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SAES-422 Format for Multistate Research Activity Accomplishments Report Annual Progress Report for Project: NC-228 Regional Research Avian Respiratory Diseases: Pathogenesis, Surveillance, Diagnosis and Control 1 October 2000 - 30 September 2001 January 21, 2002 Annual Meeting Dates: November 9-10, 2001 1. Meeting participants: AL AES CT AES DE AES IA AES IL AES IN AES MD AES MN AES NC AES OH AES Administrative Advisor CSREES Representative V. S. Panangala Report submitted by M. I. Khan J. E. Dohms and J. Gelb, Jr. D. L. Reynolds D. N. Tripathy C. C. Wu Report submitted by V. N. Vakharia and R. A. Heckert V. Kapur (report included work by D. N. Foster, S. M. Goyal, D. A. Halvorson, K. V. Nagaraja, M. K. Njenga, S. Noll, and J. M. Sharma) Report submitted by D. H. Ley Y. M. Saif (report included work by D. J. Jackwood) J. Klausner (U. Minnesota) W. Wagner (USDA) 2. Brief summary of minutes of annual meeting: The NC228 business meeting was held Nov. 9, 2001, in the Atrium C Room of the Millennium Hotel, St. Louis, Missouri. Dr. Jack Gelb, Chair of NC228 opened the meeting at 2 PM. Dr. Gelb will continue to serve as Chair and Dr. Wu as Secretary for 2001-2002. For completion of the final composite annual report, Dr. Gelb requested that each Station to submit the report by objectives via e-mail. In keeping with the regional research mission, cooperative activities among project members were to be highlighted. Dr. Klausner stated that the NE138 regional project had officially been merged into the NC228 project. A subcommittee consisting of Drs. Kapur, Reynolds and Gelb will create a NC228 website that will post the membership list, project proposal, annual station and composite reports. Drs. Saif and Dohms agreed to identify and invite new members to the NC228 project. Drs. Saif and Kapur will finish a position paper on genotypes/serotypes and submit to the journal, Avian Diseases. Dr. Saif proposed that the NC228 co-sponsor a symposium on poultry respiratory diseases with the North Central Avian Diseases Committee for the 2003 meeting in Ohio. Dr. Wagner indicated that conference grants are available from the National Research Initiative. Dr. Wagner summarized federal legislative activities as follows; funding for FY 2002 has passed; the NRI received $120 million for FY '02; the Initiative for Future Agriculture and Food 1 Systems was cancelled for FY '02; Formula, Hatch and Extension funding is the same as FY'01; Animal Health and Disease funding is similar to last year (~$5 million); indirect cost allowable charges to 35% are under consideration. The business meeting concluded at 4 PM. The remainder of the meeting was devoted to presentation and discussion of the station reports. The meeting adjourned Nov. 10 at 3:30 PM. 3. Accomplishments and Impacts: Objective 1. Determine the pathogenesis and interactions of specific agents. Alabama determined that M. gallisepticum sequences both upstream and downstream of the GAA repeats in the promoter region of the pMGA gene are important for regulation and that the number of repeats affects expression by altering the spacing between the flanking sequences. They have hypothesized that a hemagglutinin-activator protein (HAP) binds to the GAA repeat region and stimulates pMGA gene transcription if and only if 12 copies of the GAA repeat are present. Delaware. Vaccination of 1-day-old SPF chicks with Marek's disease virus (MDV) vaccines HVT + SB1 is highly effective against very virulent plus MDV (584A) challenge and did not suppress immunity to infectious bronchitis virus (IBV) vaccination at 1 and 14 days of age. Dr. Robin Morgan contributed to this work. Delaware. Continued work on the molecular pathogenesis of Mycoplasma