Progress 10/01/10 to 09/30/13
Outputs
Target Audience: The target audience of this work is other scientists interested in understanding the molecular events leading to immunopathological responses to M. gallisepticum. As all mycoplasmas are inflammatory, the results are also of interest to scientists studying other mycoplasmas (and other inflammatory pathogens). Information gleaned from these studies will ultimately guide vaccine research and may lead to better vaccines and immunotherapeutic approaches to improve the management of this organism. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project is part of a long-standing collaboration with the Center of Excellence in Vaccine Research at UCONN. This collaboration includes members from Dr. Geary's research lab, with assistance on pathology from Dr. Frasca's lab. Each of our collaborators contributes important elements to the project in microbial genetics (Geary), pathology (Frasca) and immunology (Silbart). Students and research techniciansbenefited by developing their respective careers and publication lists, and learned a great deal about host-pathogen interactions by virtue of the "cross-pollination" that has evolved over the years. How have the results been disseminated to communities of interest? Recently generated data regarding changes in gene expression profiles in chicken tracheal epithelial cell ex-vivo upon exposure to Mycoplasma gallisepticum membrane lipoproteins have been summarized in an abstract submitted at the 20th Congress of International Organization of Mycoplasmology, 2014 (S. Majumder). Her work was selected as one of the best student abstracts and she was invited for an oral presentation at the Divisionmeeting of the American Society of Microbiology general meeting, 2013. One of her oral presentations on a related project was awarded the "best student presentation" at the Conference of Research Workers in Animal Disease meeting in Chicago. She has also presented a poster of her work in American Society of Microbiology, General meeting in 2012. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Mycoplasma gallisepticum is a respiratory pathogen of chickens which after attachment to the respiratory epithelium mediates a severe dysregulated state of immune response marked by lymphoplasmacytic and heterophilic infiltration without known invasion or secretion of any known endotoxin or exotoxin. We hypothesized that mycoplasma membrane lipoproteins isolated from pathogenic wild-type strain (Rlow) or the cytoadhesion deficient high-passage avirulent mutant (Rhigh) would be capable of mediating early inflammatory changes from respiratory epithelium, ostensibly due to TLR ligation. To test this hypothesis, live Rlow , Rhigh and lipoproteins from each strain were incubated with primary chicken tracheal epithelial cells in vitro. As anticipated, lipoproteins isolated from both strains up-regulated many inflammatory cytokine and chemokine genes (including IL-12p40, IL-1β, CCL-20, IL-8, IL-6 and NOS-2) after as little as 1.5 hours of exposure at a comparable level to that observed upon incubation with the live strains. However lipoproteins from Rhigh induced somewhat lower responses than those elicited by Rlow in some instances. In presence of a TLR-2 inhibitor, lipoprotein-mediated gene expression of these genes was significantly reduced, in a dose dependent manner, and an NF-κB inhibitor abrogated the responses entirely. We also observed that lipoproteins from both strains were capable of up-regulating expression of similar inflammatory cytokines and chemokines from the tracheal epithelium upon ex-vivo exposure. Taken together we conclude that lipoproteins isolated from Both Rhigh and Rlow induced early inflammatory/chemokine responses in primary tracheal epithelial cells via TLR-2 ligation through an NF-κB dependent pathway.
Publications
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2013
Citation:
Majumder S. and Silbart L.K., (2013), Mycoplasma gallisepticum lipoproteins initiate inflammatory responses in primary
chicken tracheal epithelial cells via an NF-kB dependent pathway, May 18-21, 2013 (Denver, CO), American Society of Microbiology 113th General Meeting.
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2014
Citation:
Majumder S. and Silbart L.K.,(2014), Mycoplasma gallisepticum Lipoproteins Up-regulate Genes Encoding Inflammatory Mediators in Chicken Tracheal Epithelial Cells. Abstract submitted to the International Organization of Mycoplasmologists (IOM), June 1-5, 2014.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Recently generated data regarding changes in gene expression profiles in primary chicken tracheal epithelial cell cultures upon exposure to Mycoplasma gallisepticum membrane lipoproteins have been summarized in an abstract submitted to the American Society of Microbiology and will be presented by the doctoral student, Sanjukta Majumder at the general meeting 2013, who is supported by this award. One of her oral presentations on a related project was awarded the "best student presentation" at the Conference of Research Workers in Animal Disease meeting in Chicago. She has also presented a poster of her work in American Society of Microbiology, General meeting on 2012. PARTICIPANTS: This project is part of a long-standing collaborative interaction amongst the investigators in the Center of Excellence in Vaccine Research at UCONN. This collaboration includes members from Dr. Geary's research lab, with assistance on pathology from Dr. Frasca's lab. Each of our collaborators contributes important elements to the project in microbial genetics (Geary), pathology (Frasca) and immunology (Silbart). Students and research technicians (e.g. Debra Rood) benefit by developing their respective careers and publication lists, and learn a great deal about host-pathogen interactions by virtue of the "cross-pollination" that has evolved over the years. Debra Rood, Frank Zappulla and Sanjukta Majumder worked on various aspects of this project. TARGET AUDIENCES: The target audience of this work is, at least initially, other scientists interested in understanding the molecular events leading to immunopathological responses to M. gallisepticum. As all mycoplasmas are inflammatory, the results are also of interest to scientists studying other mycoplasmas (and other inflammatory pathogens). Information gleaned from these studies will ultimately guide vaccine research and may lead to better vaccines and immunotherapeutic approaches to improve the management of this organism. PROJECT MODIFICATIONS: For the time being we are focusing on the role of membrane lipoproteins on the inflammatory process as hydrogen-peroxide knock-out isogenic mutants had only modest effects on inflammatory gene regulation.
