Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? The US food animal economy has long been threatened by infectious diseases that could devastate the cattle, swine and poultry industries. Viral and bacterial infections affecting the airways and other mucosal tissues present the major risk to these economically important animal species. Pathogenic microbes are now, and will continue to be, the most significant cause of animal disease and economic loss. The easy pathogens have been dealt with, the difficult, intractable ones remain. New pathogens are continuously emerging and, in some cases, past disease control strategies (vaccines, antibiotics, other drugs and chemicals) are either losing effectiveness and/or public support for their use. Development and administration of new, more effective vaccines to counteract these pathogens will
increase food safety, quality and production that will strengthen our markets at home and abroad. This research falls within component 1: Biodefense Research of the NP- 103 National Program. The recent Foot and Mouth Disease Virus outbreak devastated Britain and many European countries is an unfortunate harbinger of what could happen in the US. The outbreak not only affected the food animal production industries in these countries but also severely damaged travel and tourism. Tourists were restricted from visiting sites in the countryside such as Stonehenge in England for fear of spreading the virus. This discourages tourist travel to these countries and results in lost revenues. There is a growing risk that damaging infections will be introduced and spread through the US food animal population due to several factors: enhanced opportunities for transmission of agents among host animals, rapid dispersal of infected animals during commercial distribution, and inadvertent introduction
of microbial agents through increased importation. The result could be introduction of the pathogen without immediate symptoms of overt disease, raising the prospect that massive loss in a sector of the food animal industry could occur without adequate, prior warning. Compounding this threat is the ominous emergence of new or dormant microbial diseases resulting from ever increasing global economic and societal interactions. Mycoplasma gallisepticum infection is of considerable economic importance to poultry producers throughout the world. It causes respiratory diseases in the form of tracheitis and air sacculitis in chickens and turkeys. Mycoplasma gallisepticum infection is the major cause of reduced egg production, reduced hatchability, and downgrading of carcasses. Losses to the broiler chicken industry are estimated at $588 million annually in the United States. Additionally, an estimated 37% of the commercial egg laying birds (262 million birds) in the United States are infected
with M. gallisepticum resulting in a $132 million annual loss to that industry. Transmission is through the egg or by infected airborne droplets and depending on the system of management, the infection may spread rapidly through an entire flock. Consequently there is increasing interest in new approaches for M. gallisepticum control. Finally, we must consider the possibility that clandestine introduction of the most destructive disease agents may occur through an act of bioterrorism aimed at damaging the $104 billion (annual) US, cattle, swine and poultry industries. This would have a devastating and irreversible effect on these export markets that exceed $8 billion annually. 2. List by year the currently approved milestones (indicators of research progress) FY 2006: 1. Identify virulence determinants and protective epitopes in Mycoplasma gallisepticum. These will be characterized biochemically and their encoding genes identified, cloned and sequences determined and analyzed. We will
expand the utility of a modified live strain of Mycoplasma gallisepticum both as a vaccine and vector for the expression and delivery of heterologous antigens. We will utilize the M. gallisepticum genomic data to investigate the mechanism(s) regulating gene expression and how this relates to the expression of proteins involved in virulence and host response. 2. Investigate the utility of specific amino acids of the VP1 protein of the FMDV as mucosal vaccine candidates, using both synthetic peptide and DNA vaccine approaches. 3. Investigate the use of recombinant chicken interferon-alpha (rChIFN- alpha) as an immunostimulation/adjuvant with low pathogenicity (LP) avian influenza virus. 4. Develop specific diagnostic assays for the rapid identification of the above mentioned organisms as well as to designated USDA specified threat agents. 4a List the single most significant research accomplishment during FY 2006. The completion of genome sequencing and annotations of M. gallisepticum
