Progress 07/01/09 to 09/30/15
Outputs
Target Audience:The Poultry Diagnostic and Research Laboratory conducts research to support the poultry industry.Applied and basic research programs target the diagnosis and control of economically important diseases of poultry in the USA as well as world wide. Our stakeholders also include the various support companies for the poultry industry including but not limited to, biologics companies, vaccine producers and feed additive companies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training opportunities were provided to graduate studnets (3 MS and 2Ph.D) as well as undergraduate students in the areas of nucleotide sequence generation and bioinformatics. How have the results been disseminated to communities of interest?The results of this work has been published in a number of different peer reviewed journals and presented at national and international meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1) Genomic sequence analysis of CEO and TCO vaccines for ILTV were elucidated and published. Based on that sequence data, a glycoprotein J gene-deleted "knock-out" vaccine for ILTV was created and is currently being tested in chickens for safety and efficacy. 2) A novel chicken astrovirus was isolated and characterized and its role in RSS was established. In addition, ANV-1 and ANV-2 vaccine candidates for RSS were developed using recombinant technology. Safety and efficacy testing was conducted in chickens. 3) Nothing to report. The faculty member working on this project left the University. 4) A sequence data base was developed for avianMycoplasma gallisepticum. The data base was tested an applied to routine diagnosis of avianMycoplasmas. Vaccine candidates form recently isolated avian Mycoplasmaswere identified and are being characterized.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Armour NK, VA Laibinis, SR Collett, N Ferguson-Noel. The development and application of a Mycoplasma gallisepticum sequence database. Avian Pathol, 42: 408-15. 2013.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Durairaj V, E Linnemann, AH Icard, SM Williams, HS Sellers, E Mundt. Development of a novel in vivo experimental model to differentiate antigenic variations among infectious bursal disease viruses. Avian Pathol, 42(4):309-315, 2013
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Garc�a M, J Volkening, SM Riblet, S Spatz. Genomic Sequence Analysis of the United States Infectious Laryngotracheitis Vaccine Strains Chicken Embryo Origin (CEO) and Tissue Culture Origin (TCO).Virology. 440:64-74. 2013.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Roh H-J, DA Hilt, SM Williams, MW Jackwood. Evaluation of Infectious Bronchitis Virus Arkansas-type Vaccine Failure in Commercial Broilers. Avian Dis. 57:248-259, 2013.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2014
Citation:
Durairaj V, HS Sellers E Mundt. Development of a molecular tool for antigenic characterization of infectious bursal disease viruses at the global level. Avian Path
- Type:
Journal Articles
Status:
Under Review
Year Published:
2014
Citation:
Ferguson-Noel N and SM Williams. The Efficacy of Mycoplasma gallisepticum K-Strain Live Vaccine in Broiler and Layer Chickens. Submitted to Avian Pathology,
- Type:
Journal Articles
Status:
Under Review
Year Published:
2014
Citation:
Kang KI, AH Icard, V Durairaj, E Linnemann, E Mundt, HS Sellers. Isolation and characterization of a chicken astrovirus- an etiological agent of runting and stunting syndrome in broiler chickens. Submission to PLOS Pathogen
- Type:
Journal Articles
Status:
Under Review
Year Published:
2014
Citation:
Mundt E, SR Collett, A Connolly. Effects of All-Lac; Orally After Hatch, Acid-Pak; in Water for Five Days, and Actigen; in Starter Feed on Broiler Chicks Challenged with Intestinal Homogenate or Litter from Runting-Stunting Syndrome Positive Broilers. Avian Diseases.