gallisepticum, resulting in further characterization of the cytadhesin operon consisting of mgc1, mgc2, mgc3, and mgc4. MGc3 was surface-exposed as determined by protease digestion and partitions into the insoluble triton shell suggesting that it is also associated with the cytoskeleton, a structure involved in cell division, gliding motility, and bleb organization. A tn916 transposon library containing approximately 700 MG mutants was prepared by optimizing the plasmid (pAM120) DNA and the percent PEG concentrations. A summary of all the insertions will soon be available at http://udgenome.ags.udel.edu/mgall/mutants/html. Illinois. A-type inclusion body protein was found not to be essential for replication of fowlpox virus in cultured avian cells. Replication of photolyase-deficient fowlpox virus in the chicken, was impaired when compared to that of the parent virus, but still was immunogenic and protective and thus may be a vaccine candidate. Insertional inactivation of a hemagglutinin (HA)-like gene homologue in fowlpox virus had no effect on the growth kinetics of the recombinant and parent virus. Previously unrecognized condorpox virus was determined to be a pox virus based on EM yet was biologically, antigenically, and genetically distinct from fowlpox virus and thus may represent a new species of the avipoxvirus genus. Iowa. The Colorado strain of avian pneumovirus (APV) was found to be a mild/low virulent pathogen for egg-type laying chickens based on clinical respiratory signs and egg production. Minnesota. Transmission of APV was demonstrated via vertical an airborne roues. 2 Minnesota completed the genomic sequence of Pasteurella multocida, the first major veterinary bacterial pathogen sequenced in its entirety. The filamentous hemagglutinin protein homolog, which is expressed in virulent but not avirulent isolates and whose inactivation results in more than a million-fold reduction in ability to cause mortality, was identified. Ohio. Serotype 2 infectious bursal disease virus (IBDV) that was pathogenic for chicken embryos was found to be infectious, but not pathogenic in chickens or turkeys. Ohio. Uncomplicated APV infection did not persist in birds for a long period and was usually limited to the respiratory tract. APV was shown to have limited transmissibility. Objective 2. Surveillance, occurrence and consequences of agents and host variation on disease susceptibility Minnesota. Wild passerine birds were found to be sero- and virus-positive for APV. Infectious virus persisted on premises inhabited by APV inoculated birds for a period of up to 6 weeks after initial exposure. APV infection increased T and B cell infiltration into the gland of Harder, upregulated Type I and II interferon synthesis, and reduced splenic T-cell responsiveness. Disease in young poults was more severe and associated with reduced T-cell function compared to older birds. Microarray studies showed that lymphoid tissues undergo altered gene expression in response to APV infection. Transcripts representing proteins involved in the activation of T cells, T- and B-cell signal transduction and efficient antigen processing and presentation were found to be upregulated. North Carolina. The random amplification of polymorphic DNA (RAPD) fingerprint database consisting of all Mycoplasma gallisepticum (MG) reference strains and more than 200 poultry isolates, plus ~100 songbirds isolates has been completed. The application of a computer-assisted analysis system using Gene Profiler (Scanalytics, Inc., version 4.0) is ongoing. Since January 2001, MG was isolated from only one broiler breeder and two turkey farms in the state. Each isolate was a new and unique RAPD type, which suggested that they were introduced to the farms from separate external