strains Rhigh and F which allowed us to perform comparative genetic analysis of the virulent Rlow strain versus the avirulent Rhigh and F strains. 4b List other significant research accomplishment(s), if any. The engineering and expression of a non-toxic Pseudomonas aeruginosa exotoxin A (ntPE) and assessment of its capacity to serve as a carrier/adjuvant for delivery of the G-H loop of the FMDV, in its native conformation. 4d Progress report. Mycoplasma gallisepticum Project: A) Comparative Genomics of Virulent and Vaccine Strains of Mycoplasma gallisepticum We are utilizing DNA microarray technology to identify genetic differences in Mycoplasma gallisepticum vaccine strains. This research is based on the hypothesis that the reduced virulence of vaccine strains of M. gallisepticum can be accounted for by genomic and transcriptional differences in genes involved in virulence. Therefore, comparative genetic analysis of virulent and vaccine strains of M. gallisepticum will enable us to
identify candidate virulence-related genes. B) To effectively analyze Mycoplasma gallisepticum for virulence- associated determinants, the ability to create stable genetic mutations is essential. Utillizing a procedure referred to as signature sequence mutagenesis (SSM). SSM we carried out comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. Chickens were challenged with a pool of 27 unique mutants, those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7. C) We investigated the effects of mucosal vaccination with Mycoplasma gallisepticum on the host response. Studies were
initiated that focused on characterization of chemokine and cytokine gene profile within tracheal tissue of chickens infected with a pathogenic strain of Mycoplasma gallisepticum. Our results indicate the importance of chemokines in the development of lesions in Rlow-inoculated chickens as well as immunomodulatory effects of Mycoplasma gallisepticum leading to down regulation of IL-8 and IL-12 gene expression in tracheal mucosa. Foot and Mouth Virus Project: Development of mucosal vaccines and adjuvants for FMDV: Non-toxic Pseudomonas Exotoxin A as a delivery platform for FMDV VP1 G-H loop: During the past year, our group has engineered and expressed a non-toxic Pseudomonas aeruginosa exotoxin A (ntPE) to assess its capacity to serve as a carrier/adjuvant for delivery of the G-H loop of the FMDV, in its native conformation. We constructed a chimeric ntPE-GH protein by inserting the coding sequence of the G-H loop into an expression plasmid encoding ntPE, in place of the native,
non-essential sub-domain Ib loop. ntPE-GH was evaluated for the display of the G-H loop, and tested as a mucosal immunogen by immunizing pigs intranasally (Table 1), then assessing the anti-peptide immune response (Fig 1). We show that ntPE-GH induced anti-G-H serum IgG antibodies along with anti-ntPE serum IgG and mucosal IgA antibodies, confirming the merit of this approach. In summary, we tested the response of pigs to a chimeric protein composed of a non-toxic form of pseudomonas exotoxin A containing 25 amino acid residue G-H loop of FMDV. We have shown that mucosal application of this chimera stimulated the production of ntPE and G-H loop specific serum and nasal IgA immune responses. At present, it is unclear how ntPE-GH induces all of the immune responses observed in these studies, whether by facilitating the delivery of G-H loop or enhancing the immune response by an adjuvant activity, or both. In any case, ntPE may prove useful for inducing an efficient immune response when
applied mucosally to swine. Mucosal adjuvants for chimeric FMDV peptides: Studies conducted over the past year have focused on an analysis of mucosal adjuvants administered to the upper respiratory tract of pigs. Among the mucosal adjuvants tested were two mutants of the heat-labile enterotoxin of E.coli (LT) which were rendered non-toxic through specific amino acid substitutions in the catalytic A subunit of the toxin. These two constructs, LTr72 and LTk63, provided to us through a collaborative agreement with Chiron Inc. These adjuvants were co-administered with a novel FMDV peptide vaccine that had induced virus neutralizing antibodies upon parenteral administration to mice. This peptide was comprised of two B cell epitopes (described above) plus a T cell helper derived from VP4, and has been previously reported in the literature. Based on the data analyzed to date, it appears that the pigs were most effectively primed when the TCA peptide was adjuvanted with either CT or either