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: The Poultry Diagnostic and Research Laboratory conducts research to support the poultry industry. Applied and basic research programs target the diagnosis and control of economically important diseases of poultry in the USA as well as world wide. Our stakeholders also include the various support companies for the poultry industry including but not limited to, biologics companies, vaccine producers and feed additive companies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Training opportunities were provided to graduate studnets (3 MS and 2Ph.D) as well as undergraduate students in the areas of nucleotide sequence generation and bioinformatics. How have the results been disseminated to communities of interest? The results of this work has been published in a number of different peer reviewed journals and presented at national and international meetings. What do you plan to do during the next reporting period to accomplish the goals? We plan to conduct safety and efficacy testing on knock-out ILTV vaccines in chickens and compare them to commercial vaccines. Although ANV-1 and ANV-2 vaccine candidates for RSS were efficacious against the homologous viruses, we will continue to look for other virus vaccine candidates to create a multivalent vaccine providing the best possible protection against RSS. Thein vitro system created for IBDV immunogenic proteins will be evaluated as a new platform to produce poultry vaccines. We will continue to isolate, identify, and characterize avian Mycoplasma isolates and evaluate their potential as a live vaccine.
Impacts
What was accomplished under these goals?
1) Genomic sequence analysis of CEO and TCO vaccines for ILTV were elucidated and published. Based on that sequence data, a glycoprotein J gene-deleted "knock-out" vaccine for ILTV was created and is currently being tested in chickens for safety and efficacy. 2) A novel chicken astrovirus was isolated and characterized and its role in RSS was established. In addition, ANV-1 and ANV-2 vaccine candidates for RSS were developed using recombinant technology. Safety and efficacy testing was conducted in chickens. 3) An in vitro system was created for IBDV immunogenic proteins and it was evaluated for its utility as a molecular tool to characterize IBV antigens at the global level. 4) A sequence data base was developed for avian Mycoplasma gallisepticum. The data base was tested an applied to routine diagnosis of avian Mycoplasmas. Vaccine candidates form recently isolated avian Mycoplasmas were identified and are being characterized.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Armour NK, VA Laibinis, SR Collett, N Ferguson-Noel. The development and application of a Mycoplasma gallisepticum sequence database. Avian Pathol, 42: 408-15. 2013.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2014
Citation:
Ferguson-Noel N and SM Williams. The Efficacy of Mycoplasma gallisepticum K-Strain Live Vaccine in Broiler and Layer Chickens. Submitted to Avian Pathology,
- Type:
Journal Articles
Status:
Under Review
Year Published:
2014
Citation:
Kang KI, AH Icard, V Durairaj, E Linnemann, E Mundt, HS Sellers. Isolation and characterization of a chicken astrovirus an etiological agent of runting and stunting syndrome in broiler chickens. Submission to PLOS Pathogen
- Type:
Journal Articles
Status:
Under Review
Year Published:
2014
Citation:
Mundt E, SR Collett, A Connolly. Effects of All-Lac; Orally After Hatch, Acid-Pak; in Water for Five Days, and Actigen; in
Starter Feed on Broiler Chicks Challenged with Intestinal Homogenate or Litter from Runting-Stunting Syndrome Positive Broilers. Avian Diseases.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Durairaj V, E Linnemann, AH Icard, SM Williams, HS Sellers, E Mundt. Development of a novel in vivo experimental model to differentiate antigenic variations among infectious bursal disease viruses. Avian Pathol, 42(4):309-315, 2013
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Garc�a M, J Volkening, SM Riblet, S Spatz. Genomic Sequence Analysis of the United States Infectious Laryngotracheitis Vaccine Strains Chicken Embryo Origin (CEO) and Tissue Culture Origin (TCO).Virology. 440:64-74. 2013.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Roh H-J, DA Hilt, SM Williams, MW Jackwood. Evaluation of Infectious Bronchitis Virus Arkansas-type Vaccine Failure in Commercial Broilers. Avian Dis. 57:248-259, 2013.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2014
Citation:
Durairaj V, HS Sellers E Mundt. Development of a molecular tool for antigenic characterization of infectious bursal disease
viruses at the global level. Avian Path
|
Progress 01/01/13 to 09/30/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two graduate students received training and were involved in this work. How have the results been disseminated to communities of interest? The results were published in relavent journals and sequences were uploaded to GenBank. What do you plan to do during the next reporting period to accomplish the goals? We will continue to move forward on our research focusing on the important goals of this project.
