Brief Summary of Minutes of Annual Meeting

BRIEF SUMMARY OF MINUTES OF ANNUAL MEETING. The annual NC 1019 business meeting was held on Tuesday, January 24, 2007, in room C211 in the Georgia World Congress Center, Atlanta, GA. Dr. David Suarez, Chair of NC 1019 opened the meeting at 12:30 pm. He welcomed the committee advisor Dr. Jeff Klausner, guests Dr. John Glisson from Georgia, Dr. B. Kong from Arkansas, Dr. L. Zsak from USDA/ SEPRL, and Dr. T. L. Lin from Indiana. Dr. Peter Johnson, USDA/CSREES representative, joined the group around 3 pm. Dr. Klausner reminded the Chair and Secretary that the minutes of the meeting should be submitted to his office within 30 days after the meeting is held. Dr. Peter Johnson briefed the group with the CSREES budget. The budget review panel has not met yet but some cuts will occur. Dr. Johnson also discussed with the group on the research priority list for 2006 NRI. He indicated one out of the three agents recommended by NC 1019 was included in the published priority list and that CSREES will continue to solicit input from federal, state and local partners to guide competitive programs. He requested the NC 1019 multi-state committee to again provide consensus feedback on the research priorities for 2007. In response to Dr. Johnsons request, the group discussed and prioritized diseases of importance. While there are many important diseases, three came to the top for this year: ILT>IBDV>APV. Both avian influenza virus and Newcastle disease virus are typically covered in the foreign animal disease priority, they were not included in the recommendations for the priority list. Drs. David Suarez and Ching Ching Wu will prepare a feedback letter to Dr. Johnson reflecting our consensus. NC 1019 members reached the consensus on the importance to bring industry guests and renowned speakers to the committee meeting periodically and as needed. Possible funds and revenues to support this activity were discussed. The meeting venue for the next year was discussed and it was agreed to meet in Atlanta next year in association with the International Poultry Exposition, but we will likely hold it at a hotel to provide more flexibility on times to meet. The exact date has not been set, but hopefully that will be determined soon. Drs. Giambrone and Wu will finalize the detail. Dr. Wu will check out the cost of renting meeting room at local hotels and Drs. Glisson and Giambrone will find funds to cover the room. Dr. Giambrone will inform the members of the specifics as soon as they are available. The members will reassess the meeting place in 2008. The meeting will remain closed but the Chair and Secretary can invite guests per members suggestion or as needed. The group will continue to actively recruit other stations to participate. Dr. Suarez, after completing his two year term of office, decided not to seek renewal of his Chair position. After polling the membership by email and at the meeting, Dr. Joseph Giambrone was nominated for the chair position and Dr. Ching-Ching Wu for secretary position. No additional nominations were offered from the floor, and Drs Giambrone and Wu were elected by unanimous consent. The members expressed their appreciation for the leadership that Dr. Suarez provided during his term. The business meeting end at 4:00 pm Jan 23, 2007. Station reports followed and stopped by 5:30pm. Station progress report resumed at 7:30 am Wed., Jan. 24, 2007. We had excellent discussion on the clinical aspects, pathogenesis, diagnostics, and vaccine/immunology of various poultry disease research among the stations. At the end of the meeting, Dr. Suarez requested that each station submit their annual report and the collaboration records electronically to him ASAP. The annual meeting was adjourned at 11am, Jan. 24, 2007. WORK PLANNED FOR THE COMING YEAR University of Connecticut: Development of Avian Influenza vaccine using reverse genetic system. Reverse genetics methods, i.e., methods that allow the generation of an influenza virus entirely from cloned cDNAs, have provided us with one means to address these issues. This technique allows customized construction of influenza vaccine by assembling genes that code for the desired features of the particular virus strain. We plan to use reverse genetic in order to develop a vaccine for the H7N2 strain of Avian Influenza and expect that this vaccine will be able to protect chickens against this specific low path virus infection. Infectious Bronchitis: Efficiency of recombinant IBV DNA vaccine developed earlier in our laboratory is in progress. University of Delaware: 1)We plan to continue work on AIV, IBV and ILTV . 2)Mycoplasma gallisepticum will involve confocal microscopy to more accurately evaluate the intracellular invasion in Chick Embryo Fibroblasts using MG-685 and additional strains of MG. Auburn University: The objective is to develop transgenic vaccines for poultry against AIV. There are a number of commercially available, efficacious vaccines against HP H5 AIV for use in poultry. However, they must be injected. The injection system is suitable for smaller breeder and layer flocks, which are routinely injected with killed vaccines, but is too costly for larger broiler flocks. In contrast, vaccines in plants or yeast could be propagated and given in mass to poultry. Yeast are routinely given in the drinking water as a probiotic, in place of antibiotics to kill bacteria, and plants can be given in feed to commercial poultry. The Ohio State University: 1. Completing the antigenic and genetic relatedness work using the newly obtained human and duck viruses H5 AIVS. 2. Sequencing and comparing the HA and NA genes of the different isolates of TK/OH/03 virus from the transmission studies, to see how the virus changes upon replicating in different host systems. 