of the LT mutants. This was particularly evident upon assaying serum IgG anti TCA peptide response in sac sera (above). Many of these sera samples have been transferred to PIADC, and preliminary evidence indicates that limited neutralizing activity against FMDV O1 was present in some samples, with considerably higher activity against FMDV Sat 3-2. These results are currently being re-tested to ascertain their validity. Diagnostic Platform Development: Experiments done at UCONN focus on the characterization and optimization of GCSPRI instrument capabilities for the assessment of microbial samples and the analysis of immune parameters from biological materials. We have made significant progress with the measurements of both bacterial analytes and viral analytes. We have used several approaches to compare GCSPR measures to traditional assays of antibody/antigen interactions. We have found the levels of sensitivity to be essentially equivalent between the GCSPRI and a commercially
available cytokine assay. Similarly, we have directly compared the sensitivity of the GCSPRI to the ELISA in the assessment of an in vivo response to OVA immunization. We have explored the use of the GCSPRI detection system in the measurement of porcine pathogens. Specifically, we have explored the use of affinity purified pig antibody from immunized animals to detect solubilized M. hyopneumonia. These results correlate well with parallel ELISA assays of the same samples but require far less time to accomplish, and are compatible with other simultaneous assays in the same samples. Use of Avian Interferons and dsRNA as Antiviral Agents and Immunomodulators: Several studies carried out over the past year have progressed significantly. Our studies demonstrated that influenza virus A/PR/8/34 expressing the NS gene from lethal H5N1/97 converts an IFN induction- suppressing particle (ISP) into an IFN-inducing particle (IFP) and is sensitive to the action of IFN in chicken embryonic kidney
cells. This study showed for the first time that the per-oral use of recombinant chicken interferon-? administered in drinking water functions as a potent immunostimulant/adjuvant with respect to avian influenza virus. Experiments were initiated on the quantification of cell-killing particle (CKP) activity of influenza virus have been initiated. Preliminary results indicate that there is about a 5-fold excess of CKP to plaque- forming particles when tested on Vero cells as hosts; An invited paper was published concerning the interferon-inducing capacity of adenoviruses carried out several years ago. The findings of these studies are reported briefly below, and publications cited where appropriate. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Cecchinni, K.R., Gorton, T. S. and Geary, S. J. 2005. "Transcriptome Microarray analysis of
Mycoplasma gallisepticum strain R". The 105th American Society for Microbiology Annual Meeting, Atlanta, GA. Geary, S. J. 2005. "Mycoplasma gallsepticum: Pathogenesis, Progress and Prospects". The 105th American Society for Microbiology Annual Meeting, Atlanta, GA. Invited, Divisional Lecturer. Gorton, T. S., Barnett, M. and Geary, S. J. 2005. "Comparative Genomic Hybridization Analysis of Attenuated Mycoplasma gallisepticum strains". The 105th American Society for Microbiology Annual Meeting, Atlanta, GA. May, M. A., Gorton, T. S., and Geary, S. J. 2005. "Examination of the P65-like Protein (PlP) of Mycoplasma gallisepticum for Atypical Transmembrane Domains". The 105th American Society for Microbiology Annual Meeting, Atlanta, GA. Cecchini, K. A. and S. J. Geary. 2005. "Transcriptional Differences in Mycoplasma gallisepticum Interacting with Host Cells". Conference of Researcher Workers in Animal Diseases. St. Louis, MO Geary, S. J. 2005. "Mycoplasma gallisepticum Virulence &
Vaccine". The University of Vienna School of Veterinary Medicine. Vienna, Austria Geary, S. J. 2006. "Mycoplasma gallsepticum: Pathogenesis, Progress and Prospects". Guizhou University, Guiyang, China. Invited Speaker Gorton, T. S., E. R. Tulman, X. Liao, Z. Lu, G. F. Kutish and S. J. Geary. 2006. "Comparative Genomic Analysis of Attenuated Mycoplasma gallisepticum Strains R (high) and F". The 106th American Society for Microbiology Annual Meeting, Orlando, FL May, M. A., K. R.Cecchini, T. S. Gorton and S. J. Geary. 2006. "Characterization of the Attachment Organelle of Mycoplasma gallisepticum". The 106th American Society for Microbiology Annual Meeting, Orlando, FL Cecchini, K. R., T. S. Gorton and S. J. Geary. 2006. "Microarray Analysis of Mycoplasma gallisepticum Strains R Attached to Host Cells". The 106th American Society for Microbiology Annual Meeting, Orlando, FL Geary, S. J. 2006. "Comparative Genomic Analysis of Attenuated Mycoplasma gallisepticum Strains Rhigh and F".