Impacts
What was accomplished under these goals?
A full length genome analsis was conducted on pathogenic ILTV to elucidate genetically important areas for the knock out/knock in system. The etiology of a severe enteric disease in broilers (Runting Stunting Syndrome) was investigated and progress toward identifying specific causative agents (ANV1 & 2) was made. Immunogenic proteins were identified for recombinant studies. Details on submission of the best samples for Mycoplasma detection were determined, which are important for accurate diagnosis. The procedures were used to characterize recent Mycoplasma isolates from the field.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Ferguson-Noel N, V Laibinis, and SH Kleven. Evaluation of Mycoplasma gallisepticum K-Strain as a Live Vaccine in Chickens. Avian Dis. 56:44-50. (2012)
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Kang K, M El-Gazzar, HS Sellers, F Dorea, SM Williams, T Kim, SR Collett, E Mundt. Investigation on the etiology of Runting Stunting Syndrome in chickens. Avian Pathology. 41(1):41-50. (2012)
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Phillips JE, MW Jackwood, ET McKinley, S Thor, DA Hilt, ND Acevedo, SM Williams, JC Kissinger, AH Paterson, J Robertson, C Lemke. Changes in non-structural protein 3 are associated with attenuation in avian coronavirus infectious bronchitis virus. Virus Gene. 44(1):63-74. (2012)
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Spatz SJ, JD Volkening, CL Keeler, GF Kutish, SM Riblet, CM Boettger, KF Clark, L Zsak, CL Afonso, ES Mundt, and M Garc�a. Comparative Full Genome Analysis of four Infectious Laryngotracheitis Virus (Gallid herpesvirus-1) Virulent Isolates from the United States. Virus Genes. 44:273-285. (2012)
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Williams SM and HS Sellers. Response of White Leghorn Chickens to Infection with Avian Leukosis Virus Subgroup J and Infectious Bursal Disease. Avian Diseases. 56(1):2-6. (2012)
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Williams SM, G Zavala, S Hafner, SR Collett, S Cheng. Metastatic melanomas in young broiler chickens (Gallus gallus domesticus). Veterinary Pathology. 49(2): 288-291. (2012)
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Zavala G, S Cheng, T Barbosa. Natural Infection and Transmission of a Retrovirus Closely Related to Myeloblastosis-Associated Virus Type 1 in Egg-Type Chickens. Avian Dis. 56(1):7-14. (2012)
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Ferguson-Noel N, K Cookson, V Laibinis, and SH Kleven. The Efficacy of Three Commercial Mycoplasma gallisepticum Vaccines in Laying Hens. Avian Dis .56:272-275. (2012)
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Ferguson-Noel N, V Laibinis, and M Farrar. Influence of Swab Material on the Detection of Mycoplasma gallisepticum and M. synoviae by Real-time PCR. Avian Dis. 56:310-314. (2012)
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Conduct Mycoplasma diagnostics/ surveillance in commercial poultry. Diagnostic tests conducted over the past year include 1076 cultures, 6884 HI tests, 3613 PCR tests and 141 sequencing reactions. Develop a multiplex assay to detect avian infectious bronchitis virus types. In an effort to improve on current diagnostic tests for IBV, a microsphere-based multiplex assay was developed for simultaneous detection and identification of the four most common IBV serotypes diagnosed in the USA; Arkansas (Ark), Connecticut(Conn), Delaware(DE) and Massachusetts(Mass). Biotinylated primers were used to amplify the hypervariable region of the spike glycoprotein of IBV. Using serotype-specific probes for each of the four serotypes (Ark, Conn, DE, Mass) hybridized to microspheres with different spectral addresses, we performed a hybridization reaction with the biotin labeled amplicons. A fluorescent tag (Streptavidin-R-Phycoerythrin) bound to the biotin labeled amplicons and the spectral addresses of the microspheres were measured in the Bio-Plex suspension Array System (Bio-Rad, Hercules, CA). Develop a quantitative method for detection of infectious laryngotracheitis virus (ILTV) in clinical and laboratory samples. The