3. Start cloning all the genes of: TK/IL/04, TK/OH/03, SW/NC/03 and HUM/OH/06 for the use in reverse genetics studies. 4. Studies will be conducted on the molecular evolution of IBDV in the United States. 6. The molecular basis for antigenicity in IBDV strains will be further studied using a reverse genetics system. 7. More intensive animal study using a large number of eggs and chickens will be conducted to examine the potential of each NS variant as live attenuated vaccine candidates for AIV. 8. Microsphere-based influenza diagnostic assay will be validated by comparing side by side with those obtained using traditional virus isolation and RRT-PCR. Purdue University: We will examine the pathogenesis of IBDV using reverse genetically engineered strains for the purpose of understanding the molecular events and mechanisms by which the virus interacts with bursa of Fabricius. We will also continue to study the effect of prime-boost on protection of chicken by DNA vaccination against IBD, such as boosting with transgenic algae expressing IBDV VP2. Additional studies on chicken cytokine genes (other than chicken interferon-r gene and chicken IL-2 gene) on immune response and protection of chickens against IBD by DNA vaccination. University of Minnesota: 1)Examine the feasibility of inducing protective immunity by respiratory exposure to inactivated virus or viral subunits. 2)Define the functional role of the G gene in aMPV propagation by modifying important amino acid differences by site-directed mutagenesis. 3)Identify the virulence gene of aMPV and create a safe by reverse genetics. 4)Define the role of macrophages in the immunopathogenesis of aMPV and IBDV. Minutes Submitted by C.C. Wu

Objective 1. Determine the pathogenesis and interactions of specific agents. Avian Influenza Research to determine the resistance and susceptibility of various poultry lines against AIV infection was conducted with an emphasis on the MX gene. Mice that express high levels of the MX gene are resistant to AIV and VSV challenge. Chickens have an MX gene, but it is unknown the gene provides similar resistance to chickens. We determined that there is polymorphism in the expression of the Mx gene in various chicken breeds. The amino acid residue 631, at which, Asn determines antiviral activity, and Ser renders the MX protein inactive. We've spent most of the year developing reagents (an expression vector for chicken interferon-alpha from another scientist, transfecting it, and characterizing the titer of the interferon). Also we've have refined our assays for CEF preparation, developing faster methods of Mx typing, developing quantitative PCR for Mx expression, and evaluating replication by ELISA. Now we have most all our tools and are ready to do animal experiments, and plan to correlate MX expression of various breeds with susceptibility of CEFs from their eggs to AIV replication. (Auburn U) The interspecies transmission of different H3N2 influenza viruses between turkeys and swine were examined in an aerosol transmission experimental model. Of the viruses tested viruses, TK/IL/04 (H3N2); TK/OH/03 (H3N2); TK/NC/03 (H3N2); SW/NC/03 (H3N2); A/TK/OH/88 (H1N1) and A/SW/OH/06 (H1N1), only TK/OH/03 efficiently transmitted from pigs to turkeys (replicated for two days or more in both species). All pigs seroconverted with mean HI titer of 1:360 and 50% of turkeys seroconverted with mean HI titer of 1:80. The TK/IL/04 and TK/NC/03 replicated well in pigs, but were detected in turkeys for only one day. Only pigs seroconverted. A/TK/OH/88 and A/SW/OH/06 replicated efficiently in pigs but were not detected at all in turkeys. Only pigs seroconverted. In the reverse experiment of transmission from experimentally infected turkeys to pigs four viruses were compared, TK/IL/04 (H3N2); TK/OH/03 (H3N2); TK/NC/03 (H3N2) and SW/NC/03 (H3N2). In this experiment, the TK/OH/03 was also the only virus that efficiently transmitted from turkeys to pigs. Both species seroconverted at HI titers of 1:344 and 1:320 respectively. None of the other viruses were transmitted. The TK/OH/03 influenza virus was also inoculated into chickens, and ducks. The virus replicated in the inoculated chicken for 5 days but was not detected in the contact chicken. Infected chickens seroconverted but the contact ones did not. The virus could not be detected in either inoculated nor contact ducks. (OhioStateU) The first comprehensive biological characterization of H5N1 high pathogenicity avian influenza (HPAI) virus from wild birds were completed on viruses that came from Mongolia. H5N1 HPAI virus has caused outbreaks of disease in poultry and wild birds of 50 countries in Asia, Europe and Africa. With field assistance of