sources (evidence suggests backyard flocks), and there was no evidence of transmission to or from other commercial poultry. Ohio. Selection for increased body weight in turkeys was associated with increased mortality following vaccination with live LaSota Newcastle disease virus (NDV) vaccine. Objective 3. Develop new and improved methods for the diagnosis, prevention, and control of avian respiratory diseases. Connecticut, having developed IBV DNA vaccines for chicks, is now extending the application to in ovo administration. Future work will focus on in ovo DNA and viral vector (fowlpox) vaccination and protection studies using vaccines containing the IBV surface glycoprotein S and S1 genes. 3 Delaware. Following live IBV vaccine priming, inactivated oil emulsion IBV vaccination containing the PA/Wolgemuth/98 strain provided better protection than a heterologous inactivated vaccine containing Massachusetts + Arkansas strains based upon challenge with the virulent nephropathogenic strain, PA/Wolgemuth/98. Dr. Conrad Pope contributed to this study. Illinois. Monoclonal antibodies against fowlpox virus (FPV) were used to differentiate between vaccine and field strains of fowlpox virus and may be used for diagnosis. Illinois and Delaware. A recombinant (r) infectious laryngotracheitis virus (ILTV) lacking the ability to produce glycoprotein C (gC) was generated to circumvent possible reversion to a more virulent form and also to "genetically mark" a vaccine virus. The vaccine potential of the rILTV is being evaluated by Dr. Calvin Keeler, Indiana. Three weekly DNA vaccinations of chickens using plasmids containing various fragments of large segment genome (VP2) of serotype 1 IBDV protected chickens against clinical signs and mortality following virulent challenge. Iowa. Anti NDV specific immunoglobulin administered subsequent to virulent NDV challenge protected chickens against clinical ND if the immunoglobulin was given prior to the onset of clinical signs. Maryland. Using the cRNA-based reverse genetics system developed for IBDV, residues involved in virulence and pathogenesis were identified that could lead to the development of a marked, attenuated, multi-spectrum vaccine against IBDV. Maryland. Dermal DNA vaccination of chickens using the HN gene of NDV and the VP2 of IBDV induced IgG, IgM, IgA antibody responses to the viruses in serum and tears. Plasmid DNA persisted in kidney, bone marrow and muscle. DNA vaccination in ovo using combinations of plasmid DNA, neutral lipid and DMSO resulted in gene expression in liver and muscle. Production of insert-specific mRNA and protein in a variety of tissues was evident, resulting an immune response that can provide partial protection from viral challenge with IBDV. Minnesota. Turkey turbinate and kidney cells were successfully life-span-extended to produce higher APV titers for the purposes of vaccine production. APV sequencing lead to the development of RT-PCR and Taqman diagnostic tests. ELISA assays for APV were developed using recombinant viral M and N proteins. APV isolates from the upper Midwest were sequenced and the N, P, M, F, and M2 genes were analyzed. Nucleotide substitution rates across provided evidence that only a single clone of APV exists and is widely disseminated. Formalin and UV-inactivated APV vaccines were determined to be safe but ineffective whilst a live-attenuated passage 63 vaccine was both safe and effective. In ovo vaccination did not adversely affect hatchability or livability of the hatched poults and induced earlier and longer lasting protection compared to post-hatch vaccination. Minnesota. A temperature-sensitive (Ts) mutant of the respiratory pathogen Ornithobacterium rhinotracheale (ORT) was shown to elicit an immune response in turkeys