Impacts
Mycoplasma gallisepticum is a respiratory and reproductive pathogen of chickens which attaches to the respiratory epithelium and causes a severe, dysregulated immune response marked by the infiltration of heterophils, macrophages and lymphocytes. We hypothesized that mycoplasma lipoproteins isolated from pathogenic wild-type strain (Rlow) or the cytoadhesion deficient high-passage mutant (Rhigh) would be equally capable of mediating early inflammatory changes, ostensibly due to TLR ligation. To test this hypothesis, lipoproteins were isolated from each strain and incubated with cultured primary chicken epithelial cells. As anticipated, lipoproteins isolated from both strains up-regulated many inflammatory cytokines and chemokine genes (including IL-1β, CCL-20, IL-8, IL-6 and NOS-2) after as little as 1.5 hours of exposure; however lipoproteins from Rhigh induced somewhat lower responses that those isolated from Rlow in some instances. A similar pattern of differential gene expression was observed when epithelial cells were exposed to the live organisms. Upon addition of a TLR-2 inhibitor, lipoprotein-mediated gene expression of both IL-1β and CCL-20 was only slightly reduced. Conversely, an NF-κB inhibitor abrogated the response entirely. Taken together we conclude that lipoproteins isolated from both Rhigh and Rlow induced early inflammatory/chemokine responses in primary tracheal epithelial cells through an NF-κB dependent pathway, but these inflammatory signals were not entirely TLR-2 dependent.
Publications
- Majumder S and Silbart L.K. (2013) Mycoplasma gallisepticum lipoproteins initiate inflammatory responses in primary chicken tracheal epithelial cells via an NF-κB dependent pathway. Abstract (accepted). ASM for presentation May 18-21, 2013 (Denver, CO), American Society of Microbiology 113th General Meeting.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Recently generated data regarding changes in gene expression profiles in chicken epithelial primary cultures upon exposure to Mycoplasma gallisepticum or membrane lipoproteins derived there from have been summarized in an abstract submitted to the American Society of Microbiology and will be presented by the doctoral student, Sanjukta Majumder, who is supported by this award. One of her oral presentations on related work was awarded the "best student presentation" at the Conference of Research Workers in Animal Disease meeting in Chicago. PARTICIPANTS: This project is part of a long-standing collaborative interaction amongst the investigators in the Center of Excellence in Vaccine Research at UCONN. This collaboration includes members from Dr. Geary's research lab, with assistance on pathology from Dr. Frasca's lab. Each of our collaborators contribute important elements to the project in microbial genetics (Geary), pathology (Frasca) and immunology (Silbart). Students and research technicians (e.g. Debra Rood) benefit by developing their respective careers and publication lists, and learn a great deal about host-pathogen interactions by virtue of the "cross-pollination" that has evolved over the years. Debra Rood, Frank Zappulla and Sanjukta Majumder worked on various aspects of this project. TARGET AUDIENCES: The target audience of this work is, at least initially, other scientists interested in understanding the molecular events leading to immunopathological responses to M. gallisepticum. As all mycoplasmas are inflammatory, the results are also of interest to scientists studying other mycoplasmas (and other inflammatory pathogens). Information gleaned from these studies will ultimately guide vaccine research and may lead to better vaccines and immunotherapeutic approaches to improve the management of this organism. PROJECT MODIFICATIONS: For the time being we are focusing on the role of membrane lipoproteins on the inflammatory process as hydrogen-peroxide knock-out isogenic mutants had only modest effects on inflammatory gene regulation.
Impacts
Mycoplasma gallisepticum is an important and pervasive pathogen affecting commercial poultry production. It causes respiratory and reproductive disease in chickens following attachment to the ciliated tracheal epithelium, eventually leading to severe lymphoplasmacytic and histiocytic infiltration of the tracheal mucosa. Mycoplasmal lipoproteins play an important role in the pathogenesis of many mycoplasmas, but have not been well studied in the context of M. gallisepticum infection. As M. gallisepticum does not typically invade the chicken trachea, we hypothesized that early inflammatory changes may be initiated upon interaction of bacterial lipoproteins with tracheal epithelial cells. To test this hypothesis, a primary chicken tracheal epithelial cell culture system was developed in order to examine changes in gene expression at early time points post-exposure. Bacterial lipoproteins were isolated by Triton TX-114 phase partitioning and incubated with cultured epithelial cells at varying concentrations. Changes in gene expression were compared to similar experiments using the pathogenic Rlow strain of M. gallisepticum. Alterations in gene expression patterns were observed using RT-PCR upon incubation with as little as 5 μg/mL lipoprotein, and several key inflammatory genes were expressed within 1.5 hours of exposure, including IL-1β, IL-6, IL-8 and MIP-1β. This pattern of expression was similar to that induced by the Rlow strain at an MOI of 500, peaking at 1.5 hours post exposure and waning thereafter. A more detailed analysis of altered gene expression using microarray revealed that other important genes are induced within 1.5 hours of exposure, including CCL20, nitric oxide synthase-2, prostaglandin synthase 2 precursor and plasminogen activator urokinase. Taken together, these findings indicate that M. gallisepticum lipoproteins may play an important role in pathogenesis by mediating the up-regulation of early inflammatory genes and chemokines, thereby setting the stage for subsequent inflammatory events in the chicken trachea. Ultimately, this work will lead to new approaches for rational vaccine design for this important poultry pathogen.