Wildlife Conservation Society, Food and Agricultural Organization and Government of Mongolia, H5N1 HPAI viruses were isolated from a dead Whooper Swan in Mongolia. In experimental studies, this virus expressed high lethality in chickens and young domestic ducks, and was easily passed between ducks by causal contact. The virus grew in many internal tissues including the brain and heart. Because the outbreak occurred where no poultry exist, this indicates the H5N1 HPAI virus spread into Mongolia by migrating wild birds, but is a virus that can infect poultry and cause severe disease. (SEPRL) We provided an in-depth analysis, including sequencing and animal studies, of the first highly pathogenic avian influenza virus (HPAIV) in the U.S. in the last 20 years. An outbreak of avian influenza occurred in Texas in 2004 that had the sequence of a highly pathogenic avian influenza virus, but it was not highly pathogenic when it was used to infect chickens. Our laboratory in collaboration with the National Veterinary Services Laboratories, APHIS provided in depth sequence analysis, animal studies, and mutational studies to try and understand why the sequence and animal studies did not match together. The data helped to provide evidence that the sequence definition of highly pathogenic avian influenza needs to be reconsidered based on this and other exceptions to the O.I.E. rules. It remains critical, because of the sever affects in trade, to accurately diagnosis and report correctly any HPAI outbreaks. (SEPRL) A pathogenesis experiment was performed to examine the interactions of different immunosuppressive agents and infection with a low pathogenic avian influenza virus. Two experiments were performed to evaluate the effects of exposure of chickens with and without maternal antibodies to chicken anemia virus (CAV) and infectious bursal disease virus (IBDV) to low path AIV challenge. In the first experiment, the infection of commercial broiler chickens at different ages with CAV and IBDV did not affect (enhance or decrease) the pattern of low path H7N2 AIV (A/chicken/Maryland/Minh Ma/2004) detection (virus isolation and real time RT-PCR) in tracheal swabs following challenge on day 21 days of age or in the serum antibody responses (AGID. ELISA, and HI). The second experiment was set up similarly to the first experiment except that specific pathogen free leghorn type chickens were used instead of commercial broilers. Because of the lack of maternal antibody, a 60% mortality occurred in the SPF leghorns concurrently infected with IBD and CAV prior to the planned 21 day challenge with the LP H7N2 AIV, so this treatment group was not used in the trial. Remaining CAV only and IBDV infected only birds and controls were challenged intraocularly with the H7N2 virus at 21 days of age. No affect (enhance or decrease) on the pattern of low path H7N2 AIV detection (VI or RRT-PCR)) in tracheal swab was observed between treatment groups. Compared to AI challenge controls, SPF leghorns infected with IBDV had diminished AIV antibody titers determined by HI and ELISA, as well as fewer positive AGID responders. Infection with CAV reduced HI antibody titers but had no effect on ELISA and AGID antibody responses. The results of this experiment show that early infection with CAV or IBDV reduces AIV serological responses in response to a later challenge, but not likely enough to prevent detection by standard test. (U Delaware) Avian Metapneumovirus (AMV) Turkeys exposed to aMPV showed extensive lymphoid cell infiltrations in the upper respiratory tract (URT). The cellular infiltration occurred after the first virus exposure but not after reexposure. Quantitation of the relative proportions of mucosal IgA+, IgG+ and IgM+ cells in controls and virus-exposed turkeys revealed that at 7 days following the first virus exposure, when mucosal infiltration was well pronounced, there was a significant increase (P<0.05) in the numbers of infiltrating IgA+ but not of IgG+ and IgM+ cells. Following the second virus exposure, although the overall numbers of mucosal lymphoid cells were similar in the virus-exposed and control turkeys, the relative proportions of IgA+ and IgG+ cells were significantly higher in the virus-exposed turkeys (P<0.05) than in controls. Further, elevated levels of aMPV-specific IgA were detected in the nasal secretions and the bile of virus-exposed birds after the second but not after the first virus exposure. This result suggested, for the first time, the possible involvement of local mucosal immunoglobulins in the pathogenesis of aMPV in turkeys. (U Minnesota) Two respiratory adjuvants at three dose levels (10¼g, 20 ¼g , 40 ¼g or 60¼g) were tested: poly(I:C) and holotoxin-containing cholera toxin B (hCTB). One-day-old turkeys were given daily intranasal injections of each adjuvant for 6 days. Neither of the adjuvants caused detectable gross or microscopic lesions in the URT or lasting loss in body weight gain. This result indicated that two of the most commonly used respiratory adjuvants were safe for turkeys. Poly(I:C) and hCTB were given intranasally or oculonasally alone or in combination with inactivated aMPV. IgA cells were enumerated in the turbinate tissue of treated and untreated turkeys. Poly(I:C)+inactivated