NE138: Epidemiology and Control of Emerging Strains of Poultry Respiratory Disease Agents

(Multistate Research Project)

Status: Inactive/Terminating

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SAES-422 Reports

Annual/Termination Reports:

[01/11/2001]

Date of Annual Report: 01/11/2001

Report Information

Annual Meeting Dates: 11/10/2001 - 11/11/2001
Period the Report Covers: 01/01/2000 - 12/01/2000

Participants

Meeting Attendees:
Mazhar Khan (CT),
Daral Jackwood (OH),
John Dhoms (DE),
Jack Gelb, Jr (DE),
William Saylor (DE), and
William Wagner, USDA/CSREES Coordinator;

Complete List of Tecnical Committee Members:

AL AES: F. J. Hoerr, L. H. Laurerman, L. Liu, S. Brown-Lockaby, V. Van Santen, V. Pangala, J. E. Wright

CT AES: M. I. Khan

DE AES: S. Cloud, J. E. Dohms, M. Emara, J. Gelb, Jr., C. L. Keeler, R. Morgan, C. R. Pope, and J. K. Rosenberger

GA VMES: S. Kleven, M. W. Jackwood

MD AES: V. N. Vakharia, and R. A. Heckert

NC AES: D. H. Ley

NY AES: S. A. Naqi

OH AES: D. J. Jackwood, and Y. M. Saif

TN St Jude: R. Webster

TX AES: E. Collisson

OH AES: Y. M. Saif (Permanent Chair)

USDA ARS: D. E. Swayne, and D. Suarez

Administrative Advisor: NERA, D. R. MacKenzie (Interim AA)

CSREES Rep.: W. Wagner

Brief Summary of Minutes

Steven R. Alm (stevealm@uri.edu) - University of Rhode Island;

Paul Backman (pbackman@psu.edu) - Pennsylvania Agricultural Experiment Station;

Mark J. Carroll (mc92@umail.umd.edu) - University of Maryland;

J. Marshall Clark (jclark@ent.umass.edu) - University of Massachusetts;

Bruce B. Clarke (clarke@aesop.rutgers.edu) - Rutgers University;

Richard S. Cowles (rcowles@caes.state.ct.us) - Connecticut Agricultural Experiment Station;

J. Scott Ebdon (sebdon@pssci.umass.edu) - University of Massachusetts;

Steven Fales;

Karl Guillard (karl.guillard@uconn.edu) - University of Connecticut;

Richard J. Hull (rhu6441@postoffice.uri.edu) - University of Rhode Island;

Noel Jackson;

Peter J. Landschoot (pj11@psu.edu) - The Pennsylvania State University;

Pim Larsson-Kovach (il11@cornell.edu) - Cornell University;

James Lin - USEPA;

Bill Meyer (wmeyer@aesop.rutgers.edu) - Rutgers University;

Kevin Morris - NTEP;

Bridget Ruemmele (bridgetr@uri.edu) - University of Rhode Island;

Mike Sullivan (senmike@uriacc.uri.edu) - University of Rhode Island;

