SAES-422 Multistate Research Activity Accomplishments Report

Sections

Status: Approved

Basic Information

Participants

Advisor: Saif, Yehia (saif.1@osu.edu) State Station Representatives: Haroldo, Toro (torohar@auburn.edu),Auburn University; Kong, Byung-Whi (bkong@uark.edu, University of Arkansas; Khan, Mazhar (mazhar.khan@uconn.edu) - University of Connecticut; Gelb, Jack (jgelb@udel.edu), University of Delaware; Jackwood, Mark (mjackwoo@uga.edu, University of Georgia; Tripathy, Deoki (tripath@uiuc.edu), University of Illinoise; Wu, Ching Ching (wuc@purdue.edu), Purdue University; Lee, Chang Won (lee.2854@osu.edu), Ohio State University; Johnson, Tim (joh04207@umn.edu)- University of Minnesota; Zsak, Laszlo (Laszlo.Zsak@ars.usda.gov), USDA, Southeast Poultry Research Lab. Other participants: Keeler, Calvin (ckeeler@udel.edu), University of Delaware; Erin Brannick (brannick@udel.edu), University of Delaware; Joseph Giambrone (giambjj@auburn.edu), Auburn University; Maricarmen, Garcia (gmaricar@uga.edu), University of Georgia; Naola Ferguson-Noel (nferguson@uga.edu), University of Georgia; Lin, Tsang Long (tllin@purdue.edu), Purdue University; Mo Saif (saif.1@osu.edu ), Ohio State University; Pantin-Jackwood, Mary (Mary.Pantin-Jackwood@ars.usda.gov), Yu, Qingzhong (Qingzhong.Yu@ars.usda.gov)-USDA, Southeast Poultry Research Lab.

[Minutes]