aMPV group had higher numbers of IgA+ cells than the untreated group or the groups given Poly(I:C) or inactivated aMPV alone. (U Minnesota) The availability of the complete genome information is essential for development of a reverse genetics system to study the molecular biology and rescue infectious ampv from cloned cdna. Therefore, we determined the nucleotide (nt) sequence of the complete genome of ampv-c colorado strain (ampv-c-co) propagated in vero cells in our laboratory (here designated as seprl variant). The full-length genome is comprised of 13,136 nt encoding eight genes, a 40 nt leader at its 3 end and a 45 nt trailer at its 5 end. It is two nt longer than the ampv-c-co strain propagated in the university of minnesota (umn variant, lwamba et al., 2005), and 1,014 nt shorter than the same strain of virus propagated in the university of maryland (umd variant, govindarajan and samal, 2005). The significant difference in length between these variants was found in the coding region of the g gene, where the seprl and umn variants were 1,015 nt or 333 amino acids (aa) shorter when compared with the umd variant. In addition, there were 23 nt differences scattered along the genome of the variants. Nine of them resulted in eight aa coding changes in five genes, three of which were located in the l gene. Based on the genomic sequence of the seprl variant, we developed a reverse genetics minireplicon system using a green fluorescence protein (gfp) gene as a reporter, which allowed us to assess the effects of coding differences in the l gene on viral gene expression. It was found that one of the coding differences (position 1371 leu vs phe) in the rna-dependent polymerase l gene was critical for the polymerase functionality. (SEPRL) Adherent cells from spleen, bone marrow and the circulation of normal turkeys were cultured in vitro. After 7 days, the cells were inoculated with the 63rd Vero cell passage of subtype C aMPV. At 96 hours following exposure, viral genome was detected by RT-PCR in the RNA extracts of virus exposed cells. Immunohistochemistry staining of the cells revealed the presence of intracellular viral proteins. Virus-exposed adherent cells had upregulation of nitric oxide production, iNOS gene and genes of several proinflammatory cytokines and chemokines. These results indicated that turkey macrophages were susceptible to infection and activation by aMPV63. (U Minnesota) The aMPV G protein is a major determinant for distinguishing virus subtypes and different lengths (and resulting changes in the predicted amino acid and nucleotide sequences) have been reported. Sequence analysis revealed that the complete 1.8kb G gene was found when aMPV was propagated in our immortalized turkey turbinate (TT-1) cells. In contrast, Vero cell propagated aMPV revealed an essentially deleted G gene in the viral genome, resulting in no G gene mRNA expression. The lack of expression was confirmed by Northern blot hybridization. As expected, viral G gene mRNA was not detected in the Vero-aMPV at any time post infection (p.i.), while the TT-1-aMPV showed increased levels of G gene mRNA in a time-dependent manner post infection. Both the TT-1-aMPV and Vero-aMPV templates were examined for the existence of splicing mRNA variants containing any partial G gene fragments using an RNase protection assay. After RNase digestion, the TT-1-aMPV showed a single mRNA transcript of approximately 1.8kb, while the Vero-aMPV did not show any detectable G gene fragments. The functional role of viral genes may be different depending on the species of the cellular host substrate. While the G protein may function as a key attachment protein in TT-1 cells, it appears not to be required for Vero cell infection. (U Minnesota) Infectious Bronchitis Virus From 1997 to 2002, the ARK type of IBV was the most dominant strain isolated from birds with respiratory disease submitted to the AL State Lab in Auburn. S1 gene analysis and challenge studies of these isolates indicated that they were closely related to vaccine strains. Experimental IBV vaccine and challenge studies with CAV and IBDV infections indicated that these 2 pathogens compromised IBV vaccine efficacy. (Auburn U) Newcastle Disease Virus A system to make Newcastle disease viruses with specific genetic changes was established to study Newcastle disease virus (NDV) infection in chickens. Different NDV strains cause variable clinical disease in chickens that ranges from severe to mild or in some cases inapparent infections. We have constructed a full-length copy of the genome of the NDV anhinga strain by combining each of the virus genes in an artificial system that allows virus replication to generate a virus that can be propagated and infect chickens just like the original field isolate. This system is being utilized to replace NDV anhinga genes with genes from other NDV strains that cause different clinical outcomes to identify which genes are important in controlling the severity and form of the clinical disease resulting from an NDV infection. The findings from application of this system will impact vaccine development and the identification of the role of the genes controlling the different clinical disease forms may impact other control strategies. (SEPRL) Domestic