Publications
- Majumder S, Zappulla F, Silbart L.K. (2012) Mycoplasma gallisepticum lipoproteins induce differential expression of early response genes in cultured chicken tracheal epithelial cells. Abstract 12-A-2422-GM-ASM for presentation June 16-19, 2012 (San Francisco, CA), American Society of Microbiology 112th General Meeting.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Recent data regarding the in situ expression of inflammatory genes in Rlow infected chicken tracheas was communicated through an oral presentation by the doctoral student, Sanjukta Majumder, who is working on this project. Her presentation was awarded the "best student presentation" at the Conference of Research Workers in Animal Disease meeting in Chicago. This work was also presented at the Regional American Society of Microbiology meeting in Albany, NY. This work will also be published as an extended abstract in the AAVI proceedings. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: As one of the target audiences is research scientists working on similar problems, the presentation of this data at two important research conferences is notable. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
( 553 characters) This project is part of a long-standing collaborative interaction amongst the investigators in the Center of Excellence in Vaccine Research at UCONN. Drs. Geary, Frasca and Silbart each contribute important elements to the project in microbial genetics and pathogenesis (Geary), pathology (Frasca) and immunology (Silbart). Students and research technicians benefit by developing their respective careers and publication lists, and learn a great deal about host-pathogen interactions by virtue of the "cross-pollination" that has evolved over the years.
Publications
- Majumder S., Mohammed J., Rood D., Szczepanek S.M., Geary S.J., Frasca Jr. S. , Silbart L.K. (2010); Mycoplasma gallisepticum Infection Causes Up-Regulation of Inflammatory Mediators, Cytolytic Molecules and Pro-Apoptotic genes in Chickens. American Society of Microbiology, Regional Meeting , November 9-10,2010, Albany, NY
- Majumder S., Mohammed J., Rood D., Szczepanek S.M., Geary S.J., Frasca Jr S. , Silbart L.K. (2010); Expression of Inflammatory Mediators and Pro-apoptotic Genes During Mycoplasma gallisepticum Infection. Conference of Research Workers in Animal Diseases, December 5-7, 2010, Chicago, IL
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: This project is part of a long-standing collaborative interaction amongst the investigators in the Center of Excellence in Vaccine Research at UCONN. Drs. Geary, Frasca and Silbart each contribute important elements to the project in microbial genetics and pathogenesis (Geary), pathology (Frasca) and immunology (Silbart). Students and research technicians benefit by developing their respective careers and publication lists, and learn a great deal about host-pathogen interactions by virtue of the "cross-pollination" that has evolved over the years. Currently a Ph.D. student in Animal Science is investigating the temporal expression of key cytokine/chemokine, TLR and apoptotic gene trascripts to better understand their cellular context. These include genes that were identified as substantially upregulated in microarray studies (verified by RT-PCR)following M. gallisepticum infection. After classifying these genes into families, five probes have been developed for in situ analysis of chicken tracheal tissues: CCL-19, IL-10, IFN-gamma, granzyme A and MIP-2. The results of these studies will be presented at the international organization of mycolasmology annual meeting. PARTICIPANTS: This project is part of a long-standing collaborative interaction amongst the investigators in the Center of Excellence in Vaccine Research at UCONN. Drs. Geary, Frasca and Silbart each contribute important elements to the project in microbial genetics and pathogenesis (Geary), pathology (Frasca) and immunology (Silbart). Students and research technicians benefit by developing their respective careers and publication lists, and learn a great deal about host-pathogen interactions by virtue of the "cross-pollination" that has evolved over the years. Debra Rood and Sanjukta Majumder worked on this project to move the in situ analyses discussed in other sections, forward. TARGET AUDIENCES: The target audience is, for the most part, other scientists working in the field of animal health. Since the research is aimed at understanding virulence determinants of an important poultry pathogen with an eye toward vaccine development, it will ultimately be of interest to veterinary vaccine manufacturers and end users in the poultry industry. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
It is always difficult to assess the impact of research publications on the scientific community until years later when similar studies build upon those which preceded it. However, publication in high-impact scholarly journals (such as Vaccine) is certainly an indicator that the editorial staff and reviewers believe that the experiments are valid and that the results and conclusions are of interest to their readers. Ultimately, this work will lead to new approaches for rational vaccine design for this important poultry pathogen. As mentioned above, the results of these studies will be presented at the international organization of mycolasmology annual meeting.