Accomplishments

Objective I: Identify reservoirs of infectious respiratory disease agents in wild birds and poultry. 1. Isolation and characterization of avian influenza viruses (AIV) from wild birds and commercial poultry flocks which include live bird markets and backyard flocks were accomplished. The surveillance data obtained from different states (AL, CT, DE) were shared. No AIV activity using USDA NAHLN-approved agent detection (real time RT-PCR and antigen capture on oropharyngeal swabs) were seen in commercial flocks (CT, DE). 2. Surveillance activities on the Delmarva Peninsula have yielded infectious laryngotracheitis (ILT) virus and infectious bronchitis virus isolates from commercial broiler chickens and Newcastle disease virus isolates from wild birds. 3. Delmarva has continued to observed ILT activity in 2010 and 2011. The severity of ILT clinical signs and lesions are mild to moderate, very similar to that seen in adverse CEO vaccine reactions. 4. 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. 5. SEPRL (USDA) obtained virulent Newcastle disease viruses from Mexico, Africa, China, Pakistan, and from U.S. wild birds which have been sequenced and characterized phylogenetically. The sequence data has allowed the improvement of the current diagnostic tests for NDV to ensure that the circulating viruses can be diagnosed. Objective II. Develop improved diagnostic capabilities including real-time PCR as well as other rapid on-farm tests for economically important respiratory diseases. 1. AL developed a TaqMan® real time polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assays. Both assays were specific, sensitive, and reproducible for ILTV detection. Although the sensitivity of LAMP was lower than real time PCR, it was faster, had a lower cost, and did not require a temperature cycler. This was the first report comparing these methods for ILTV DNA detection. 2. CT in collaboration with Guangxi Veterinary Institute, China developed loop-mediated isothermal amplification (LAMP) assays to detect the H3 subtype AIVs visually and rapid detection of group I avian adenoviruses. The newly developed H3-RT and group I avian adenoviruses LAMP assays are simple, sensitive, rapid and can identify H3 subtype AIVs and group I avian adenoviruses visually. Consequently, they will be very useful screening assays. 3. DE, SEPRL evaluated the effect of pooling 11 or 5 oropharyngeal swabbings on detecting avian influenza virus by real time reverse transcription PCR . 4. DE, GA and SEPRL using next generation sequencing technologies for rapid determination of the primary sequence of the infectious laryngotracheitis virus ( ILTV) genome. 5. GA 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. 6. GA optimized duplex real-time PCR method for relative quantification of ILTV. 7. GA developed and validated N1 and N2 ELISAs as the assays that will be required for the implementation of a DIVA control strategy for H5N1, H5N2, H9N2, H3N2 and H1N1 poultry infections that will be required for the implementation of a DIVA control strategy for H5N1, H5N2, H9N2, H3N2 and H1N1 poultry infections. 8. GA developed and validated N1 and N2 ELISAs as the assays that will be required for the implementation of a DIVA control strategy for H5N1, H5N2, H9N2, H3N2 and H1N1 poultry infections. 9. IL developed a photolase gene specific PCR. Based on sequence information, avian pox viruses could be differentiated into four different groups. 10. OH developed 19-plex assay which can differentiate different HA subtypes of avian influenza viruses. Objective III. Investigate the pathogenesis and polymicrobial interactions of specific infectious agents associated with poultry respiratory diseases (this includes interactions with underlying immunosuppressive agents). 1. AL investigated venereal transmission of IBV by artificially inseminating old hens either with semen from IBV infected roosters or with IBV suspended in naïve semen. IBV RNA was detected in the trachea of all hens inseminated with IBV-spiked semen and in 50% of hens inseminated with semen from IBV-infected males. These results provide experimental evidence for IBV venereal transmission. 2. AL investigated that the dominant genotype of the vaccine strain of IBV was rapidly negatively selected in all chicken groups [CAV, IBDV, CAV+IBDV, and immunocompetent]. These results suggest that the generation of genetic diversity in IBV is constrained. This finding constitutes further evidence for phenotypic drift occurring mainly as a result of selection. 3. GA examined and compared the genomes of pathogenic and attenuated strains of IBV and measure evolution of IBV by examining virus diversity and mutation rates. 4. GA investigated embryo lethal dose50 for a reliable indication of the virulence of MG isolates. The ELD50 of ts-11 like isolates tested correlated well with the history and previous pathogenicity testing. 5. IL genetically characterized a vaccine strain of fowlpox virus showed complete homology with the corresponding gene sequences and indicated absence of full-length REV in the genome of this virus. 6. MN identified molecular mechanisms enabling APEC to survive and grow in this critical host environment. 7. OH investigated the replication of swine and human influenza A viruses in juvenile and layer turkeys. 8. OH studied maternal immunity in limiting the spread or reducing the severity of the clinical disease caused by very virulent infectious bursal disease virus (vvIBDV). 9. OH studied amino acid sequence data acquired from sequencing of IBDV collected from bursa samples during a ten year period from 2002  2011. 10. OH investigated Fas/FasL and perforin systems as important mechanisms of T cell-mediated cytotoxicity in infectious bursal disease virus infected chickens. 11. SEPRL evaluated the pathogenicity of H5N1 HPAI viruses isolated in Egypt in domestic ducks. 12. SEPRL characterized pathogenicity of new NDV viral isolates from South America, Africa and US waterfowl in chickens. Objective IV. Develop new prevention and control strategies for poultry respiratory diseases. 1. AL evaluated protection conferred by mucosal vaccination with replication competent adenovirus (RCA)-free recombinant adenovirus expressing a codon-optimized avian influenza (AI) H5 gene fromA/turkey/WI/68 (AdTW68.H5ck). 2. AL developed a DNA vaccine consisted of the entire HA gene of an AIV H1N1 subtype (A/bluewinged teal/ AL/167/2007) cloned into the eukaryotic expression vector. The immunological responses induced by DNA vaccine against AIV were also investigated. 3. AR made comparison of ILTV genome sequences of two US CEO vaccines. 4. CT evaluated the level of protection of M2e-nanopartle based vaccine using quantitative real time PCR at 4, 6, and 8 days post-challenge with H5N2 LPAI by measuring virus shedding from trachea and cloaca. 5. IN conducted studies to determine if the combination of chicken calreticulin (CRT) gene and infectious bursal disease virus (IBDV) large segment (VP243) gene in a fusion gene or a chimeric DNA could enhance protection against IBD by DNA vaccination. 6. MN Correlated between virulence and MDR in avian E. coli and characterized the biology of the emergent IncA/C plasmid group. 7. OH in collaboration with the University of Cincinnati utilized flexible norovirus P particle as a novel influenza vaccine platform in vitro analysis of virus particle subpopulations in candidate live-attenuated influenza vaccines which could distinguish effective from ineffective vaccines. 8. SEPRL showed that a single vaccination can protect ducks and geese from avian influenza virus if the virus and vaccine are related. Reduction of pandemic H1N1 avian influenza growth with use of chicken interferon was investigated. 9. SEPRL generated and evaluated a bivalent vaccine against avian metapneumovirus and Newcastle disease viral diseases.