pigeons and other hobby birds can be infected with exotic Newcastle disease (END) virus, and this presents a risk of spread of the virus to poultry. The potential role of racing pigeons in that dissemination was examined by evaluating their susceptibility to infection and disease. Susceptible and Newcastle disease (ND) vaccinated pigeons were infected by eye drop and intranasally with an END virus isolate recovered during the 2002-03 END outbreak in the Southwestern U. S. Pigeons were readily infected and shed virus from both the respiratory and intestinal tract, but they were more resistant to disease with a virus dosage that would cause high mortality in chickens. Vaccination reduced the virus shed from infected pigeons and thereby reduced but didnt eliminate the risk of transmitting virus to other birds. The results provide a basis for establishing regulations concerning the vaccination as well as the movement and flying of racing pigeons in a quarantine zone during an END outbreak. (SEPRL) Infectious Bursal Disease Virus SPF chickens were exposed to virulent IBDV and bursal adherent cells were examined by immunohistochemisrty and RT-PCR for virus infection and by real-time quantitative RT-PCR (qRT-PCR) for mRNA transcripts of proinflammatory cytokines and iNOS. Viral genome was detected in bursal macrophages at 3, 5 and 7 days post-infection (dpi). Immunohistochemical staining revealed double positive cells for KUL01 (macrophage marker) and intracellular viral proteins, showing viral replication in bursal macrophages of infected chickens. We noted a significant decrease in the total number of bursal macrophages in infected chickens, probably due to the lysis of infected cells Inflammatory cytokines (IL-6, IL-1b and IL-18) were upregulated. These data suggested that B cells may not be the sole targets for the virus; macrophages and possibly other cells may serve as host for IBDV. (U Minnesota) Infection with infectious bursal disease virus (IBDV) causes activation of macrophages, the key cells involved in inflammatory and immune-regulatory functions. Exposure of cells of avian macrophage line, NCSU and cultured spleen macrophages (SM) from SPF chickens to IBDV resulted in the production of nitric oxide (NO). In addition, there was upregulation of gene expression of inducible nitric oxide synthase (iNOS), IL-8 and cyclooxygenase-2 (COX-2). The signal transduction pathways involved in macrophage activation were examined. The role of mitogen- activated protein kinases (MAPKs) and nuclear factor-ºB (NF-ºB) was tested by using specific pharmacological inhibitors. Addition of p38 MAPK inhibitor, SB-203580, and NF-ºB inhibitor Bay 11-7082, suppressed IBDV-induced NO production and mRNA expression of iNOS, IL-8 and COX-2. The results suggest that IBDV uses cellular signal transduction machinery, in particular the p38 MAPK and NF-ºB pathways, to elicit macrophage activation. The increased production of NO, IL-8 and COX-2 by macrophages may contribute to bursa inflammatory responses commonly seen during the acute IBDV infection. (U Minnesota) Objective 2. Surveillance, occurrence and consequences of agents and host variation on disease susceptibility. Avian Influenza The surveillance of commercial poultry, backyard poultry, wild birds and live poultry markets in New England for avian influenza continues as part of a USDA initiated program. A summary of results for the sampling period of Jan 1, 2006 to Dec 31, 2005 are as follows: 1632 samples were tested by real-time RT-PCR (RRT-PCR) from birds from live bird markets (LBMs) and backyard flocks; 1723 wild bird samples were tested by RRT-PCR; 3,779 blood samples from birds from LBMs were tested serologically. All samples were negative for avian influenza. Additional samples from LBMs were also tested by virus isolation in embryonating chicken eggs. (UConn) Surveillance of 200 samples from non-migrating and migrating wild water fowl for AIVs from Alabama, Georgia and Florida were conducted in 2006 using both net caught ducks and hunter killed ducks. Samples were from non-migrating wood ducks, and migrating hooded mergansers, blue-winged teal, gadwall, and ring-necked ducks. Twenty samples were from net caught adults ducks, whereas the rest were hunter killed ducks. Six of the samples produced HA positive results. However, only one sample was positive for AIV using real time RT-PCR and antigen capture ELISA. This sample was sent to the NVSL in Ames, Iowa and found to be a low pathogenic H10N7. We isolated and amplified the H10 gene and it is being sequenced. (Auburn U) Four viruses, three from turkeys and one from swine, were tested for their antigenic relatedness using Hemagglutinin Inhibition (HI) test and Virus Neutralization (VN) test in cell culture. The viruses are: TK/IL/04 (H3N2); TK/OH/03 (H3N2); TK/NC/03 (H3N2) and SW/NC/03 (H3N2). The formula of Archetti and Horsfall was employed to express the antigenic relatedness of the different isolates. Results showed that turkey isolates are highly related (71-100 % similar), however the swine isolate was distantly related from the others (< 30% similar to the turkey isolates). The genetic analysis revealed a high degree of similarity between the turkey virus isolates which were less similar to