Source: AGRICULTURAL RESEARCH SERVICE submitted to
IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF AGENTS...ENTERITIS MORTALITY SYNDROME OF TURKEYS
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0404971
Grant No.
(N/A)
Project No.
6612-32000-036-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Nov 15, 2001
Project End Date
Nov 14, 2006
Grant Year
(N/A)
Project Director
SPACKMAN E
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
(N/A)
ATHENS,GA 30613
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113230104025%
3113230109025%
3113230110120%
3113230116020%
3113230202010%
Goals / Objectives
Isolate, identify, and characterize the viral agent(s) associated with Poult enteritis mortality syndrome (PEMS). Develop rapid and accurate diagnostic techniques for PEMS-associated pathogens. Determine the pathogenesis of PEMS infections for development of control strategies. Evaluate the prevalence and pathogenesis of PEMS associated agents for the development of control strategies. Based on epidemiology and pathogenesis studies determine what agents are playing the most significant role in the disease.
Project Methods
PEMS is a complex disease that may involve numerous infectious agents. This project will focus on isolating and identifying PEMS-associated viruses by classical virologic and molecular techniques, producing molecular and immunologic tools to diagnose the viruses in commercial turkey flocks, and understand the role of individual and combinations of viruses in PEMS pathogenesis. The development of diagnostic tests will aid in the identification of viruses causing clinically significant disease in commercial turkey flocks and understand the epidemiology of enteric viruses of poultry. The characterization of enteric viruses will also increase the genomic information available on enteric diseases of poultry. BL-2 and BSL-3AG; 05/23/2002.

Progress 11/15/01 to 11/14/06

Outputs
Progress Report Objectives (from AD-416) Isolate, identify, and characterize the viral agent(s) associated with Poult enteritis mortality syndrome (PEMS). Develop rapid and accurate diagnostic techniques for PEMS-associated pathogens. Determine the pathogenesis of PEMS infections for development of control strategies. Evaluate the prevalence and pathogenesis of PEMS associated agents for the development of control strategies. Based on epidemiology and pathogenesis studies determine what agents are playing the most significant role in the disease. Approach (from AD-416) PEMS is a complex disease that may involve numerous infectious agents. This project will focus on isolating and identifying PEMS-associated viruses by classical virologic and molecular techniques, producing molecular and immunologic tools to diagnose the viruses in commercial turkey flocks, and understand the role of individual and combinations of viruses in PEMS pathogenesis. The development of diagnostic tests will aid in the identification of viruses causing clinically significant disease in commercial turkey flocks and understand the epidemiology of enteric viruses of poultry. The characterization of enteric viruses will also increase the genomic information available on enteric diseases of poultry. BL-2 and BSL-3AG; 05/23/2002.

Impacts
(N/A)