Patricia Vittum (pvittum@ent.umass.edu) - University of Massachusetts

Accomplishments

Progress of the Work and Principal Accomplishments: a. Project Objective:Develop and evaluate rapid diagnostic capabilities for the identification of emerging agents causing Mycoplasmosis, infectious bronchitis, avian influenza, and infectious laryngotracheitis. AL AES has determined that mycoplasma colonies that were blue (lacZ-positive) contained 12 GAA repeats and colonies that were white had a GAA repeat number of more (14-16) or less (6-10) than 12. The goal of current work is to determine the basis for how the GAA repeats regulate adhesin gene expression CT AES has developed and optimized a respiratory disease multiplex RT-PCR to identify six major avian respiratory pathogens. This multiplex RT-PCR identified and differentiated chickens infected with the following respiratory avian pathogens: infectious bronchitis virus, Avian Influenza, Mycoplasma gallisepticum, Mycoplasma synoviae, Infectious laryngotracheitis virus and Newcastle disease virus. NC AES completed RAPD DNA fingerprints of all MG strains and isolates in their archival storage. One hundred field isolates and reference strains, plus 50 isolates from songbirds, mainly house finches but also including American goldfinches, and grosbeaks (evening and pine). TX AES is using real-time PCR to identify and quantify infectious bronchitis viruses. b. Project Objective: Characterize immunosuppressive viruses (IBDV) and assess interaction of underlying immunosuppressive agents (IBDV and CAV) on emerging respiratory agents. MD AES evaluated the persistence of a variant strain of infectious bursal disease virus (IBDV) in commercial broilers. The disease as produced by this strain appeared later than typically seen and little if any recovery from infection was seen by 6 weeks of age. The implications of this study are that commercial broilers are susceptible to variant strains of IBDV, become infected and are potential virus shedders for much longer than previously thought. This would allow the older birds to seed the environment with infectious virus for the next flock, thereby perpetuating the infection. OH AES has used the RT/PCR-RFLP assay to place IBDV strains into molecular groups. The predicted amino acid sequences corresponding to the VP2 variable sequence region for 14 strains of IBDV from different molecular groups and 11 IBDV strains from one molecular group. The results indicated that viruses within a molecular group defined by the RT/PCR-RFLP assay have similar VP2 gene sequences compared to viruses from different molecular groups. OH AES examined three IBDV strains with unique RT/PCR-RFLP patterns. One of these viruses, T1, broke through maternal immunity in broiler chicks and caused disease the other two viruses did not. The VP2 nucleotide sequences of all three viruses were determined. Amino acid mutations in the T1 strain (not found in the other strains) were consistent with the ability of this virus to break through maternal immunity to classic and variant IBDV strains. OH AES has identified a genetic marker for wild-type potentially pathogenic IBDV strains. This marker was validated using known vaccine (attenuated) and wild-type (pathogenic) IBDV strains. c. Project Objective: Delineate epizootiology of emerging Mycoplasma and the viruses that cause infectious bronchitis, avian influenza, and infectious laryngotracheitis. DE AES Infectious bronchitis virus isolates, found previously to be a subtype of the Ark serotype, were found to be present as subpopulations in the Ark DPI prevaccine laboratory stocks and two commecial attenuated vaccines. These findings provide evidence that the field isolates may have originated in the vaccines and reverted to virulence, perhaps by inconsistent, seasonal use of the vaccine. In addition, they may have become established as a challenge because of infrequent poultry house clean-out and disinfections. A genotypic change has become evident in IBV associated with nephropathogenic IB in Pennsylvania. DE AES During the week of May 15, 2000, an egg-layer operation housing over 1 million chickens became MG infected. Although there were no overt signs of respiratory disease, egg production decreased from 6-10% in seven of the eight houses and the affected houses were serologically positive as detected by the MG plate agglutination test. Chickens within the complex have historically been MS positive, but no MG vaccination program was in use. MG isolations were made and determined to be MG S6 using RAPD at the University of Georgia (GA AES). NC AES is continuing to monitor the ongoing outbreak of M. gallisepticum-conjunctivitis in house finches, American goldfinches and other newly identified host-species by DNA fingerprinting. Since October 1999, North Carolina has experienced unprecedented outbreaks of MG in commercial poultry. RAPD analysis on these MG isolates found that they had fingerprints different than the MG vaccine strains (F, ts11 and 6/85) and the house finch strain, which indicates that these strains were not involved in this epidemic. d. Project Objective: Design and implement novel immune and genetic prophylactic strategies for effective control of respiratory disease caused by emerging agents. CT AES A recombinant fowl poxvirus (rFPV) containing a cDNA copy of the infectious bronchitis virus S1 gene (rFPV-S1) was constructed and its immunogenicity and vaccine potential were evaluated. The recombinant virus elicited anti-IBV protective immunity that was recognized by the relatively mild clinical signs of disease, decreased titers of recovered challenge virus and less severe histological changes of the tracheas in virulent IBV-challenged chickens previously receiving rFPV-S1 as compared to parental FPV vaccinated control birds. Thus, rFPV has potential as a poultry vaccine against both fowl pox and infectious bronchitis. DE AES Additional sequence within the MG cytadhesin operon revealed two additional genes (mgc3 and mgc4) with homology to cytadhesin accessory genes found in the human pathogen M. pneumoniae. The mgc3 gene was expressed and antiserum to it was prepared in goats. DE AES An interdependence between the University of Illinois (IL AES) and the University of Delaware (DE AES) was established to construct novel live recombinant attenuated strains of infectious laryngotracheitis virus. The formation of recombinant viruses is in progress. MD AES Infectious bursal disease viruses in which VP4, VP4+VP3 and VP1 coding sequences of the IM strain were substituted for those genes of the D78 strain, failed to induce a hemorrhagic lesion in the bursa. A