the swine isolate. All eight genes were more than 99% similar between the three different turkey isolates, however, genes from swine isolate were 94-96% similar to the turkey isolates genes. (Ohio State U) We conducted monitoring for avian influenza viruses in poultry and wild birds from samples from the U.S. and around the world. Avian influenza viruses are present in various wild birds and poultry throughout the world. Southeast Poultry Research Laboratory worked with several laboratories to monitor and study avian influenza viruses. No viruses were identified by molecular tests or were isolated from wild birds in Tunisia and Canada Geese in the USA. Some avian influenza (AI) viruses and Newcastle disease viruses (NDV) were obtained from samples from Iraq, Yemen and Nigeria. The Iraqi viruses included both virulent and non-virulent NDV, but antibodies to low pathogenic AI H9N2 viruses were detected. The Nigerian viruses were H5N1 high pathogenicity AI viruses and virulent Newcastle disease viruses. A virulent Newcastle disease virus was isolated from the Yemeni samples. These studies emphasize the need to continue to monitor poultry and wild birds worldwide for AI virus and NDV. (SEPRL) Avian influenza virus (AIV) surveillance in poultry (commercial and backyard) and wild birds is ongoing at University of Delaware's Lasher Laboratory and Allen Laboratory, respectively. No AIV activity using USDA NAHLN approved agent detection (real time RT-PCR and antigen capture on oropharyngeal swabs) or antibody detection assays was observed in over 15,000 active (pre-slaughter) or passive (clinical disease cases) surveillance samples in commercial broilers. One backyard duck flock was found to be positive by real time RT PCR. Wild bird surveillance was initiated at the Allen Lab in Newark in October 2006 in cooperation with the Delaware Department of Natural Resources & Environmental Control. Testing has yielded many real time RT-PCR positive cloacal samples. Only one H5 sample was identified but was determined by NVSL to be a non-Nl neuraminidase subtype. Virus isolation attempts on these samples are now being performed. Our ongoing collaboration with Dr. Richard Slemons (Ohio State University) and his research group yielded several AIV isolates from waterfowl and shorebirds from the Delmarva Peninsula region. The isolates will be characterized in poultry in laboratory trials. (U Delaware) Infectious Bursal Disease Virus (IBDV) Infectious bursal disease virus (IBDV) exists in several different antigenic and pathogenic forms. The immune suppression caused by this virus in young chickens is not always associated with clinical signs of disease. The antigenic Variant viruses originally described in the United States, typically do not cause clinical signs of disease but can cause a marked immune suppression via the destruction of B lymphocytes. Using a reverse-transcriptase polymerase chain reaction (RT-PCR) assay we conducted a survey of asymptomatic chicken flocks in Europe for IBDV. Restriction fragment length polymorphisms in the VP2 gene of four viruses from Spain and four viruses from France indicated they may be different from the Classic and very virulent (vv) IBDV strains found throughout Europe. Nucleotide sequence and phylogenetic analysis of the hypervariable region of the VP2 gene indicated that all eight viruses were more similar to U.S. Variant viruses than Classic viruses. In two viruses, one from France and one from Spain, Threonine was observed at amino acid position 222 and Serine was found at position 254. These two substitution mutations are characteristic of the Delaware Variant viruses. In addition, all eight viruses had mutated amino acid position 318 from Glycine to Aspartic acid; another substitution mutation commonly found in U.S. Variant viruses. Although importation restrictions prevented us from directly testing the antigenicity of these viruses, their nucleotide and predicted amino acid sequences strongly suggest they may be antigenically unique compared to Classic and vvIBDV commonly found in Europe. (Ohio State U) Infectious Bronchitis Virus Routine virus isolation attempts from respiratory disease accessions from Delmarva commercial broiler chickens yielded four isolates of a variant of IBV based on S1 gene sequencing. This variant (Fig. 1) is similar to a 2004 cecal tonsil isolate (K0401737 ct) from commercial broilers in California, recovered by Dr. Peter Woolcock's laboratory and sequenced by Dr. Mark Jackwood. The potential role of this variant to cause disease in vaccinated or unvaccinated chickens under laboratory conditions has not been established. Other field isolates from broilers were Arkansas, Massachusetts or Connecticut S 1 genotypes. (U Delaware) Objective 3. Develop new and improved methods for the diagnosis, prevention, and control of avian respiratory diseases. Avian Influenza Real time multiplex RT-PCR for avian influenza for subtypes H5, H7, and H9 and multiplex PCR or RT-PCR tests for Mycoplasmas and Infectious bronchitis infections were developed at the Guangxi Veterinary Research Institute Nanning, China. Plans are to continue to optimize the test and collaborate with the National Veterinary Services Laboratory (Ames, IA) and other experiment stations