The Sixteenth International Congress of the International Organization for Mycoplasmology, Cambridge, UK Invited Symposium speaker. Marcus, P.I. Cauthen, A.N., Swayne, D. E., Sekellick, M.J., and Suarez, D.L. (2006). Avian influenza pathogenesis in chickens: Amelioration attributed to interferon-inducing capacity of the virus. American society for Virology, 25th Annual Meeting, University of Wisconsin, July 15-19, 2006. Abstracts, W2-1, p.69. OBrian, K.B., Moser, L.A., Marcus, P.I., Sekellick, M.J., and Schultz- Cherry, S.L. 2006. Type-1 interferon regulation by human astrovirus-1? American Society for Virology, 25th Annual Meeting, University of Wisconsin, July 15-19, 2006. Abstracts, P15-15, p.251. Sekellick, M.J., Mohni, K.N., and Marcus, P.I. (2006). Influenza virus A/PR/8/34 expressing the NS gene from H5N1/97 converts an ISP to IFP and is sensitive to the action of IFN in chicken embryonic kidney cells. American Society for Virology, 25th annual Meeting, University of Wisconsin,
July 15-19, 2006. Abstracts, W2-2, p69. Barrette RW, Urbonas J, Silbart LK. 2006. Quantifying specific antibody concentrations by enzyme-linked immunosorbent assay using slope correction. Clin Vaccine Immunol. Jul;13(7):802-5. Gorton, T. S., Papazisi, L, and S. J. Geary. 2005. "Development of a molecular diagnostic system for the identification of Mycoplasma mycoides subsp. mycoides sc." Vet Microbiol. 2005 Nov 30;111(1-2):51-8. Epub 2005 Oct 27 Javed, M., Frasca Jr., S., Rood, D., Cecchini, K., Gladd, M., Geary, S. J. and L.K. Silbart. 2005. "Correlates of Immune Protection in GT5 Vaccinated Chickens Challenged with Pathogenic Mycoplasma gallisepticum Rlow" .Infect Immun. 2005 Sep; 73(9):5410-9. Hudson, P., Papazisi, L., Gorton, T. S., Cecchini, K., Liao, X., Gladd, M., and S. J. Geary. 2006. "Identification of novel virulence determinants in Mycoplasma gallisepticum through in vivo screening of transposon mutants." Infect. Immunity 2006 Feb; 74(2):931-9. May, M., Gorton, T.,
Papazisi, L. and Geary, S.J. 2006. "Identification of Fibronectin-Binding Proteins in Mycoplasma gallisepticum Strain Rlow" Infec. Immun. 2006 Mar; 74(3):1777-85. Burnett, Tracey, Dinkla, Katrin, Manfred, Rohde, Gurcharan, S. Chhatwal, Srivasta, Mukesh, Cordwell, Stuart. Geary, Steven, Minion, F. Chris, Walker, Mark and Steven Djordjevic. 2006. "P159 is a proteolytically processed, surface adhesion of Mycoplasma hyopneumoniae: defined domains of P159 bind heparin and promote adherence to eukaryotic cells". Mol Micro 60(3), 669-686 Marcus, P.I., Rojek, J.M., and Sekellick, M.J. 2005. Interferon induction by viruses. XXIV. Interferon induction and/or production and its suppression by influenza viruses. J. Virology 79:2880-2890. Marcus, P.I. and Sekellick, M.J. 2005. Interferon induction by viruses. XXV. Adenoviruses as inducers of interferon in developmentally aged primary chicken embryo cells. Acta Microbiologica et Immunologica Hungarica 52:273-290. [Invited article on the
occasion of the 80th birthday of Professor Ilona Beladi]. Marcus, P.I., Girshick, T., van der Heide, L., and Sekellick, M.J. 2006. Chicken IFN-? in drinking water functions as an adjuvant for influenza virus. Sixth International Cytokine Conference, Vienna, Austria, August 27-31, 2006. Special abstract issue of European Cytokine Network, vol. 17:69 (05-33/P). Sekellick, M.J., Mohni, K.N., and Marcus, P.I. 2006. Both host cell and NS gene origin determine the interferon action and induction phenotype of influenza virus H5N1(NS). Sixth International Cytokine Conference, Vienna, Austria, August 27-31, 2006. Special abstract issue of European Cytokine Network, vol. 17:69 (05-32/P). Jin G. B., Unfricht D. W., Fernandez S. M., Lynes, M. A. 2006. Cytometry on a chip: Cellular phenotypic and functional analysis using grating- coupled surface plasmon resonance. Biosens Bioelectron. Aug 15;22(2):200- 6. Epub 2006 Feb 7.
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