Publications
- No publications reported this period
|
Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: This project is part of a long-standing collaborative interaction amongst the investigators in the Center of Excellence in Vaccine Research at UCONN. Drs. Geary, Frasca and Silbart each contribute important elements to the project in microbial genetics and pathogenesis (Geary), pathology (Frasca) and immunology (Silbart). Students and research technicians benefit by developing their respective careers and publication lists, and learn a great deal about host-pathogen interactions by virtue of the "cross-pollination" that has evolved over the years. PARTICIPANTS: This project is part of a long-standing collaborative interaction amongst the investigators in the Center of Excellence in Vaccine Research at UCONN. Drs. Geary, Frasca and Silbart each contribute important elements to the project in microbial genetics and pathogenesis (Geary), pathology (Frasca) and immunology (Silbart). Students and research technicians benefit by developing their respective careers and publication lists, and learn a great deal about host-pathogen interactions by virtue of the "cross-pollination" that has evolved over the years. Debra Rood and Sanjukta Majumder worked on this project. TARGET AUDIENCES: It is always difficult to assess the impact of research publications on the scientific community until years later when similar studies build upon those which preceded it. However, publication in high-impact scholarly journals (such as Vaccine) is certainly an indicator that the editorial staff and reviewers believe that the experiments are valid and that the results and conclusions are of interest to their readers. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
It is always difficult to assess the impact of research publications on the scientific community until years later when similar studies build upon those which preceded it. However, publication in high-impact scholarly journals (such as Vaccine) is certainly an indicator that the editorial staff and reviewers believe that the experiments are valid and that the results and conclusions are of interest to their readers. Ultimately, this work will lead to new approaches for rational vaccine design for this important poultry pathogen. In addition, we have recruited a new doctoral student who will receive assistance from this financial support.
Publications
- Gates A.E., Frasca, S. Jr, Nyaoke, A., Gorton, T.S., Silbart, L.K., Geary, S.J. 2008. Comparative Assessment of a Metabolically Attenuated Mycoplasma gallisepticum Mutant as a Live Vaccine for the Prevention of Avian Respiratory Mycoplasmosis. Vaccine Apr. 7;26(16):2010-9. Epub 2008 Feb 22
|
Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Two manuscripts have been prepared that communicate the major findings of our research group on this project. In addition, Dr. Silbart presented these findings at an invited talk for the American Society of Microbiology's annual meeting (Division G).
PARTICIPANTS: This project was part of a long-standing collaborative interaction amongst the investigators in the Center of Excellence in Vaccine Research at UCONN. Drs. Geary, Frasca and Silbart each contribute important elements to the project in microbial genetics and pathogenesis (Geary), pathology (Frasca) and immunology (Silbart). Students and research technicians benefit by developing their respective careers and publication lists, and learn a great deal about host-pathogen interactions by virtue of the "cross-pollination" that has evolved over the years.
TARGET AUDIENCES: The target audience for this project is, initially, the scientific and vaccine manufacturing communities. As the project develops, certain live attenuated strains may become vaccine candidates and attract the interest of our technology transfer office for possible commercialization. If this occurs, the target audience would be expanded to include the farm community, agricultural extension agents, sales and marketing professionals and others.
Impacts
It is always difficult to assess the impact of research publications on the scientific community until years later when similar studies build upon those which preceded it. However, publication in high-impact scholarly journals (such as Vaccine) is certainly an indicator that the editorial staff and reviewers believe that the experiments are valid and that the results and conclusions are of interest to their readers. Based on the comments and questions raised after the presentation of our results at an international meeting (ASM), our work seemed to engender a great deal of interest and discussion. Ultimately, this work will lead to new approaches for rational vaccine design for this important poultry pathogen. In addition, a doctoral student completed and defended his dissertation based upon these studies and is now completing a post-doctoral fellowship. These outputs would not have been possible without the financial resources and facilities that these funds help to
support.
Publications
- Mohammed, J., Frasca, S. Jr., Cecchini, K., Rood, D., Nyaoke, A.C., Geary, S.J., Silbart, L.K. 2007. Chemokine and cytokine gene expression profiles in chickens inoculated with Mycoplasma gallisepticum strains R(low) or GT5. Vaccine. 25(51):8611-21 Epub 2007 Oct 16.