chimeric virus, in which the VP2 gene of the GLS strain was substituted for that gene of D78, caused rapid bursal atrophy as typically seen with the GLS strain. This information suggests that the residues involved in virulence and pathogenesis reside in the VP2 region. TX AES has examined immune mechanisms against infectious bronchitis virus in chickens. To this end they cloned genes of the IL-2 and IFN-gamma. They were able to demonstrate protection against IBV acute infection following transfer of immune T cells from infected chicks. Genetic differences in susceptibility to IBV illness in birds with distinct MHC haplotypes were examined. Chicks that have B haplotypes 2, 5, 8, 12 and 19 were challenged with the Gray strain of IBV. The order of clinical illnesss in these preliminary studies from most to least susceptible was 12>8>19>5>2. They have also shown that type I interferon, produced by splenocyte preparations, inhibits IBV replication in vivo and in vitro. Usefulness of findings: a. Project Objective: Develop and evaluate rapid diagnostic capabilities for the identification of emerging agents causing Mycoplasmosis, infectious bronchitis, avian influenza, and infectious laryngotracheitis. AL AES Studies on the GAA repeat region in Mycoplasma will be important because the regulation of gene expression by a trinucleotide repeat region located upstream of the gene!/s transcription site is a novel finding and possibly a novel mechanism for regulating bacterial gene expression. The analysis of gene regulation in M. gallisepticum should lead to understanding the events that are central for pathogenesis, and are important for targeting future therapeutic approaches. CT AES Multiplex RT-PCR specific for six respiratory disease agents will be highly advantageous for simultaneous detection and differentiation of respiratory pathogens. This multiplex PCR is more economical and requires less time as compared to single PCR assays for each pathogen. DE AES The presence of highly related, multiple subpopulations in viral stocks is a characteristic of viral quasispecies which are created by the high mutation caused by the error-prone nature of RNA polymerases. Vaccines still retain a virulent subpopulation(s) of the original virulent field isolated following attenuation, although, it is a much smaller fraction of the population. IBV vaccines with residual virulent subpopulations have inherent risks that may be realized by intermittent (seasonal) use of the modified live vaccine. b. Project Objective: Characterize immunosuppressive viruses (IBDV.) and assess interaction of underlying immunosuppressive agents (IBDV and CAV) on emerging respiratory agents. OH AES Assessment of the virulence of IBDV field strains is useful because more than one virus can be isolated in a flock diagnosed with infectious bursal disease. Not all viruses isolated from these outbreaks are pathogenic. OH AES Using the RT/PCR-RFLP assay to examine the epidemiology of IBDV strains requires viruses within a molecular group be relatively similar genetically and phenotypically. Studies in this area should provide data on the utility of molecular diagnostic assays. c. Project Objective: Delineate epizootiology of emerging Mycoplasma and the viruses that cause infectious bronchitis, avian influenza, and infectious laryngotracheitis. NC AES The successful application of the RAPD fingerprinting technique to MG field isolates has validated the utility of strain identification. This work has allowed scientists to make important hypotheses and conclusions regarding the spread of this disease. d. Project Objective: Design and implement novel immune and genetic prophylactic strategies for effective control of respiratory disease caused by emerging agents. CT AES Recombinant Fowl poxvirus containing the S1 gene of infectious bronchitis virus has potential as a poultry vaccine against both fowl pox and infectious bronchitis. MD AES Information gained by identifying the residues involved in virulence and pathogenesis would greatly facilitate the development of a recombinant subunit vaccine against infectious bursal disease virus. Work planned for next year by participating AES: The NE-138 is seeking to join the NC-228 project. Both committees share common objectives and have been meeting jointly to discuss poultry disease research results for several years. AL AES will examine the regulatory elements involved in M9/pMGA gene expression in MG with the extension of transposon-mutagenesis studies. Additional studies are being conducted to characterize the chicken tracheal ciliary receptor(s) for the M9/pMGA adhesin of MG. DE AES will continue work on nephropathogenic IB by evaluating efficacy of killed vaccination. They will assess the relationship between VN serotyping, S-1 sequencing and cross-challenge results for characterizing antigenic and genotypic properties of IBV. They will evaluate efficacy of Marek!/s disease virus vaccination to prevent potential immunosuppressive effects of very virulent Marek!/s disease challenge on IBV immunity. Studies will continue on the evaluation of recombinant MG cytadhesins as single and combined entities and incorporating expressed portions of MGC3 and MGC4 with previously described MGC1 and MGC2. Work will continue on the construction of live attenuated recombinant ILTV vaccines. Efforts will continue on characterizing potential biological adjuvants identified through the sequencing of an avian macrophage cDNA library. CT AES will examine IBV DNA vaccines (fowl poxvirus vector S1 recombinant, plasmid S1 and S gene DNA) under field conditions. Vaccination trials using in ovo IBV DNA and viral vector vaccines will be conducted. MD AES will prepare chimeric cDNA clones of IBDVs that could be used as a DNA vaccine or to generate attenuated, marked vaccines using the reverse genetics system. They will evaluate protection against IBDV by DNA vaccines. NC AES will continue evaluation and application of various PCR-based diagnostic methods for avian Mycoplasmosis. They will continue to develop and apply RAPD fingerprinting of pathogenic avian Mycoplasma species. The pathogenic potential of M. sturni and other emerging avian Myoplasma species will be investigated. Investigations will continue on vaccines for avian Mycoplasma and work will begin on the dynamics of an emerging pathogen in an introduced host. OH AES will continue studies on the genetic basis for pathogenicity and antigenicity in IBDV strains. They will examine methods for the molecular detection and identification of IBDV strains with the goal to reduce the cost and improve the information generated in these assays.

