Advanced Animal Vaccines and Diagnostic Applications

APPROACH: Comparative genomic analysis will be used to identify optimal genomic targets. Genomic targets will be used to develop rapid PCR-based and immuno-assays for key agents. Comparative genomics and genetic techniques will be used to identifiy and characterize pathogen virulence, host range and immune evasions mechanisms. Identified pathogen targets will be exploited to improve vaccine design or to develop novel interaction/ theraputic approaches. BSL-3; 01/2002. Recertification currently being requested.
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PROGRESS: 2005/10 TO 2006/09<BR>
Progress Report<BR> Foreign animal diseases (FAD) continue to be a highly significant animal health concern. Today, the threat posed by these diseases is unprecedented due to the increasing globalization of trade, movement of people and agricultural products and changes in farm practices including movement to more intensive agriculture. Recent FAD outbreaks in Europe and Asia (1997-2001) have been catastrophic resulting in devastating economic consequences for the countries involved. A similar scenario would be expected should these diseases be introduced into the United States. New disease control strategies are needed to reduce this threat. New disease control strategies are needed to reduce the threat posed by high consequence FAD. Functional pathogen genomics offers an unprecedented opportunity for addressing this critical issue. High throughput pathogen genome sequencing together with detailed genome analysis will be used to provide improved tools for pathogen detection, disease diagnosis and epidemiological disease investigations, improved vaccines and therapeutics, and in all likelihood, completely novel disease control strategies with increased efficacy and utility. <P>
Specifically the goal of this research is to: <ol>
<li> Develop methods for rapid strain identification and epidemiological investigations for key FAD agents; <li> Develop rapid field diagnostic tests for key FAD agents; <li> Develop improved vaccines for key FAD; <li> Develop new methods on strategies for efficient and effective disease control. </ol> This research addresses NPS-103 Microbial genomics, pathogen detection, and Epidemiology of disease components. <P>
2.FY 2006.<BR> 1-Develop rapid diagnostic tests based on microbial genomics, nucleic acid sequences and immuno-assays for key Foreign Animal Disease Agents (FAD)<BR> FY 2007. 2-Develop improved vaccines and vaccine delivery vectors for key FAD 3-Develop improved methods for pathogen detection on microbial genomics, nucleic acid sequences, and immuno-assays. 4a List the single most significant research accomplishment during FY 2006. <BR>
1940-32000-045-01S - University of Missouri: The most significant accomplishment from this program over the past year is the successful completion (in collaboration with TIGR) of three genomes from important mycoplasma pathogens, including the hot (disease- causing) Gladysdale strain of the OIE "A-list" and potentially devastating FAD organism M. mycoides SC, the related pathogen Mycoplasma bovine serogroup 7 (currently nearing completion), which causes mastitis, abortion and arthritis in cattle, and the emerging pathogen Mycoplasma bovis (causing serious, economically damaging diseases of cattle including pneumoniae, mastitis and arthritis). The complete annotation of these data sets, in comparison to four other mycoides subgroup genomic sequences (including another virulent strain and an avirulent strain of mycoides SC, and two sequences from M. capricolum), will identify distinctive genome signatures and expressed gene products for these serious pathogens.

<p>Impact: As a result, sequence-specific and structure- specific reagents will be generated that are applicable to a variety of diagnostic platforms for the detection and control of corresponding CBPP and mastitis-pneumonia caused by these respective mycoplasmal pathogens. 4d Progress report. This report documents activities in 2006 under CRIS project 1940-32000- 039-00D. In-house ARS research objectives associated to this CRIS were carried out and are reported under CRIS 1940-32000-040. Progress of collaborating institutions: University of Missouri and University of Connecticut are reported in CRIS 1940-3200-045-01S and 1940-32000-045-02S respectively. In June 2006 this CRIS project expired and a bridge CRIS 1940-32000-045 plan was prepared in collaboration with U. of Connecticut and U. of Missouri. A new project plan is being developed and will undergo OSQR review during 2007. This CRIS project has made significant progress on the planned research for 2006 including agents causing important animal disease including viral, bactrerial and parasitic agents. Important contributions have been made to the knowledge of full-length genomic sequences of complex bacterial agents such as various mycoplasma species affecting poultry and livestock. Progress report from the University of Missouri, CRIS Project 1940-32000- 045-01S: It is clear from these descriptions that significant progress has been made in Objective 1, where three mycoplasma genomes have been completed and are being annotated and finalized for release to the scientific public within the next year, where they will provide an important foundation for developing the next generation of research strategies to control and eradicate these important food animal diseases. The major component of Objective 2, to develop genetic models for PRRSV pathogenesis was cut short by the recruitment of our principal scientist to the private veterinary pharmaceutical sector. We were able however to continue investigation of two important PRRSV co-pathogens, porcine Circovirus and Mycoplasma hyopneumoniae. Although these projects will not be continued, they were successful in providing useful new information about the biology of these organisms. We have continued the development of several strategies and platforms for identifying and characterizing useful tools for the detection and diagnosis of important veterinary pathogens. <p>Most of these studies have been pursued with funding sources outside of PPAID and are not described in detail in this report. Several publications describing these efforts with technologies such as RT-PCR, multiplex bead-based (Luminex) serologic assays, multiplex bead-based nucleic acid assays, PCR-on-a-CHIP assays, and FRET-based biosensors have been released in the past year by investigators at MU. For the duration of this agreement, these efforts will continue and they will take advantage of new nucleic acid and protein sequence information for both bacterial and viral pathogens continually released to the scientific community, some as a result of these PPAID efforts. Progress in two distinct Discovery projects funded with PPAID funds was described in Question 4b above and in several publications listed in Question 8 below. <P> The proof of principle experiment using RNA interference to sterilize a model nematode has been reported and this experiment has turned its focus to investigation of the pathogenic nematode. 2) In the zoonotic pathogen, Francisella tularensis, allelic replacements have identified several important virulence genes in the organisms secretome, including three important acid phosphatases. Structural analysis of these proteins has led to a clearer understanding of their function and possible therapeutic targets. Progress report from the University of Connecticut, CRIS Project 1940- 32000-045-02S: 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. <li>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. 5. Describe the major accomplishments to date and their predicted or actual impact. This is a Bridge Project which is currently involved in the NP 103 Review Process. See the report for 1940-32000-039-00D.