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Progress 01/01/06 to 12/31/06
Outputs
Colonization of the avian respiratory tract with Mycoplasma gallisepticum results in a profound inflammatory response in the air sacs, conjunctiva, trachea and lungs. The tracheal inflammation is characterized by the infiltration of the mucosa by macrophages, heterophils, and lymphocytes, which leads to considerable immunopathology. In the first study completed during the project period, we investigated changes in cytokine and chemokine gene expression profiles in tracheas of chickens inoculated with the virulent M. gallisepticum strain (Rlow) vs. the attenuated GT5 strain using real-time RT-PCR. Expression of lymphotactin mRNA was found to be higher in Rlow-inoculated chickens, while CXCL13/BCA1 mRNA expression levels were higher in both Rlow- and GT5-inoculated chickens on day 1 post-inoculation (when comparted to PBS-inoculated controls). However, both the GT5 and Rlow strains induced a down-regulation of mRNA levels for CCL20, IL-1β, IL-8 and IL-12 genes (by
10-, 2-, 10- and 20-fold, respectively), with CCL20 and IL-12 mRNA levels remaining lower on days 4 and 8 post-inoculation. On day 4, Rlow-inoculated chickens exhibited higher tracheal lesion scores and mRNA levels for lymphotactin, CXCL13, CXCL14, RANTES, MIP-1β, IL-1β and IFN-γ mRNA (when compared to PBS-inoculated controls). GT5-inoculated chickens had a modest increase in lymphotactin and CXCL13 and a 5-fold decrease in IL-8 mRNA levels compared to PBS-inoculated controls. In the second study, the mRNA expression profiles of chemokines, cytokines and other molecules involved in innate and acquired immune response during M. gallisepticum infection were investigated. Significantly higher mRNA levels of lymphocyte chemoattractant molecules such as CCL19, CXCL13, lymphotactin and IL-16 were detected in tracheal mucosa of infected chickens. In addition, IFN-γ and IL-10 mRNA levels were significantly higher, while IL-12p40 mRNA expression was down-regulated in M.
gallisepticum-infected chickens. These results reflect the importance of lymphocyte and monocyte chemotactic factors in the development of tracheal lesions in chickens inoculated with pathogenic strains of M. gallisepticum.
Impacts
Mycoplasma gallisepticum is an important avian pathogen that leads to significant losses in productivity. Current vaccines are beneficial, but have several problems, including pathogenicity in turkeys. Further improvements and refinements of these vaccines will require a better understanding of the correlates of immune protection induced by vaccines. This information will be critical to designing more effective vaccines for Mycoplasma gallisepticum as well as other respiratory disease of poultry.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
Colonization of the avian respiratory tract with Mycoplasma gallisepticum results in a profound inflammatory response in the air sacs, conjunctiva, trachea and lungs. A live-attenuated M. gallisepticum vaccine strain GT5 was previously shown to be protective in chickens upon challenge; however, the mechanisms by which it confers protection remains unknown. Our research group evaluated several potential correlates of GT5 vaccine-mediated immune protection following challenge with the pathogenic M. gallisepticum Rlow. The GT5 vaccinated chickens developed mild tracheal lesions, with few and scattered, discrete, lymphofollicular aggregates occurring in the lamina propria. In addition, low numbers of aggregated B-cells, CD4+ and CD8+ cells infiltrated the trachea, in contrast to large numbers found in sham-vaccinated chickens. Lymphofollicular aggregates were only occasionally observed before day 12 post-challenge in sham-vaccinated chickens. Instead, the tracheas
contained an increasingly more cellular inflammatory response resulting in expansion of the lamina propria by lymphoplasmacytic and histiocytic infiltrates. This correlated with expansion of interfollicular zones by large numbers of infiltrating CD8+ and CD4+ T cells and a large population of IgG- and IgA-secreting plasma cells. GT5 vaccinated chickens had higher serum IgG concentrations and significantly higher numbers of M. gallisepticum-specific IgA- and IgG-secreting plasma/B cells in the trachea than sham vaccinated chickens. These responses were observed as early as day 4 post-challenge, indicating the importance of antibody-mediated clearance of mycoplasma in GT5 vaccinated chickens. A second study to assess the safety of the Mg7 vaccine was recently completed to determine the relationship between dose, immune response and respiratory tract lesions. At high doses (e.g. 3 X 108), strong serum IgG responses were observed, with very low levels of concomitant inflammation. A
vaccinate/challenge study will be scheduled in the near future to assess the protective capacity of this putative vaccine.
Impacts
Mycoplasma gallisepticum is an important avian pathogen that leads to significant losses in productivity. Current vaccines are beneficial, but have several problems, including pathogenicity in turkeys. Further improvements and refinements of these vaccines will require a better understanding of the correlates of immune protection induced by vaccines. This information will be critical to designing more effective vaccines for Mycoplasma gallisepticum as well as other respiratory disease of poultry.