vaccinated via the 4 drinking water and resulted in reduced clinical signs, gross lesions, and re-isolation rates following experimental challenge with pathogenic ORT. Ohio. Single and multiple nucleotide polymorphisms of IBDV were detected using real-time RT-PCR. Analysis revealed the presence of multiple genetic sub-populations of virus within a vaccine. Studies will be conducted to determine if plaque purification creates single and stable genetic viral populations. Real-time RT-PCR will be used to examine vaccines and laboratory samples for polymorphisms encoding major neutralizing epitiopes. A genetic marker for wildtype potentially pathogenic IBDV strains was identified making it possible to differentiate between strains that are the potential cause of the disease and those viruses that are attenuated. Monoclonal antibodies to the very virulent IBDV will be developed. Work Planned for the Coming Year Avian paramyxoviruses (pneumovirus and Newcastle disease virus): Iowa will develop new and improved methods for the diagnosis, prevention and control of APV and NDV. Minnesota will further study the pathogenesis and transmission of APV, and with Illinois will develop and evaluate recombinant fowl pox vectored APV-N and F protein vaccines. Minnesota will construct an infectious clone of APV to develop targeted mutants for evaluation as vaccines, develop temperature sensitive mutants and will sequence APV genes (G, SH and L). Ohio will continue to evaluate antigenic relationships of avian paramyxoviruses. Infectious bronchitis: Connecticut will evaluate IBV DNA vaccines (Fowlpox vector S1 recombinant and plasmid S1 and S gene DNA) and will continue In ovo IBV DNA and viral vector vaccination trials and protection studies. Delaware will assess the antigenic characteristics of IBV DE/072/92-related field isolates from Georgia (GA 98) and Alabama broiler chickens, provide IBV isolates to Indiana, and with Maryland, evaluate Newcastle disease virus as a vector for delivering IBV S antigen. Infectious bursal disease: Indiana will continue work on DNA vaccines for IBD. Maryland will prepare chimeric cDNA clones of IBDV for use as DNA vaccines or to generate live, attenuated, multivalent IBDV vaccines. Ohio will develop monoclonal antibodies to very virulent IBDV, use real-time RT/PCR to examine IBDV strains for genetic polymorphisms in sequences that encode major neutralizing epitopes, and attempt to characterize viruses present in vaccine and laboratory samples. Mycoplasmosis: Delaware will continue to characterize the M. gallisepticum cytadhesin operon. North Carolina will continue to evaluate PCR-based diagnostic methods for avian mycoplasmosis and develop RAPD fingerprinting of pathogenic Mycoplasma spp. Furthermore, NC will assess the pathogenicity of emerging Mycoplasma spp. isolates for poultry, investigate mycoplasma vaccines, and study the ongoing MG outbreak in house finches. Alabama will use transposon mutagenesis to obtain pMGA- negative mutants with a disrupted HAP gene. Gel mobility shift experiments will be performed to examine interactions between the putative HAP and the promoter region of the pMGA genes. 5 6 4. Publications: Abdel-Alim, G.A. and Y. M. Saif: Immunogenicity and antigenicity of very virulent strains of infectious bursal disease viruses. Avian Dis. 45:92-101, 2001. Alkhalaf, A.N., R.N. Dearth, and Y.M. Saif. Pathogenicity, infectivity, and tissue distribution of avian pneumovirus in turkey poults. Proc. 52nd North Central Avian Dis. Conf., p. 38, 2001. Bautista DA, Elankumaran S, Heckert RA, Oshop GL, Moura LC, Wilson JD. Interaction of Salmonella typhimurium and infectious bursal disease virus (IBDV) in broiler chickens. Proc. 138th Am. Vet. Med. Assn. Mtg, Boston, MA, July 14-18, 2001. Brandt, M., Yao, K., Liu, M., Heckert, R.A., and Vakharia,V.N. Molecular determinants of virulence, cell tropism and pathogenic phenotype of infectious bursal disease. J. Virol. 