Publications


    Progress 10/01/05 to 09/30/06

    Outputs
    Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Poult enteritis mortality syndrome (PEMS) is a highly infectious disease of young turkeys. PEMS was first reported in North Carolina in 1991. Since then, PEMS and similar disease conditions have been reported in most regions where turkeys are commercially produced including; the Southeastern United States, Texas, California, Arkansas, Missouri, and Israel. PEMS has been called one of the most devastating emergent disease to strike the poultry industry in recent years. Since its emergence in the early 1990s, outbreaks of PEMS have cost the turkey industry millions of dollars in losses annually. Although there are no detailed financial studies, its estimated that PEMS outbreaks cost the North Carolina turkey industry $34 million dollars in losses in 1995 alone, making it the most economically devastating disease of turkeys. Another recent outbreak occurred in 1999 when turkey flocks in Missouri were depopulated because of PEMS. PEMS represents an emerging disease. The major impact of PEMS is due to mortality and decreased production as turkeys are stunted and grow poorly when affected by the disease. Currently the agent or agents that cause PEMS are unknown. PEMS appears to be a complex disease, possibly involving multiple pathogens and some anecdotal evidence suggests that immunosuppression may be involved. Developing a better understanding of PEMS will be beneficial to the turkey industry. By understanding what agents are involved in causing PEMS, management practices may be modified to minimize the impact of the disease. There are three specific goals for this project: 1. Isolate and identify agents that are associated with PEMS. Novel molecular techniques will be used to sensitively and specifically identify these agents. 2. Develop rapid and accurate diagnostic tools and reagents for PEMS associated pathogens. 3. Evaluate the pathogenesis of PEMS associated agents for the development of control strategies. The goals of this project fall under National Program 103- Animal Health and addresses the following goals described in the National Programs Action Plan: Pathogen detection and diagnostics, microbial genomics, mechanism of disease and strategies to control infectious and non- infectious disease. 2. List by year the currently approved milestones (indicators of research progress) Year 1 FY 2002 1. Perform a field epidemiological study to help determine what agents involved with causing PEMS. 2. Characterize a novel strain of turkey astrovirus 3. Develop a standard RT-PCR test for turkey astrovirus detection in clinical samples Year 2 FY 2003 1. Design real-time reverse-transcriptase polymerase chain reaction (RT- PCR) primers and probes for three viruses, turkey astrovirus, turkey coronavirus and turkey reovirus, associated with PEMS 2. Collect clinical specimens from commercial turkeys experiencing PEMS to isolate turkey astrovirus, turkey coronavirus and turkey reovirus. Year 3 FY 2004 1. Perform a bench validation of the real-time RT-PCR test for turkey astrovirus, turkey coronavirus and turkey reovirus and determine the optimal sample type for detection of each virus 2. Evaluate the pathogenesis of turkey reovirus in turkeys and determine if these viruses have the potential to replicate and cause disease on chickens. 3. Initiate development of a database of gene sequence information for viral agents associated with PEMS, focusing on turkey astrovirus and turkey reovirus Year 4 FY 2005 1. Evaluate the pathogenesis of simultaneous turkey astrovirus and turkey reovirus infection 2. Evaluate the antigenic variation and pathogenesis of numerous turkey astrovirus isolates selected because of genetic variation from the reference isolate 3. Continue development of a database of turkey astrovirus and turkey reovirus gene sequences and perform phylogenetic analysis on genes sequences from each virus Year 5 FY2006 1. Develop and bench validate an RT-PCR test for avian rotavirus 2. Perform a nationwide survey of commercial poultry for enteric viruses 3. Evaluate the pathogenesis of avian rotavirus in specific pathogen free and commercial-line turkeys. 4a List the single most significant research accomplishment during FY 2006. This project addresses component 6, countermeasures to prevent and control of enteric diseases, problem statement 6B, enteric viruses of poultry, of NP-103, Animal Health. A nationwide survey for avian astrovirus, avian reovirus and avian rotavirus in commercial chicken and turkey flocks was conducted. This survey evaluated the distribution of avian enteric viruses in commercial poultry populations and provided data to establish a molecular epidemiological map for enteric viruses of poultry. Commercial poultry producers nationwide participated and submitted clinical samples which were tested for enteric viruses by RT-PCR tests. Selected viruses were isolated and gene sequence data was collected and analyzed. This study established the widespread presence of enteric viruses in poultry and revealed that chickens ad turkeys may be infected with similar viruses. Additionally, strains of avian astroviruses and avian reoviruses not previously identified in the US were detected. 4b List other significant research accomplishment(s), if any. This project addresses component 6, countermeasures to prevent and control of enteric diseases, problem statement 6B, enteric viruses of poultry, of NP-103, Animal Health. A multiplex RT-PCR test for the simultaneous detection and differentiation of the four types of avian astrovirus and avian rotavirus has been developed. This test detects avian astrovirus strains not detected by other tests and allows for the rapid identification of specific strains in a clinical sample. Once validated, this test can be used by the poultry industry to detect infection of specific enteric viruses in poultry in a rapid and cost effective manner. Viruses involved in causing runting-stunting syndrome (RSS) of broiler chickens, a recently re-emerging disease of chickens that is clinically very similar to poult enteritis, will cause similar disease in turkeys. This was demonstrated with pathogenesis studies. This showed that enteric viruses in poultry are not necessarily species specific in their ability to infect chickens or turkeys and cause similar disease in each specie. Understanding the host range of enteric viruses in poultry is critical for developing and targeting diagnostic tests and developing control programs. 