Publications
- Javed, M.A., Frasca, Jr., S., Rood, D., Cecchini, K.,Gladd, M., Geary, S.J., Silbart, L.K. 2005. Correlates of Immune Protection in Chickens Vaccinated with Mycoplasma gallisepticum Strain GT5 following Challenged with Pathogenic M. gallisepticum Strain Rlow. Infection and Immunity 73:5410-5419
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Progress 01/01/04 to 12/31/04
Outputs
Colonization of the avian respiratory tract with Mycoplasma gallisepticum results in a profound inflammatory response in the air sacs, conjunctiva, trachea and lungs. A live-attenuated M. gallisepticum vaccine strain GT5 was previously shown to be protective in chickens upon challenge; however, the mechanisms by which it confers protection remains unknown. Our research group evaluated several potential correlates of GT5 vaccine-mediated immune protection following challenge with the pathogenic M. gallisepticum Rlow. The GT5 vaccinated chickens developed mild tracheal lesions, with few and scattered, discrete, lymphofollicular aggregates occurring in the lamina propria. In addition, low numbers of aggregated B-cells, CD4+ and CD8+ cells infiltrated the trachea, in contrast to large numbers found in sham-vaccinated chickens. Lymphofollicular aggregates were only occasionally observed before day 12 post-challenge in sham-vaccinated chickens. Instead, the tracheas
contained an increasingly more cellular inflammatory response resulting in expansion of the lamina propria by lymphoplasmacytic and histiocytic infiltrates. This correlated with expansion of interfollicular zones by large numbers of infiltrating CD8+ and CD4+ T cells and a large population of IgG- and IgA-secreting plasma cells. GT5 vaccinated chickens had higher serum IgG concentrations and significantly higher numbers of M. gallisepticum-specific IgA- and IgG-secreting plasma/B cells in the trachea than sham vaccinated chickens. These responses could be observed as early as day 4 post-challenge, indicating the importance of antibody-mediated clearance of mycoplasma in GT5 vaccinated chickens. Having a better understanding of these correlates of immunity will lead to advances in vaccine design, and a faster response to vaccine failures should they occur in the future.
Impacts
Mycoplasma gallisepticum is an important avian pathogen that leads to significant losses in productivity. Current vaccines are beneficial, but have several problems, including pathogenicity in turkeys. Further improvements and refinements of these vaccines will require a better understanding of the correlates of immune protection induced by vaccines. This information will be critical to designing more effective vaccines for Mycoplasma gallisepticum as well as other respiratory disease of poultry.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
In order to better understand the correlates of mucosal immune protection conferred to chickens vaccinated with a live attenuated Mycoplasma gallisepticum (M. gal) vaccine, we embarked upon an immunopathological time course study. This study focused exclusively on the course of infection and immune response in tracheas of SPF chickens infected with the pathogenic R-low strain. We found that initial attachment of M. gal to mucus occurred within one day of challenge, and that colonization of respiratory epithelium occurred within 3 days. By day seven, nearly the entire surface of the tracheal epithelium was colonized, but few if any microorganisms were present in the sub-epithelial tissues until days 9 and 14 post-infection. Curiously, only a mild inflammatory infiltrate was observed within the first 3- to 7-days of infection. This inflammation was characterized by a diffuse heterophilic infiltrate, with a more focal clustering of lymphocytes. Immunohistochemical
staining revealed the presence of clearly defined B cell follicles and interfollicular T cell zones, reminiscent of mammalian secondary lymphoid follicles. These follicles stained weakly for IgG, yet contained few, if any, IgA+ B cells. By day 9 post-infection, dramatic changes in tissue morphology had occurred. The most obvious changes involved mucosal thickening resulting from moderate to severe diffuse lymphocytic infiltrates and expansion of the lamina propria, with or without luminal and intra-epithelial heterophilic infiltrates. The lymphocyte staining pattern revealed large numbers of IgA+ B or plasma cells distributed throughout the sub-epithelial connective tissue, with very few IgG positive cells. M. gal specific ELISA assays confirmed these observations, with approximately 8-fold more M. gal-specific IgA secreting cells and a comparable ratio of M. gal specific IgA/IgG titers in tracheal washings. Flow cytometric analysis confirmed the large number of lymphocytes present in
the tissues (both B and T cells), as well as the overwhelming number of granulocytes, presumably heterophils, in tissues harvested at days 9 and 14 days post-infection. A follow-up study is planned in which comparisons will be made between the live attenuated GT5 vaccinated and control chickens challenged with the pathogenic R-low strain.
Impacts
Mycoplasma gallisepticum is an important avian pathogen that leads to significant losses in productivity. Current vaccines are beneficial, but have several problems, including pathogenicity in turkeys. Further improvements and refinements of these vaccines will require a better understanding of the correlates of immune protection induced by vaccines. This information will be critical to designing more effective vaccines for Mycoplasma gallisepticum as well as other respiratory disease of poultry.
Publications
- Orenstein, S. M., S. Levisohn, S. J. Geary, D. Yogev. 2003. Cytadherence-Deficient Mutants of Mycoplasma gallisepticum Generated by Transposon Mutagenesis, Infect. Immunity, Jul;71(7):3812-20.