74. In press. Chang, H. C., Lin, T. L., and C. C. Wu. DNA-mediated vaccination against infectious bursal disease in chickens. Vaccine 20: 328-335, 2002. Chiang, S., A. M. Dar, S. M. Goyal, M. A. Sheikh, J. C. Pedersen, B. Panigrahy, D. Senne, D. A. Halvorson, K. V. Nagaraja, and V. Kapur. A modified enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies J Vet Diagn Invest. 12:381-4. 2000. Dar, A. M., K. Tune, S. Munir, B. Panigrahy, S. M. Goyal, and V. Kapur. PCR-based detection of an emerging avian pneumovirus in US turkey flocks J Vet Diagn Invest. 13:201-5. 2001. Dar, A. M., S. Munir, S. M. Goyal, M. S. Abrahamsen, and V. Kapur. Sequence analysis of the nucleocapsid and phosphoprotein genes of avian pneumoviruses circulating in the US Virus Res. 79:15-25. 2001. Davison, S., A. F. Ziegler, J. Gelb, Jr., P. A. Dunn, and R. J. Eckroade. Infectious bronchitis in Pennsylvania. Proc. AAAP Symposium. Respiratory Diseases of Poultry. AVMA/AAAP Ann. Mtg. Boston, Massachusetts, July 14-18, 2001. Elankumaran, S, Heckert, RA, Moura, L. Persistence and tissue distribution of a variant strain of infectious bursal disease virus in commercial broiler chickens. Proc. 2nd International symposium on infectious bursal diseases and chicken infectious anemia. Rauischholzhausen, Germany, June 2001. Gelb, J., Jr., C. L. Keeler, B. S. Ladman, and B. F. Kingham. S1 sequence analysis; a tool for understanding IBV outbreaks. Proc. AAAP Symposium. Respiratory Diseases of Poultry. AVMA/AAAP Ann. Mtg. Boston, Massachusetts, July 14-18, 2001. Goyal, S. M., S. J. Chiang, A. M. Dar, K. V. Nagaraja, D. P. Shaw, D. A. Halvorson, and V. Kapur. Isolation of avian pneumovirus from an outbreak of respiratory illness in Minnesota turkeys J Vet Diagn Invest. 12:166-8. 2000. 7 Gulati, B. R., D. P. Patnayak, A. M. Sheikh, P. E. Poss, and S. M. Goyal. Protective efficacy of high-passage avian pneumovirus (APV/MN/turkey/1- a/97) in turkeys Avian Dis. 45:593-7. 2001. Gulati, B. R., K. T. Cameron, B. S. Seal, S. M. Goyal, D. A. Halvorson, and M. K. Njenga. Development of a highly sensitive and specific enzyme-linked immunosorbent assay based on recombinant matrix protein for detection of avian pneumovirus antibodies J Clin Microbiol. 38:4010-4. 2000. Gulati, B. R., S. Munir, D. P. Patnayak, S. M. Goyal, and V. Kapur. Detection of antibodies to US. isolates of avian pneumovirus by a recombinant nucleocapsid protein-based sandwich enzyme-linked immunosorbent assay J Clin Microbiol. 39:2967-70. 2001. Hartup B. K., Kollias GV, Ley DH. Mycoplasmal conjunctivitis in songbirds from New York. J. Wildlife Dis., 36:257-264. 2000. Hartup BK, Bickal JM, Dhondt AA, Ley DH, Kollias GV. Dynamics of conjunctivitis and Mycoplasma gallisepticum infections in house finches. The Auk,118:327-333, 2001. Jackwood, D. J. Diagnosis of infectious bursal disease viruses using the RT/PCR-RFLP assay: Advantages, limitations and comparison to other molecular assays. Proc. of the Partnerships in Poultry Symposium. Fort Dodge Animal Health. Paris, France. July 9-11. 2001. Jackwood, D. J. Genotypic and phenotypic diversity among wild-type IBDV strains. Proc. of the Partnerships in Poultry Symposium. Fort Dodge Animal Health. Paris, France. July 9-11. 2001. Jackwood, D. J. Molecular diagnosis of infectious bursal disease viruses: Practical applications and significance of the results. Proc. of the Symposium on Molecular identification and epidemiology of avian pathogens. Amer. Assn. of Avian Pathol.. 137th Am. Vet. Med. Assn.. Mtg., Salt Lake City, Utah. 2000. Jackwood, D. J. Molecular diagnosis of infectious bursal disease viruses. Proc. of the Intervet Workshop on Immune Suppression and Emerging Diseases in Broilers. Baltimore, Maryland. 