to test the new protocols. (UConn) A replication-defective adenovirus recombinant vaccine to protect chickens against avian influenza virus was developed that encoded the hemagglutinin gene from the low pathogenic avian influenza virus Turkey/Wisconsi/68 H5N9. The vaccine was given to SPF leghorns in ovo or at day of age by SQ route. The AdTW68.H5 vectored vaccine induced measurable HI (log29) titers against the LP turkey H5N9, but no titers using IDEXX ELISA, when given in ovo at 18 days or SQ at day of age. Thirty one day old vaccinated birds were challenged at the USDA SEPRL lab in Athens, GA with either the Mongolian HP H5N1 (89% hemagglutinin sequence homology) or Mexican H5N2 (94% hemagglutinin sequence homology) AIVs. The vaccine induced 68 % protection against the Asian and 100% against the Mexican virus. (Auburn U) Live-virus vaccines have distinct advantages over inactivated vaccines such as triggering mucosal immune responses and inducing a cell-mediated immunity, which may give the animal a more cross-protective and longer-lasting immunity. From the TK/OR/71-del (H7N3) virus, we previously found that several variants with different sizes of the NS gene can be generated by serial passage of the virus in embryonating chicken eggs. To create a H5 vaccine strain (since the selected variants are H7 subtype) that contains the selected NS gene, we utilized a traditional reassortment method. Briefly, D-del var1 and TK/WI/68 (H5N9) viruses were co-infected into 10-day-old embryonating eggs for reassortment. After 48 hrs of co-infection, infectious allantoic fluid was harvested, followed by intensive plaque purification of derivatives in CEF cells. Individual clones were examined for their gene composition by RT-PCR and sequencing. We obtained a H5-D-del-v1 variant which has the NS gene of D-del var1 and other remaining genes of TK/WI/68 virus. (Ohio State U) We are developing a microsphere-based multiplex assays as an alternative to RRT-PCR for the detection and subtyping of H5 and H7 subtype avian influenza virus. To accomplish this, we utilized branched DNA (bDNA) signal amplification technology (a sandwich nucleic acid hybridization assay) and microsphere-based assay for the detection of influenza viral RNA. The microshpere-based array system is a newly emerging technology that provides the multiplexing of up to 100 different assays within a single sample. In this study, we utilized this system coupled with branched DNA (bDNA) signal amplification technology (a sandwich nucleic acid hybridization assay) to detect and subtype H5 and H7 influenza virus. In our 3-plex assay, we were able to detect different HA subtype of influenza virus and differentiate H5 and H7 HA subtype at the same time based on capture probes specific for the M, H5, and H7 gene. In addition to multiplex capacity, this system does not require an RNA extraction step and samples can simply be treated with lysis buffer for the assay. (Ohio State U) In the last two years our laboratories have developed and successfully evaluated an avian influenza DNA microarray. This array contains 21 elements representing various avian influenza hemagglutinin (HA) and neuraminidase (NA) subtypes, as well as a pan-influenza probe, based on the matrix (M) gene sequence. These 21 elements are spotted in duplicate (42 spots) creating a "subarray". As a result of the subarray being spotted four times on each slide, each element is represented by 8 individual spots. Each subarray consists of a number of hermagglutinin, matrix, and neuraminidase genes ). The three matrix elements were derived from AIV strains containing three different HA subtypes. Six elements on the array represent three neuraminidase subtypes ( N1,N2, and N3). A DNA product of the Newcastle disease virus (NDV)fusion (F) gene is also included as a negative control. The majority of the array elements (9) correspond to hemagglutinin subtypes (HAS, HA7, HA9). The microarray was evaluated with a panel of 10 coded samples provided by Dr. Suarez. The results of the unknown panel test indicated 80% of the HA and NA subtypes were correctly identified, and all of the isolates were correctly identified as type A influenza. All of the neuraminidase subtypes were correctly identified with the exception of a N7. No N7 gene elements are present on the array. The H1 strain (A) was also incorrectly identified, and was also not represented on the array. (U Delaware) Fowlpox We continue to use polymerase chain reaction (PCR) and immunoblotting for differentiation of avianpox virus strains isolated from domestic poultry or wild birds. The presence of A-type inclusion (ATI) gene was detected in genomes of all strains. Common as well as different antigens were detected among various strains during immunoblotting analysis. (U Illinois) Avian Metapneumovirus (AMV) A sequencing project is almost complete to try and identify the genes related to virulence in aPMV. Once the genes are identified, a virus will be created by reverse genetics to remove that gene and create an improved vaccine based on reverse genetics. Infectious clones for aMPV have been developed by our collaborator Dr. Siba Samal at the University of Maryland. The viruses sequenced are: 1. MN-1a 9p: This virus was isolated from an outbreak