- Papazisi, L., Gorton, T., Kutish, G., Markham, P., Browning, G., Nguyen, D., Swartzell, S., Madan, A., Mahairas, G. and S. J. Geary. 2003. "The complete genome sequence of the avian pathogen Mycoplasma gallisepticum strain Rlow". Microbiology. 2003 Sep;149(Pt 9):2307-16
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Progress 01/01/02 to 12/31/02
Outputs
A study comparing two attenuated Mycoplasma gallisepticum (M.gallisepticum) strains was completed in which each was assessed for its ability to protect chickens against homologous challenge. Both strains were based upon a high-passage M. gallisepticum (R-high) that contains two point mutations in an important cytadhesion molecule, termed GapA. These mutations result in premature termination of the gapA gene product, as well as other downstream genes, with no detectable protein product expressed on the cell membrane. Reconstitution(complementation) of R-high with gapA alone resulted in the M. gallisepticum strain termed GT5. Although GT5 expressed gapA on its surface, it exhibited poor binding properties to epithelial cells in comparison to the R-high stain. This may have been due to the lack of expression of the gene downstream of gapA termed crmA ("cytadhesion related molecule A"). Based upon these results, we hypothesized that expression of gapA on the surface of
GT5 would result in immune recognition of this epitope, without increasing its pathogenicity. Intratracheal administration of either R-high or GT5 conferred significant levels of protection in two separate experiments. Birds immunized with the GT-5 strain produced a significant amount of IgG anti-M. gallisepticum in both serum and in tracheal wash samples. Little, if any M. gallisepticum specific IgA was detected in nasal, tracheal or lung wash samples collected two weeks after immunization, however very high titers were measured in all groups challenged with the R low strain (1X10(7) organisms). Surprisingly, the highest titers of mucosal antibodies were observed in sham immunized/challenged animals, which in some cases were 40-fold higher than those observed in vaccinated birds. No discernable lesions were observed in tracheal or air sac tissues harvested from GT5 or R-high vaccinated birds that were subsequently challenged with R-low. In contrast, sham immunized birds that received
the R-low challenge demonstrated gross pathological lesions characteristic of M. gallisepticum infection. This observation was confirmed by histopathological examination. Based upon these studies, it appears as though the GT5 attenuated strain is quite capable of conferring protection to birds challenged with the homologous strain. This protection borders on sterilizing immunity, as only very low numbers of organisms were isolated two weeks after challenge, and none after four weeks. Protection may be mediated by either serum or mucosal antibodies, although these studies do not rule out the possible role of cell mediated or inducible elements of the innate immune system. Expression of the recombinant GapA protein has been completed and will be used for mucosal vaccination of chickens. If sufficiently high antibody titers are observed, challenge studies will follow.
Impacts
Mycoplasma gallisepticum is an important avian pathogen that leads to significant losses in productivity. Current vaccines are beneficial, but have several problems including pathogenicity in turkeys. Improved attenuated and subunit vaccines are needed, and the two strains being evaluated are potential candidates. Further testing of these vaccines is needed, both in the field, and in turkeys as this species has had significant problems with currently available vaccines.
Publications
- Papazisi, L., Silbart, L.K., Frasca Jr., S., Rood, D., Liao, X., Gladd, M. Javed, M.A. and S. J. Geary (2002) A Modified Live Mycoplasma gallisepticum Vaccine to Protect Chickens From Respiratory Disease. Vaccine 20:3709-19.
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Progress 01/01/01 to 12/31/01
Outputs
A study comparing two attenuated Mycoplasma gallisepticum (M.gallisepticum) strains was completed in which each was assessed for its ability to protect chickens against homologous challenge. Both strains were based upon a high-passage M. gallisepticum (R-high) that contains two point mutations in an important cytadhesion molecule, termed GapA. These mutations result in premature termination of the gapA gene product, as well as other downstream genes, with no detectable protein product expressed on the cell membrane. Reconstitution (complementation) of R-high with gapA alone resulted in the M. gallisepticum strain termed GT5. Although GT5 expressed gapA on its surface, it exhibited poor binding properties to epithelial cells in comparison to the R-low strain. This may have been due to the lack of expression of the gene downstream of gapA termed crmA ("cytadhesion related molecule A"). Based upon these results, we hypothesized that expression of gapA on the surface of
GT5 would result in immune recognition of this epitope, without increasing its pathogenicity. Intratracheal administration of either R-high or GT5 conferred significant levels of protection in two separate experiments. Birds immunized with the GT5 strain produced a significant amount of IgG anti-M. gallisepticum in both serum and in tracheal wash samples. Little, if any M. gallisepticum specific IgA was detected in nasal, tracheal or lung wash samples collected two weeks after immunization, however very high titers were measured in all groups challenged with the R low strain (1X10(7) organisms). Surprisingly, the highest titers of mucosal antibodies were observed in sham immunized/challenged animals, which in some cases were 40-fold higher than those observed in vaccinated birds. No discernable lesions were observed in tracheal or air sac tissues harvested from GT5 or R-high vaccinated birds that had been challenged with R-low. In contrast, sham immunized birds that received the R-low
challenge demonstrated gross pathological lesions characteristic of M. gallisepticum infection. This observation was confirmed by histopathological examination. Based upon these studies, it appears as though the GT5 attenuated strain is quite capable of conferring protection to birds challenged with the homologous strain. This protection borders on sterilizing immunity, as only very low numbers of organisms were isolated two weeks after challenge, and none after four weeks. Protection may be mediated by either serum or mucosal antibodies, although these studies do not rule out the possible role of cell mediated or inducible elements of the innate immune system.