2000. Jackwood, D. J. New molecular techniques for the diagnosis and control of infectious bursal disease virus. Proc. of the 51st North Central Avian Dis. Conf., Columbus, Ohio. 2000. Jackwood, D. J. Standardization and quality control of diagnostic reverse transcription polymerase chain reaction (RT-PCR) assays: Detection and identification of single nucleotide polymorphisms in poultry pathogens using new fluorescence hybridization based technology. Proc.of the X International Symposium of Vet. Laboratory Diagnosticians and OIE Seminar on Biotechnology. Salsomaggiore-Parma, Italy, July 4-7. 2001. 8 Jackwood, D. J. and S. Sommer. Detection of single and multiple nucleotide polymorphisms in infectious bursal disease viruses using real-time RT/PCR. Abstr. 138th AVMA Mtg. 2001. Jackwood, D. J., E. H. Byerley and S. E. Sommer. Molecular marker for attenuation in infectious bursal disease viruses. Avian Dis. 45:701-705. 2001. Jackwood, D. J., S. E. Sommer and H. V. Knoblich. Amino acid comparison of infectious bursal disease viruses placed in the same or different molecular groups using RT/PCR-RFLP. Avian Dis. 45:330-339. 2001. Jirjis, F. E., S. L. Noll, D. A. Halvorson, K. V. Nagaraja, and D. P. Shaw. Immunohistochemical detection of avian pneumovirus in formalin-fixed tissues J Vet Diagn Invest. 13:13-6. 2001. Jirjis, F. E., S. L. Noll, D. A. Halvorson, K. V. Nagaraja, E. L. Townsend, A. M. Sheikh, and D. P. Shaw. Avian pneumovirus infection in Minnesota turkeys: experimental reproduction of the disease Avian Dis. 44:222-6. 2000. Khan, M. I. Avian Pathogenic Mycoplasmas. PCR detection of Microbial Pathogens. Methods in Molecular Biology. eds. J. Frey and K. Sachse. Humana Press Inc. Totowa, NJ. In press. Khan, M. I. Avian respiratory tract infections and their control strategies. World Poultry Science Assn. in collaboration with Pakistan Poultry Assn., Islamabad, Pakistan, July 7, 2001. Khan, M. I. Recombinant fowlpox virus containing the S1 gene of Massachusetts 41 strain of infectious bronchitis virus, Institute of Poultry Science, Shandong Academy of Agricultural Science, Jinan City, Shandong, People’s Republic of China, March 26, 2001. Khan, M., Wang, X., Schnitzlein, W. and Tripathy, D.N. A recombinant fowlpox virus containing IBV-S1 gene and its potential for a vaccine. Abst. AAAP/AVMA Scientific Program, Boston, MA. p. 39. 2001. Khan. M. I. Are your turkeys healthy? 37th Annual New England Turkey Growers Conf., Sturbridge, Massachusetts. March 8, 2000. p. 9. Kim, H., S. You, B. W. Kong, L. K. Foster, J. Farris, and D. N. Foster, Necrotic cell death by hydrogen peroxide in immortal DF-1 chicken embryo fibroblast cells expressing deregulated MnSOD and catalase Biochim Biophys Acta. 1540:137-46. 2001. Kim, H., S. You, I. J. Kim, J. Farris, L. K. Foster, and D. N. Foster. Increased mitochondrialencoded gene transcription in immortal DF-1 cells Exp Cell Res. 265:339-47. 2001. Kim, H., S. You, I. J. Kim, L. K. Foster, J. Farris, S. Ambady, F. A. Ponce de Leon, and D. N. Foster. Alterations in p53 and E2F-1 function common to immortalized chicken embryo fibroblasts Oncogene. 20:2671-82. 2001. Kim, H., S. You, J. Farris, L. K. Foster, Y. J. Choi, and D. N. Foster. Gonad-specific expression 9 of two novel chicken complementary DNA isoforms Biol Reprod. 64:1473-80. 2001. Kim, H., S. You, L. K. Foster, J. Farris, and D. N. Foster. The rapid destabilization of p53 mRNA in immortal chicken embryo fibroblast cells Oncogene. 20:5118-23. 2001. Kim, H., S. You, L. K. Foster, J. Farris, Y. J. Choi, and D. N. Foster. Differential expression of chicken dimerization cofactor of hepatocyte nuclear factor-1 (DcoH) and its novel counterpart, DcoHalpha Biochem J. 354:645-53. 