Impacts

  1. Avian influenza subtype H5 and H7 were negative from the LBM and domestic poultry birds in New England states and in Delaware commercial farms. However wild birds do carry H5 subtypes in their population.
  2. Infectious laryngotracheitis virus and infectious bronchitis virtues circulating in commercial broiler chickens flocks in Delaware.
  3. Molecular Epidemiology reinforces the importance of surveillance for MG and MS isolates in poultry for the control of avian mycoplasmas.
  4. The sequence data has allowed the improvement of the current diagnostic tests for NDV to ensure that the circulating viruses can be diagnosed.
  5. A new diagnostic tests developed for ILTV, AIV and avian adenoviruses using loop-mediated isothermal amplification (LAMP) techniques will be faster, specific, sensitive and cost effective will not require sophisticated equipment.
  6. Utilization of next generation sequencing technologies now permits the relatively rapid determination of the primary sequence of the ILTV genome.
  7. Multiplex microsphere assay for detection of avian influenza viruses provides a rapid tool to identify multiple avian influenza types in the same sample.
  8. Development of faster high-throughput serological assays for avian influenza (AI) that can complement a vaccination strategy to allow the rapid identification of infected flocks within large populations of vaccinated poultry. Identification of infected flocks is critical for control of AI outbreaks especially when vaccines are used.
  9. Successfully developed 19-plex assay which can differentiate different HA subtypes of avian influenza viruses. With the multiplex capacity and feasibility of the assay, the multiplex branched DNA assay has a great potential in influenza research in addition to rapid diagnosis.
  10. The egg internal and external quality was negatively affected in hens inseminated with semen containing IBV. These results provide experimental evidence for IBV venereal transmission.
  11. Chickens infected with IBV and co-infected with CAV+IBDV will generate genetic diversity in IBV. This finding constitutes further evidence for phenotypic drift occurring mainly as a result of selection.
  12. Poor vaccination against IBV infection contributes to the emergence of new IBV strains via mutation and/or selection. Under these conditions only IBV virus populations identical to the challenge virus was identified. From a broad perspective it indicates that selection is an important force driving IBV evolution.
  13. Examine and compare gammacorona virus genomes for recombination, comparison data indicate that reticulate evolutionary change due to recombination in IBV, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus. These data constitute a significant step forward in identifying pathogenicity genes in avian coronavirus infectious bronchitis.
  14. In vitro expression of avian pathogenic Escherichia coli (APEC ) genes . This genome-wide analysis provides novel insight into processes that are important to the pathogenesis of APEC O1. Overall, these results indicate that a number of novel APEC virulence factors exist in APEC O1 that mediate systemic infection in the chicken host.
  15. It was confirmed the susceptibility of both juvenile and layer turkeys to swine influenza viruses (SIVs) while the viruses replicated more efficiently in the reproductive tract of turkey hens compared to respiratory or digestive tracts.
  16. Studies indicate the ability of vvIBDV to infect chickens is not affected by maternal immunity to IBDV strains typically found in commercial U.S. chickens. However maternal immunity did reduce the severity of the clinical signs and macroscopic lesions. These data suggest vvIBDV might be infecting chickens in California and other regions of the U.S. but they are going unnoticed because maternal immunity affects the clinical picture which does not include mortality and macroscopic lesions typical of a vvIBDV infection.
  17. Data indicated that activated T cells may be involved in antiviral immunity and mediation of virus clearance from the bursa and spleen of IBDV-infected chickens. The findings of this study will help understanding the role of T cells in the pathogenesis of IBDV and designing effective control strategies against this immunosuppressive viral disease of chickens.
  18. An increase in pathogenicity of AI in ducks observed with H5N1 HPAI viruses has implications for the control of the disease since vaccinated ducks infected with highly virulent strains shed more viruses and for longer periods of time, perpetuating the virus in the environment and increasing the possibility of transmission to susceptible birds.
  19. Further comparison of US CEO vaccines to several ILTV genome sequences revealed that US CEO vaccines are genetically distinct from the two Australian-origin CEO vaccines, SA2 and A20, which showed close similarity. This information can be used to discriminate between vaccine ILTV strains and further, to identify newly emerging mutant strains of field isolates.
  20. Preliminary studies suggest that the self-assembling polypeptide nanoparticle shows promise as a potential platform for a development of a universal vaccine against avian influenza type A.
  21. Experimental studies indicated that live vaccines and bacterian can protect against ovarian regression as well as air sac and tracheal lesions.
  22. It was shown that recombinant vaccines against ILTV provide some protection but do not prevent shedding, which can lead to continued spread of the virus, whereas the chicken embryo origin vaccine protected against both disease and virus shedding. This study is extremely important in the control of ILTV especially in the face of an outbreak.
  23. Determining the unique sequences for chicken embryo origin (CEO) vaccines will enhance our ability to control the re-emerging epidemics ILTV in commercial chickens caused by CEO-related vaccines.
  24. Study validated that the use of glycoprotein specific ELISAs as a tool to discriminate ILTV sero-conversion due to vaccination from infection. This work involves the serological differentiation of vaccinated and field virus exposed chickens which is critical for controlling ILTV epidemics.
  25. IBDV large segment gene-based DNA can elicit specific immune response and provide protection of specific-pathogen-free and broiler chickens against infection challenge. The impact is that IBDV large segment gene-based DNA vaccine has the potential for practical application in providing protection of chickens against IBD in the poultry industry.
  26. Evidence is mounting that IncA/C plasmids are widespread among enteric bacteria of production animals and these emergent plasmids have flexibility in their acquisition of MDR-encoding modules, necessitating further study to understand the evolutionary mechanisms involved in their dissemination and stability in bacterial populations.
  27. Studies demonstrate that chicken interferon is biologically active against the pandemic H1N1 virus, is active in other avian species, and may be useful as therapy against avian influenza infection.
  28. Potential bivalent recombinant vaccine candidate for NDV and aMPV was safe, stable and provided a complete protection against virulent NDV challenge and decreased the aMPV disease severity following experimental aMPV-C infection in turkeys.

Publications

[Publications]
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