estimation of the viral load is an important tool to determine the stage of ILTV infection. A molecular method intended to replace methodologies that are poorly suited for relative quantitation of virus in biological samples was devised. This method, which is based on qPCR, was designed and validated to detect quantitatively ILTV genome copies simultaneously to quantitation of an internal control, which in this case is a housekeeping gene (collagen-α2 gene from Gallus gallus domesticus). This methodology allows for the quantitation of virus copy numbers In a given clinical sample and it also allows for elimination of cumbersome classical virology and serology methods. Quantification of viral genomes was based on the amplification of the ILTV UL44 gene and sample variability was normalized using the collagen-α2 gene (Gallus gallus domesticus) as an endogenous control in a duplex reaction. Examine the dynamics of IBV vaccination and protection against Arkansas. In this study we examine routes of vaccine administration using multiple IBV types including Ark in an effort to understand why Ark vaccines do not provide good protection and persist in commercial broilers. We found that interference between different types of IBV vaccines was not occurring when combined and administered using a commercial hatchery spray cabinet. Also, Ark vaccine virus was not efficacious in 1-day old broilers when sprayed using a hatchery spray cabinet but gave good protection when it was administrated by eye drop. Develop a deletion mutant vaccine against infectious laryngotracheitis. Infectious laryngotracheitis virus glycoprotein J is a major viral antigen but dispensable for replication in cell culture. This work is directed at examining a gJ deleted ILTV mutant for infectivity and immunogenicity in chickens. Since gJ deleted virus is egress impaired it potentially would reduce the transmissibility of the virus preventing reversion to virulence. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Conducting Mycoplasma surveillance is key for early diagnosis and the control of this economically important pathogen in poultry. A multiplex assay for IBV provides a rapid tool to identify multiple avian IBV types in the same sample. This increases the timely identification of this economically important pathogen which is important for vaccine selection and control. A quantitative tool to detect ILTV in birds can be used to establish the viral load in chickens, which provides valuable data for estimating transmission and control. Transmission of ILTV allows it to "heat up" and cause severe disease in chickens. This work is in important step in preventing ILTV outbreaks. Examining the dynamics of IBV vaccines confirms that the delivery method for Ark vaccines affects their ability to fully protect broilers. This is important for control of IBV Ark type viruses in the field. Current ILTV vaccines are less than adequate. Safe and effective vaccines are desperately needed for ILTV, making development of a gene deleted vaccine extremely important for the future control of ILTV.
Publications
- Liu, Y, E. Mundt, A. Mundt, M. Sylte, D. L. Suarez, D. E. Swayne, and M. Garcia. Development and Evaluation of an Avian Influenza (AI) Neuraminidase Subtype 1 (N1) Based ELISA for Poultry. Avian Dis. 54: 613-621. 2011.
- Wang, L., Z. Qin, M. Pantin-Jackwood, M. Garcia, B. Lupiani, Y. M. Saif, and C-W. Lee. Development of a DIVA vaccine for the Control of Triple Reassortant H3N2 Influenza in Turkeys. Vaccine. 45: 7966-7974. 2011
- Gharaibeh S, Laibinis V, Wooten R, Stabler L and Ferguson-Noel N. Molecular Characterization of Mycoplasma gallisepticum isolates from Jordan. Avian Dis 2011; 55:212-216.
- McKinley, E. T., M. W. Jackwood, D. A. Hilt, J. C. Kissinger, J. S. Robertson, C. Lemke, and A. H. Paterson. Attenuated live vaccine usage affects accurate measures of virus diversity and mutation rates in avian coronavirus infectious bronchitis virus. Virus Research. 158:225-234, 2011.
- Franca, M., P. R. Woolcock, M. Yu, M. W. Jackwood and H. L. Shivaprasad. Nephritis associated with infectious bronchitis virus Cal99 variant in game chickens. Avian Dis. Accepted 55:422-428, 2011.