2001. Kim, I. J., and J. M. Sharma. IBDV-induced bursal T lymphocytes inhibit mitogenic response of normal splenocytes Vet Immunol Immunopathol. 74:47-57. 2000. Kim, I. J., S. K. You, H. Kim, H. Y. Yeh, and J. M. Sharma. Characteristics of bursal T lymphocytes induced by infectious bursal disease virus J Virol. 74:8884-92. 2000. Kim, T-J. and Tripathy, D.N. Reticuloendotheliosis virus integration in the fowlpox virus genome: not a recent event. Avian Dis. 45: 663-669. 2001. Kim, T-J., Pessier, A.P. and Tripathy, D.N. Condor poxvirus with biological differences from fowlpox virus. Poster Presentation at the AAAP/AVMA Scientific Program, Boston, MA. p. 54. 2001. Kinde, H., H. L. Shivaprasad, B. M. Daft, D. H. Read, A. Ardans, R. Breitmeyer, G. Rajashekara, K. V. Nagaraja, and I. A. Gardner. Pathologic and bacteriologic findings in 27week-old commercial laying hens experimentally infected with Salmonella enteritidis, phage type 4 Avian Dis. 44:239-48. 2000. Knoblich, H. V., S. E. Sommer and D. J. Jackwood. Antibody titers to infectious bursal disease virus in broiler chicks following vaccination at one day-of-age with infectious bursal disease virus and Marek’s disease virus. Avian Dis. 44:874-884. 2000. Kwon, H. M., D. K. Kim, T. W. Hahn, J. H. Han and D. J. Jackwood. Sequence of precursor polyprotein gene (segment A) of infectious bursal disease viruses isolated in Korea. Avian Dis. 44:691-696. 2000. Ley DH, Martinez A, Vaillancourt J-P, Smith C. DNA fingerprints of Mycoplasma gallisepticum isolates from the 1999-2000 North Carolina experience. Proc. 35th Natl. Mtg. on Poultry Health and Processing. p. 31-33, 2000. Ley DH, Vaillancourt J-P, Martinez A. Molecular and field epidemiological investigations of Mycoplasma gallisepticum outbreaks in commercial poultry. Proc. XXI World’s Poultry Congress, Abstracts and Proc. CD. 2000. 10 Ley DH, Vaillancourt J-P, Martinez A. Mycoplasma gallisepticum in North Carolina: 19992000. Convention Notes from the 138th Ann. Convention of the Amer. Vet. Med. Assn., CDROM produced by Veterinary Software Publishing, Inc., O’Fallon, IL., July 14-18, 2001. Ley DH, Vaillancourt J-P, Martinez A. Mycoplasma gallisepticum in North Carolina: 1999-2000 (and beyond). Proc. Respiratory Diseases of Poultry, Amer. Assn. of Avian Pathol. Symposium 2001, Amer. Vet. Med. Assn Ann. Convention, Boston, MA (4 pp.). July 15, 2001. Ley DH, Vaillancourt J-P, Martinez A. Mycoplasma gallisepticum in North Carolina: 19992000. Amer. Assn. Avian Pathol./Amer. Vet. Med. Assn. Scientific Program, Boston, MA; (abstract, p. 10). July 2001. Ley DH. Identification of Avian Mycoplasma Strains by Random Amplification of Polymorphic DNA (RAPD). Zootechnica No. 6, pp. 46-48. June 2001. Li, Z., K. E. Nestor, Y. M. Saif, J. W. Anderson, and R. A. Patterson. Effect of selection for increased body weight in turkeys on lymphoid organ weights, phagocytosis, and antibody responses to fowl cholera and Newcastle Disease-inactivated vaccines. Poultry Sci. 80:689-694, 2001. Liu, L., K. Dybvig, V. S. Panangala. Sequences 5' of the GAA repeats and the spacing between GAA repeats and the transcription start site are important for pMGA gene expression in Mycoplasma gallisepticum. 101" General Mtg. Amer. Soc. Micro., (abstract # G23). May-June, Orlando, Florida. 2001. Liu, M, Brandt, M., and Vakharia,V.N. (2001). Phylogenetic analysis of infectious bursal disease virus strains of different pathotypes. 20th Ann. Mtg. of Amer. Soc. for Virol. July 21-25, Madison, WI. 2001. Liu, M., and Vakharia,V.N. (2001). Expression of infectious bursal disease virus structural and Newcastle disease virus HN protein genes in a baculovirus insect/cell system. 