Publications

Li Liu, K. Dybvig, V.S. Panangala, V.L. van Santen. 2000. GAA trinucleotide repeat region regulates M9/pMGA gene expression in Mycoplasma gallisepticum. Infect. Immun. 68:871-876. Ruffin, D.C., V.L. van Santen, Y. Zhang, L.L. Voelker, V.S. Panangala and K. Dybvig. 2000. Transposon mutagenesis of Myccoplasma gallisepticum by conjugation with Enterococcus faecalis and determination of insertion site by direct genomic sequencing. Plasmid 44:191-195. Fiorentin, L., Y. Zhang, and V.S. Panangala. 2000. Phenotypic variation of Mycoplasma iowae surface antigen. Avian Dis. 44:434-438. Kingham, B.F., C.L. Keeler, Jr., W.A. Nix, B. S. Ladman, and J. Gelb, Jr. 2000. Identification of avian infectious bronchitis virus by direct automated cycle sequencing of the S-1 gene. Avian Dis. 33:325-335. Nix, W.A., D.S. Troeber, B.F. Kingham, C.L. Keeler, Jr. and J. Gelb, Jr. 2000. Emergence of subtype strains of the Arkansas serotype of infectious bronchitis virus in Delmarva broiler chickens. Avian Dis. 44:568-581. Wang, X. and M.I. Khan. 2000. Use of RT-PCR-RFLP to examine the interaction between infectious bronchitis viruses in ovo. Avian Pathol. 29(5). Wang, X., and M.I. Khan. 2000. Molecular characterization of an infectious bronchitis virus strain isolated from an outbreak in vaccinated layers. Avian Dis. 44(4). Hartup, B.K., J.M. Bickal, A.A. Dhondt, D.H. Ley, and G.B. Kollias. 2000. Dynamics of mycoplasmal conjunctivitis in eastern house finches. The Auk (in press). Jackwood, D. J., S. E. Sommer, and E. Odor. 1999. Correlation of ELISA titers with protection to infectious bursal disease virus. Avian Dis. 43:189-197. Jackwood, D. J., and S. E. Sommer. 1999. Restriction fragment length polymorphisms in the VP2 gene of international infectious bursal disease viruses. Avian Dis. 43:310-314. Sellers, H. S., P. N. Villegas, B. S. Seal and D. J. Jackwood. 1999. Antigenic and molecular characterization of three infectious bursal disease virus field isolates. Avian Dis. 43:198-206. Smiley, J. R., S. E. Sommer, and D. J. Jackwood. 1999. Development of a ssRNA internal control reagent for an infectious bursal disease virus RT/PCR-RFLP diagnostic assay. J. Vet. Diag. Invest. 11:497-504. Zhou, M. and E.W. Collisson. 2000. Amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with 3‘ genomic RNA. Virus Res. 67:31-39. Seo, S.H., J. Pei, D.E. Briles, W. Briles, and E.W. Collisson. 2000 Adoptive transfer of infectious bronchitis virus primed %a), T cells bearing CD8 antigen protects chicks from acute infection. Virol. 269:183-189. Collisson, E.W., J. Pei, J. Dzielawa, and S.H. Seo. 1999. Cytotoxic T lymphocyte responses to infectious bronchitis virus. In Avian Immune Responses to Infectious Diseases. Developmental and Comparative Immunology, 24:187-200.