of respiratory illness in turkeys in Minnesota in 1997. The virus was passaged in CEF for seven times and then twice in Vero cells; 2. MN-1a 41p: The virus MN-1a was passaged seven times in CEF cells and then 34 times in Vero cells; 3. MN-1a 63p: The virus MN-1a was passaged seven times in CEF cells and then 56 times in Vero cells; 4. MN-1a 65/Cp: The aMPV MN-1a after 41 passages in Vero cells was adapted to grow at cold temperature. It was passaged eight times each at 35ºC, 33ºC and 31ºC; and 5. MN-2a 7p: This virus was isolated from an outbreak of respiratory illness in turkeys in Minnesota in 1997 from a farm different from where MN-1a was isolated. This was passaged in CEF for seven times. (U Minnesota) Infectious Laryngotracheitis Virus ILTV is highly contagious pathogen of poultry that is often controlled by vaccination. For broilers mass vaccination techniques results in environmental contamination that can result in persistence in the house leading to back passage (bird to bird transfer) of the virus resulting in increased virulence. With reduced down time between lots, 5 days or less, and the use of built up litter, this condition is causing serious losses in the broiler belt in SE USA. In addition, there is not sufficient vaccine produced in the US on a yearly basis to vaccinate all the broilers in affected areas. Therefore, management practices to reduce the ILTV concentrations in chicken houses are needed. We developed a natural challenge method, using sentinel chickens reared in isolation units on reused litter contaminated with ILT back passed vaccine virus and a nested polymerase chain reaction (PCR) to determine the presence of ILT vaccine virus in the feces and tracheas of the chickens. Using these methods, we determined that several commercially available poultry litter treatments (Poultry GuardTM, Al+Clear TM, PLTTM), heating the litter to 38C0 (1000 F) for 24 h, and in house composting for 5 days inactivated ILT vaccine virus. This information is of immediate use to the poultry industry for controlling ILT vaccine virus induced disease in broilers and may reduce other important viral pathogens as well. (Auburn U) Infectious Bursal Disease Virus (IBDV) The effect of prime-boost on protection of chickens against infectious bursal disease by DNA vaccination was examined. Multiple intramuscular injections with a large dose of DNA carrying a large segment gene of the infectious bursal disease virus (IBDV) have been shown to provide effective protection to chickens against infectious bursal disease (IBD). The present study was conducted to determine if priming with DNA carrying a large segment gene of the IBDV and boosting with killed IBD vaccine could adequately confer protection of specific pathogen free (SPF) chickens against IBD. One-day-old chickens were intramuscularly injected with DNA plasmid coding for a large segment gene of the IBDV strain variant E (VE) (P/VP243/E) followed by an intramuscular injection of killed IBD vaccine containing both standard and variant IBDV at 1 or 2 weeks of age. Chickens were orally challenged with IBDV strain VE or standard challenge strain (STC) at 3 weeks of age and observed for 10 days. Bursal lesion scores, bursa weight/body weight (B/B) ratios, protection efficacy, IBDV antigen in bursae, enzyme-linked immunosorbent assay (ELISA) titers to IBDV, and virus neutralization (VN) titers to IBDV were determined. Chickens primed with 50, 100, 200, or 400 mg of P/VP243/E at 1 day of age and boosted with 0.5 ml of killed IBD vaccine at 1 or 2 weeks of age had 80 to 100% protection against challenge by IBDV strain VE or 71 to 100% protection against challenge by IBDV strain STC. Chickens in the groups primed with P/VP243/E and boosted with killed vaccine had significantly higher (P<0.05) B/B ratios and significantly lower (P<0.05) bursal lesion scores than chickens in the challenge control (CC) groups and groups primed with vector plasmid and boosted with killed IBD vaccine or only primed with P/VP243/E. No IBDV antigen was detected by immunofluorescent antibody assay (IFA) in bursae of chickens protected by the DNA vaccine prime and killed vaccine boost vaccination. Prior to challenge, chickens (21 days of age) in the groups primed with P/VP243/E and boosted with killed IBD vaccine had significantly higher (P<0.05) ELISA and VN titers to IBDV. These results indicate that SPF chickens at 1 day of age primed with a DNA vaccine and boosted with killed IBD vaccine can be adequately protected against challenge by homologous variant or heterologous classical IBDV. A prime-boost strategy may be useful in enhancing immunity and protection of chickens against IBD by DNA vaccination. (PurdueU) Mycoplasma Last year we reported the unusual Mycoplasma gallisepticum -685 highly attenuated vaccine strain, in which MG-685 percent invasion rate was 18.8 percent in to Chick embryo Fibroblast cells. In contrast MG-S6, MG-PG31, MG-IOIO and MG-r strain ranged from 0.45-5.6%. In order to more carefully document these phenomena we will use confocal microscopy using propidium iodide and cytodye 119 to gain more accurate information on M. gallisepticum 685 invasion of Chick embryo fibroblast cells. (U Delaware)

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