publications
- Brockmeier, S.L., Loving, C.L., Nelson, E.A., Miller, L.C., Nicholson, T.L. , Register, K.B., Grubman, M.J., Brough, D.E., Kehrli, Jr., M.E. 2012. The presence of alpha interferon at the time of infection alters the innate and adaptive immune responses to porcine reproductive and respiratory syndrome virus. Clinical and Vaccine Immunology. 19(4):508-514.
- Mullins, M.A., Register, K.B., Bayles, D.O., Dyer, D.W., Kuehn, J.S., Phillips, G.J. 2011. Genome sequence of Haemophilus parasuis strain 29755. Standards in Genomic Sciences. 5(1):61-68.
- Nicholson, T.L., Brockmeier, S.L., Loving, C.L., Register, K.B., Kehrli, Jr., M.E., Stibitz, S.E., Shore, S.M. 2012. Phenotypic modulation of the virulent Bvg phase is not required for pathogenesis and transmission of Bordetella bronchiseptica in swine. Infection and Immunity. 80(3):1025- 1036.
- Register, K.B., Sukumar, N., Palavecino, E.L., Rubin, B.K., Deora, R. 2012. Bordetella bronchiseptica in a paediatric cystic fibrosis patient: possible transmission from a household cat. Zoonoses and Public Health. 59(4):246-250.
- Loving, C.L., Vincent, A.L., Pena, L., Perez, D.R. 2012. Heightened adaptive immune responses following vaccination with a temperature- sensitive, live-attenuated influenza virus compared to adjuvanted, whole- inactivated virus in pigs. Vaccine. 30(40):5830-5838.
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