- Keeler, S. P., P. J. Ferro, J. D. Brown, X. Fang, J. El-Attrache, R. Poulson, M. W. Jackwood and D. E. Stallknecht. Use Of FTA Sampling Cards For Molecular Detection Of Avian Influenza Virus in Wild Birds. Avian Dis. Accepted, October 2011.
- Phillips, J. E., M. W. Jackwood, E. T. McKinley, S. W. Thor, D. A. Hilt, N. D. Acevedol, S. M. Williams, J. C. Kissinger, A. H Paterson, J. S. Robertson, C. Lemke. Changes in nonstructural protein 3 are associated with attenuation in avian coronavirus infectious bronchitis virus. Virus Genes. Accepted, August 2011.
- Thor, S. W., D. A. Hilt, J. C. Kissinger, A. H. Paterson, and M. W. Jackwood. Recombination in Avian Gamma-Coronaviruses. Viruses. 3:1777-1799, 2011.
- Kuriakose, T., D. A. Hilt, and M. W. Jackwood. Multiplex microsphere assay for simultaneous detection of all avian influenza viruses and differentiation of H5, H7, N1 and N2 subtypes. Avian Dis. Accepted, October 2011.
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Gene targeted sequencing was used to determine the source and spread of MG and MS isolates in the field. Approximately 123 MG and MS were analyzed in 2011 and the circulation of field strains within complexes and companies was identified. We developed a rapid multiplex microsphere assay for the simultaneous detection of all avian influenza viruses (AIV) as well as differentiation of H5, H7, N1 and N2 subtypes. A molecular method intended to replace methodologies that are poorly suited for relative quantitation of virus in biological samples was devised. This method, which is based on qPCR, was designed and validated to detect quantitatively ILTV genome copies simultaneously to quantitation of an internal control, which in this case is a housekeeping gene (collagen-α2 gene from Gallus gallus domesticus). We have optimized the expression and production of avian influenza virus N1 and N2 antigens using the baculovirus expression system. The availability of these antigens allowed the development of an ELISA platform where no infectious virus needs to be propagated. We have developed and validated N1 and N2 ELISAs using baculovirus recombinant expressed proteins. The assays' sensitivity and specificity have been determined as well as the ability of these indirect ELISAs to differentiate infected from vaccinated animals under experimental conditions. Full-length genome sequencing of pathogenic and attenuated (for chickens) avian coronavirus infectious bronchitis virus (IBV) strains of the same serotype was conducted to identify genetic differences between the pathotypes. The full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (IBV) isolates, were sequenced and along with other full-length IBV genomes available from GenBank were analyzed for recombination. Evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. The objective of this study was to determine the efficacy of protection induced by viral vector vaccines as compared to live attenuated ILTV vaccines.The chicken embryo origin (CEO) vaccine provided optimal protection by significantly mitigating the disease and contributing to the clearance of the challenge virus in chickens vaccinated via eye drop. While the viral vector vaccines, applied in ovo and subcutaneously at 1 day of age, provided partial protection reducing to some degree clinical signs but reduction of the challenge virus in the trachea was not detected. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This work reinforces the importance of surveillance for MG and MS isolates for the control of avian mycoplasmas. It also provides a rapid tool to identify multiple avian influenza types in the same sample.In addition, this work will provide a quantitative tool to detect ILTV in birds that can be used to establish the stage of ILTV infection which provides valuable data for the control of the disease. These data constitute a significant step forward in identifying pathogenicity genes in avian coronavirus infectious bronchitis. That knowledge will help us design better control measures for this economically important disease. In this work we also found that recombinant vaccines against ILTV provide some protection but do not prevent shedding.
Publications
- Ferguson-Noel, N., V. Laibinis, and S. H. Kleven Evaluation of Mycoplasma gallisepticum K-Strain as a Live Vaccine in Chickens. Avian Dis. Accepted 2011.
- El Gazzar, M., V. Laibinis, and N. Ferguson-Noel Characterization of a ts-11-like Mycoplasma gallisepticum Isolate From Commercial Broiler Chickens. Avian Dis. Accepted 2011.