73rd Northeastern Conf. on Avian Dis., College Park, MD, June 2001. Liu, Y., and Vakharia,V.N. (2001). Molecular determinants of virulence in variant infectious bursal disease. 73rd Northeastern Conf. on Avian Dis., College Park, MD, June 2001. Lopes, V., G. Rajashekara, A. Back, D. P. Shaw, D. A. Halvorson, and K. V. Nagaraja. Outer membrane proteins for serologic detection of Ornithobacterium rhinotracheale infection in turkeys Avian Dis. 44:957-62. 2000. Martinez A, Vaillancourt J-P, Ley DH. The epidemiology of Mycoplasma gallisepticum in North Carolina: an update. Convention Notes from the 138th Ann. Convention of the Amer. Vet. Med. Assn. CD-ROM produced by Veterinary Software Publishing, Inc., O’Fallon, IL., July 14-18, 2001. 11 Martinez A, Vaillancourt J-P, Ley DH. The epidemiology of Mycoplasma gallisepticum in North Carolina: an update. Amer. Assn. Avian Pathol./Amer. Vet. Med. Assn. Scientific Program, Boston, MA; (abstract, p. 25). July 2001. May, B. J., Q. Zhang, L. L. Li, M. L. Paustian, T. S. Whittam, and V. Kapur. Complete genomic sequence of Pasteurella multocida,Pm70 Proc Natl Acad Sci U S A. 98:3460-5. 2001. Meir, R., D. J. Jackwood and Y. Weisman. Molecular typing of infectious bursal disease virus of Israeli field and vaccine strains by the reverse transcription/polymerase chain reaction/restriction fragment length polymorphism assay. Avian Dis. 45: 223-228. 2001. Mikaelian I, Ley DH, Claveau R, Lemieux M. Mycoplasma gallisepticum from evening grosbeaks (Coccothraustes vespertinus) and pine grosbeaks (Pinicola enucleator) with conjunctivitis in Quebec, Canada. Proc. 49th Ann. Wildlife Dis. Assn. Conf., 91, Abstr. 2000. Moura L, Elankumaran S, Oshop GL, Bautista DA, Wilson JD, Heckert RA. Determination of the most effective route for in ovo delivery of DNA vaccines. Proc. 138th Am. Vet. Med. Assn. Mtg., Boston, MA, July 14-18, 2001. Oshop GL, Elankumaran S, Vakharia VN, Wilson JD, Moura LC, Bautista DA, Heckert RA. In ovo nucleic acid immunization of the chicken against infectious bursal disease and Newcastle disease, Proc. 138th Am. Vet. Med. Assn. Mtg, Boston, MA, July 14-18, 2001. Oshop GL, Heckert RA, Elankumaran S. DNA and mRNA persistence and tissue distribution following topical delivery of a DNA vaccine in chickens. 73rd Northeastern Conf. on Avian Dis., College Park, MD, June 2001. Pang, Yao-shan., M. I. Khan, H. Wang, Z. Xie and T. Girshick. Multiplex PCR and its application in experimentally infected SPF chickens with respiratory pathogens. 50th Western Poultry Dis. Conf.,.March 23-26, Davis, California p. 141. 2001. Paustian, M. L., B. J. May, and V. Kapur. Pasteurella multocida gene expression in response to iron limitation Infect Immun. 69:4109-15. 2001. Pedersen J. C., D. A. Senne, B. Panigrahy and D. L. Reynolds. Detection of avian pneumovirus in tissues and swab specimens from infected turkeys. Avian Dis. 45:581-92. 2001. Pitts, G. R., S. You, D. N. Foster, and M. E. El-Halawani. Evidence for multiple prolactin receptor transcripts in the turkey Poult Sci. 79:355-62. 2000. Qureshi, M. A., Y. M. Saif, R. A. Ali, F. W. Edens, C. L. Heggen-Peay, and G. B. Havenstein. Comparison of PEMS-associated and classical astroviruses-mediated effects on performance and immune functions of turkey poults. Poultry Sci., Vol. 80, supplement 1, abstract 538, 2001. Rajashekara, G., E. Haverly, D. A. Halvorson, K. E. Ferris, D. C. Lauer, and K. V. Nagaraja. Multidrug-resistant Salmonella Typhimurium DT104 in poultry J Food Prot. 63:155-61. 2000. 12 Rajashekara, G., S. Munir, M. F. Alexeyev, D. A. Halvorson, C. L. Wells, and K. V. Nagaraja. Pathogenic role of SEF14, SEF17, and SEF21 fimbriae in Salmonella enterica serovar enteritidis infection of chickens Appl Environ Microbiol. 66:1759-63. 2000. 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