Impacts
Mycoplasma gallisepticum is an important avian pathogen that leads to significant losses in productivity. Current vaccines are beneficial, but have several problems including pathogenicity in turkeys. Improved attenuated and subunit vaccines are needed, and the two strains being evaluated are potential candidates. Further testing of these vaccines is needed, both in the field, and in turkeys as this species has had significant problems with currently available vaccines.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
Two attenuated Mycoplasma gallisepticum (M.gallisepticum) strains were evaluated for their ability to protect chickens from homologous challenge. These strains were based upon a high-passage M. gallisepticum (R-high) that contains two point mutations in an important cytadhesion molecule termed gapA. These mutations result in premature termination of the gapA gene product, as well as other downstream genes, with no detectable protein product expressed on the cell membrane. Reconstitution (complementation) of R-high with gapA alone resulted in the second M. gallisepticum strain, termed GT-5. Surprisingly, GT-5 expressed gapA on its surface, but exhibited poor binding properties to epithelial cells, analogous to the R-high stain. This may be due to the lack of expression of the gene downstream of GapA termed crmA ("cytadhesion related molecule A"). We hypothesized that expression of GapA on the surface of GT-5 would result in immune recognition of this epitope, without
increasing its pathogenicity. Intratracheal administration of either R-high or GT-5 conferred significant levels of protection in two separate experiments. High titer serological and mucosal IgG and IgA anti-M. gallisepticum titers were observed in all immunized animals prior to challenge with wild type organisms (R-low). When these birds were challenged with wild-type M. gallisepticum (1 X 10 exp 7, R-low), no discernable lesions resulted when tracheal and air sac scores were compared with unchallenged control animals. In contrast, sham immunized control birds that received the R-low challenge demonstrated gross pathological lesions characteristic of M. gallisepticum infection. This observation was confirmed by histopathological examination. In addition, M. gallisepticum was only isolated from sham immunized birds challenged with R low. To determine if the R-high strain could serve as an expression vector for chickens, the avian influenza virus (AIV) hemagglutinin (H5HA) was selected
as a heterologous vectored antigen, and the sequence inserted using transposon mediated transformation. Chickens immunized with the resulting strain (GTHA) or R-high showed undetectable lesions in tracheas and air sacs and no significant changes in gross and histopathology. Antisera from immunized birds recognized the HA antigen when probed via Western blot. These birds were not protected upon challenge with homologous avian influenza virus. Modifications to the GTHA construct are planned and repeat challenge experiments will be performed in collaboration with the USDA unit in Atlanta, GA.
Impacts
Mycoplasma gallisepticum is an important avian pathogen that leads to significant losses in productivity. Current vaccines are beneficial, but have several problems incuding pathogenicity in turkeys. Improved attenuated and subunit vaccines are needed, and the two strains being evaluated are potential candidates. Further testing of these vaccines is needed.
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
The overall goal of this research project is to develop a mucosal vaccine to Mycoplasma gallisepticum (M. gal) that is capable of conferring protection to chickens and turkeys upon exposure at respiratory surfaces. Our approach to this problem involves mucosal and parenteral immunization of chickens with a recombinant GapA molecule that is expressed as a fusion protein with maltose binding protein (MBP). The GapA molecule is expressed on the bacterial surface, and is involved in adherence to the chicken respiratory epithelial surface (trachea, lungs and air sacs). A truncated form of this cytadhesin molecule, approximately 30 kD in size, was genetically fused to the coding sequence for MBP. The recombinant GapA-MBP fusion protein was expressed in E. coli, then purified using a maltose-based affinity resin. The purified fusion protein (80 ug) was injected intramuscularly into the breast muscle of five chickens as an emulsion in RIBI adjuvant. Two control groups also
received either a sham immunization (saline) or a low dose of GapA (20 ug) that was purified from gels. Sera and nasal secretions were collected from each chicken on a weekly basis, and all birds were given a booster immunization, identical to their priming immunization, three weeks after the priming dose. After five weeks, the birds were challenged with 5 X 10(9) live S6 organisms, and were sacrificed two weeks thereafter. ELISA titers indicated that the truncated GapA was a potent immunogen, with all five chickens attaining serum IgG anti-GapA titers exceeding 1000 prior to boosting. After the booster immunization, the responses increased by approximately four-fold, then dropped off dramatically by week seven. As anticipated when using a parenteral route of immunization, little if any IgG- or IgA anti-M. gal specific antibodies were present in nasal secretions. Histopathological analysis of the respiratory tissues harvested from these animals revealed moderate to severe lesions in
all three treatment groups, but no discernable differences between the vaccinated groups and controls. These results prompted a series of experiments to define the appropriate challenge dose and strain. After four dose-response studies, we have identified a challenge dose of 2 X 10(6) of the R-low strain, which yielded reproducible lesions in a large proportion of the birds. This challenge dose and strain will be used in subsequent experiments designed to test the efficacy of the MBP-GapA fusion protein to be administered both mucosally and parenterally. Our research group is also performing site-directed mutagenesis on the GapA gene so as to express the entire construct in E. coli. This requires changing one base pair in all 17 codons that encode tryptophan in M. gal, but a stop codon in E. coli. Once these alterations in codon usage have been made, the full-length construct will be expressed in E. coli as an MBP fusion protein, then purified and tested for immunogenicity and
protective efficacy in chickens as described above.
Impacts
Although the experiments described above have not yet confirmed the utility of the MBP-GapA fusion protein as a vaccine candidate, they have laid the groundwork for meaningful immunization/challenge studies in years 2 and 3 of this project.
Publications
- No publications reported this period
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