- Ferguson-Noel, N., K. Cookson, V. Laibinis, and S. H. Kleven The Efficacy of Three Commercial Mycoplasma gallisepticum Vaccines in Laying Hens. Avian Dis. Accepted. 2011.
- Mundt, A., E. Mundt, R. J. Hogan and M. Garcia. Glycoprotein J of infectious laryngotracheitis virus us required for efficient egress of infectious virus from cells. J. Gen. Virol. 92:2586-2589. 2011
- Spatz, S., J., J. D. Volkening, C. L. Keeler, G. F. Kutish, S. M. Riblet, C. M. Boettger, K. F. Clark, L. Zsak, C. L. Afonso, E. S. Mundt, and M. Garcia. Comparative Full Genome Analysis of four Infectious Laryngotracheitis Virus (Gallid herpesvirus-1) Virulent Isolates from the United States. In Press Virus Genes. 2012.
- Vagnozzi, A., G. Zavala, S. M. Riblet, A. Mundt, and M. Garcia. Protection induced by Infectious laryngotracheits virus (ILTV) Live-attenuated and Recombinant Viral Vector Vaccines in Broilers. In Press Avian Pathology. 2012.
- Wang, L., Z. Qin, M. Pantin-Jackwood, M. Garcia, B. Lupiani, Y. M. Saif, and C-W. Lee. Development of a DIVA (Differentiation of Infected from Vaccinated Animals) Vaccines for the Control of Triple Reassortant H3N2 Influenza in Turkeys. Vaccine. 45: 7966-7974. 2011
|
Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: 1) Gene deleted mutants of infectious laryngotracheitis virus (ILTV) were created as described in previous reports. Vaccine candidates were developed and evaluated for safety and efficacy. 2) A novel chicken astrovirus (CKAstv) was isolated from chickens with Runting-Stunting Syndrome (RSS). The disease signs and lesions can be reproduced with this virus indicating that the novel CKAstv is the likely etiologic agent of RSS. 3) Recombinant vaccines expressing ILTV antigens were evaluated for efficacy and safety in broiler chickens vaccinated in-ovo. Recombinant HVT-ILT and POX-ILT commercial vaccines were found to provide a protective immune response but the immune response only provide protection after 28-35 days. The efficacy was not as good as that provided by conventional modified live ILTV vaccines. 4) The molecular epidemiology of the spread of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) was studied. Gene targeted sequencing and random amplified polymorphic DNA (RAPD) analyses were used to determine the source and spread of MG and MS isolates in the field. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1. The gene deleted ILTV vaccines candidates are being further evaluated as potential commercial vaccines. Commercial vaccine companies have shown a keen interest in this project. Garcia, M. Genetically Modified Vaccines against Infectious laryngotracheitis. Georgia Veterinary Medical Association Meeting. San Destin, Florida. June 1, 2010. A. Mundt, E. Mundt, and M. Garcia. Generation of a Glycoprotein J Deletion Mutant of Infectious Laryngotracheitis Virus (ILTV) as Potential Vaccine for a DIVA Approach. 2010 AVMA/AAAP conference, August 1-4, 2010, Atlanta, GA. 2. The novel astrovirus is being evaluated for its use as the basis for either a live vaccine, inactivated vaccine, or both. Various inoculation routes are being evaluated. This project holds promise to provide the first effective control method for RSS. T. Kim and E Mundt. Establishment of Vaccine Candidates to Runting Stunting Syndrome of Chicken Using the Metagenomic Approach. 2010 AVMA/AAAP conference, August 1-4, 2010, Atlanta, GA. 3. The commercially available recombinant ILTV vaccines were found to safe and efficacious. Optimum days of incubation were identified for inoculation of each vaccine in-ovo. Results of this study have been used by the poultry industry to optimize the use of these new products. Vagnozzi A. E., S. M. Riblet, G. Zavala, and M. Garcia*. Evaluation of the Protection Induced by Recombinant Infectious Laryngotracheitis Virus (ILTV) Vaccines in Broiler Chickens. American Association of Avian Pathologists/American Veterinary Medical Association. Atlanta, GA, August 1-4, 2010. 4. Approximately 170 MG and MS isolates were analyzed in 2010. The data were used to determine the routes and sources of spread for several outbreaks of avian mycoplasmosis in the US. A Global Survey of Mycoplasma gallispeticum Genotypes Using Multilocus Sequence Typing of the Cytadhesin mgc2 Gene and the 16S-23S rRNA Intergenic Spacer Region. 18th Congress of the International Organization for Mycoplasmolgy.
Publications
- Pantin Jackwood, M., J., Strother, K. O., Mundt, E., Zsak, L., Day, M., and E. Spackman (2010). Molecular Characterization of Avian Astroviruses. Arch of Virology. Nov 11. [Epub ahead of print]
- Sellers, H., Linneman, E., Icard, A. H., and E. Mundt (2010). A purified recombinant baculovirus expressed capsid protein of a new astrovirus provides partial protection to runting-stunting syndrome in chickens. Vaccine. 28:1253-1263.
- Vagnozzi A., M. Garcia, S. M. Riblet, and G. Zavala*. Protection Induced by Infectious Laryngotracheitis Virus Vaccines Alone and Combined with Newcastle Disease Virus and/or Infectious Bronchitis Virus Vaccines.
Avian Diseases December 2010, Vol. 54, No. 4: 1210-1219.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: 1) The coding region for the glycoprotein J (gJ) of the ILT virus was deleted from the virulent ILTV USDA reference strain (RS) by insertion of a GFP expression cassette and a gJ deletion virus (ADgJ) was obtained. Consequently, a rescue mutant (gJR) was generated. A true gJ deletion mutant (BDgJ) was generated by deletion of the GFP cassette. BDgJ was attenuated in chickens and did not induce protection when challenged with ILTV USDA-RS. gJR was as virulent as the parental virus USDA-RS demonstrating that attenuation of BDgJ was due to gJ deletion. Currently, attenuation and induction of immunity of ADgJ and BDgJ are evaluated. 2) One vaccine candidate is based on a recombinant baculovirus (rBV) expressing the capsid protein of a new chicken astrovirus and a commercial adjuvant. Using the open reading frames of capsid proteins (two astroviruses, one parvovirus) rBV's were generated and three more experimental vaccines were formulated for vaccination of commercial broiler breeder hens. The result of the vaccination will be monitored by ELISA using purified recombinant protein. The offspring will be challenged in a colony house model. 3) Continuous avian cell lines were created that express various antigens of important avian pathogens using recombinant avian retroviruses and are being evaluated as vaccines. 4) Strain typing. Mycoplasma gallisepticum (MG) isolates and DNA samples from the US and 21 other countries worldwide were collected and more than 92 MG genotypes were identified by sequencing. Based on the data it seems that MG does not frequently move across geographical lines with breeding stocks. We found that at least 41 MG genotypes were distinguishable from live vaccines and unique to the region. The information will improve diagnostics. Characterization of atypical MS isolates. Mycoplasma synoviae (MS) were isolated from diseased broilers and broiler breeders in Arkansas. Three different MS genotypes (GT) from this outbreak have been identified by vlhA sequencing. Pathogenicity of two MS Arkansas isolates (S-10 GT, S-17 GT) has been compared with the virulent MS reference strain K1968. The S-10 isolate was of similar virulence as K1968. The S-17 isolate was more attenuated. This MS outbreak has a significant impact on the approach to control MS in the US. An MS outbreak in broiler breeders in North Carolina was atypical due to a slow seroconversion on the serum plate agglutination (SPA) test which resulted in late outbreak detection. Broilers were challenged with outbreak isolates and contact birds were added. The birds reacted on all serological tests (also SPA test). These results indicate that the observed unusual field results were likely caused by the performed tests due to a variability of the MS SPA commercial antigen. Live MS Vaccine. Three MS isolates were tested as potential live vaccines.Virulence was determined by infection of chickens with each isolate and the virulent reference strain (K1968) and a concurrently challenge with other respiratory viruses. Two isolates were mildly virulent. Further tested in subsequent vaccination and challenge experiments revealed that one isolate was a promising vaccine candidate. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1.Methods were established for the deletion of glycoprotein genes. These first experiments with the deletion of glycoprotein j will be repeated for the other glycoprotein genes. The resulting mutants will be evaluated for their pathogenicity and immunogenicity. 2.An experimental vaccine was produced using recombinant baculovirus protein. This process has been standardized and will now be used to produce a series of different baculovirus recombinant vaccines for evaluation against runting-stunting syndrome. Presentations: Mundt, E, Zavala, G, and H. Sellers. Development of a recombinant vaccine against runting and stunting syndrome in chickens. Development of a competitive elisa for detection of h5 antibodies in multiple species. Southern Conference on Avian Diseases, World Congress Center, Atlanta, GA - January 26-27, 2009. Kim, T., Thurber, R.V., Sellers, H.S., Mundt, E. Metagenomic analysis of intestinal contents from runting stunting syndrome diseased broiler. 2009 AVMA/AAAP conference, July 11-15, 2009, Seattle, Washington. Mundt E., H. S. Sellers, and G. Zavala. Development of a recombinant vaccine against runting and stunting syndrome in chickens. American Association of Avian Pathologists, American Veterinary Medical Association, Seattle, Washington, July 12-15. 3.Methods were developed that allow the stable expression of selected antigens in continuous avian cell lines. These methods can serve as the platform for a new family of avian vaccines. Presentations: Barbosa T., S. Cheng, and G. Zavala. Expression of viral immunogenic proteins in stable cell culture using a retroviral-based gene delivery system. American Association of Avian Pathologists, American Veterinary Medical Association, Seattle, Washington, July 12-15. REED RUMSEY AWARD FOR BASIC RESEARCH. 4.Isolates from important avian mycoplasma outbreaks in the US were characterized using molecular techniques. Information from these studies provided the information needed for proper molecular epidemiological analysis of the outbreaks and subsequent control of the outbreaks by the poultry industry. Also the basic work was done on the development of a new live MS vaccine. Presentations: GVMA Summer Convention 2009. Destin FL. "MG and MS Infection in the Southeastern U.S." AVMA/AAAP Annual Convention 2009. Seattle, WA. "The Displacement of Mycoplasma gallisepticum Strains by Live Vaccines" Delmarva Poultry Industry 44th National Meeting on Poultry Health & Processing. Ocean City, MD. September 29th - 30th 2009."Laboratory Detection of MS - Problems and Solutions"
Publications
- Sellers, H., Linneman, E., Icard, A. H., and Mundt, E (2009). A purified recombinant baculovirus expressed capsid protein of a new astrovirus provides partial protection to runting-stunting syndrome in chickens. Vaccine. 2009 Nov 23. (Epub ahead of print).
- Callison, S. A, S. M. Riblet, A. Rodriguez-Avila, and M. Garcia. Reverse Restriction Fragment Length Polymorphism (RRFLP) Assay: A novel technique and its application to the rapid genotyping of infectious laryngotracheitis virus (ILTV) live attenuated vaccines. Journal Virol. Methods. 160:119-124. 2009.
- Waidner, L., R. Morgan, M. Garcia, A. Anderson, E. Bernberg, S. Kamboj, M. Ouyang, G. Isaacs, M. Markis, B. Meyers, P. Green, and J. Burnside. Novel ILTV and HVT microRNAs have conserved genomic locations with those of other Gallid and Meleagrid herpesvirus microRNAs. Virology. 388:128-36. 2009.
- Oldoni,I, A. Rodriguez-Avila, S. M. Riblet, G. Zavala, and M. Garcia. Pathogenicity and Growth Characteristics of Infectious Laryngotracheitis Virus (ILTV) Isolates from United States. Avian Pathology. 38:47-53. 2009.
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