5. Describe the major accomplishments to date and their predicted or actual impact. This project addresses component 6, countermeasures to prevent and control of enteric diseases, problem statement 6B, enteric viruses of poultry, of NP-103, Animal Health. Accomplishments over the life of this project have included: A unique strain of astrovirus circulating in commercial turkey flocks with PEMS was isolated, identified and characterized. PEMS-like clinical disease could be reproduced by this virus in experimentally infected poults indicating that this virus may be involved in causing PEMS. Characterization of this virus revealed that the virus caused systemic infection and affected organs in the immune system suggesting that turkey astrovirus may cause immune dysfunction in turkeys. Addresses the action plan component: mechanism of disease. Numerous disinfectants were evaluated for their ability to inactivate turkey astrovirus, two (formaldehyde and Virkon S) were found to be effective at inactivating the virus. Cleaning and disinfection are often effective helping to control infectious agents, however turkey astrovirus is an unusually environmentally stable agent and most commercial disinfectants fail to inactivate it. Therefore use of these disinfectants would not effectively control the virus. Addresses the action plan component: strategies to control infectious and non- infectious disease. A standard RT-PCR test was developed and transferred to veterinary diagnostic laboratories to detect the avian astroviruses; and a multiplex standard RT-PCR test was developed and to detect astrovirus, reovirus, and turkey coronavirus from commercial turkey flocks using a single sample. Turkey astrovirus and turkey coronavirus are difficult to culture, therefore molecular detection methods are ideal for detection of these agents. Multiplexing reduces costs and time. Addresses the action plan component: pathogen detection and diagnostics. A state-of-the-art diagnostic technique, multiplex real-time RT-PCR was developed to detect turkey astrovirus, turkey coronavirus and turkey reovirus. Real-time RT-PCR is a relatively new technology and offers numerous advantages over standard RT-PCR. This method is extremely rapid, highly specific and sensitive. Additionally, optimal samples types and times for collecting samples were determined for each virus. Historically, consistency of diagnostic results for these viruses has been problematic for the industry, this test should greatly improve this. The procedure for this test has been transferred to state veterinary diagnostic laboratories. Addresses the action plan component: pathogen detection and diagnostics. Turkey reoviruses collected from clinical cases of poult enteritis were evaluated for their ability to cause disease in poults in the absence of other viruses and for their ability to replicate and cause disease in chickens. Turkey reoviruses represent a previously un-described type of reovirus, which although related to chicken origin avian reoviruses are genetically distinct based on preliminary studies. Although, both chickens and turkey may be infected, clinical disease was only produced in turkeys, indicating that; 1) turkey reoviruses may not be a disease threat for the commercial chicken industry and 2) turkey reoviruses may be involved in causing PEMS. Importantly, it was shown that these reoviruses have the ability to cause immunosuppression in turkeys, therefore may contribute to disease as predisposing agents. This data will help the industry determine whether control measures for avian reoviruses need to be developed. Addresses the action plan component: mechanism of disease. A database of gene sequences from viruses associated with PEMS has been developed and will be maintained for the life of the project. Virus gene sequences from any viruses identified in PEMS specimens will be included. Currently the database contains sequence from turkey astrovirus, turkey reovirus, turkey coronavirus and avian rotavirus. Information from this database is being used for diagnostic test development which can be used directly by the industry, and molecular epidemiological studies. Sequence data will also be used for evaluating the mechanism of pathogenesis of viruses associated with PEMS. Addresses the action plan components: microbial genomics. A survey for enteric viruses was performed to determine which enteric viruses are present at what age. The age at which the birds are exposed to specific enteric viruses has not been determined. Samples were collected bi-weekly from each of six commercial turkey flocks through the life of each flock and screened by RT-PCR for astrovirus, rotavirus, coronavirus and reovirus. Viruses from positive samples were subsequently sequenced for a molecular epidemiological study. Since, age at exposure can affect the clinical manifestation of disease and may be controlled to some degree by management, understanding the age profiles of enteric virus infection in commercial turkeys will aid in mitigating disease impact. Addresses the action plan component: mechanism of disease. Gene sequences from turkey reoviruses, turkey astroviruses and avian rotaviruses, which have not previously been determined were obtained. Determination of markers for pathogenicity and development of diagnostic tests relies upon having sequence data from numerous virus strains. Selected genes, such as those expected to be associated with virulence, were sequenced and analyzed for, their relationships with previously characterized sequences, for molecular epidemiological analysis and to determine the target sites for molecular detection tests. The data obtained for the rotaviruses was used to develop a diagnostic test for avian rotavirus. Data from astroviruses and reoviruses was used for epidemiological purposes. Addresses the action plan components: microbial genomics. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Information regarding the presence of enteric viruses in commercial chickens and turkeys has been communicated to the industry and poultry health professionals through technical meetings. The multiplex RT-PCR for avian astroviruses should be available for use by the industry and veterinary diagnostic laboratories during FY2007.

    Impacts
    (N/A)

    Publications

    • Rives, D., Pantin Jackwood, M.J. 2006. Comparison of enteric virus RT-PCR results with production data from commercial turkey hens. In: American Association of Avian Pathologists Annual Meeting Scientific Program, July 16-19, 2006, Honolulu, Hiwaii. 2006 CDROM.
    • Pantin Jackwood, M.J., Spackman, E., Day, J.M. 2006. Molecular characterization of enteric viruses circulating in the United States. In: American Association of Avian Pathologists Annual Meeting Scientific Program, July 16-19, 2006, Honolulu, Hiwaii. 2006 CDROM.
    • Pantin Jackwood, M.J. 2006. Molecular characterization of chicken and turkey astroviruses [abstract]. International Poultry Scientific Forum Scientific Program. p.32.
    • Pantin Jackwood, M.J., Spackman, E., Woolcock, P.R. 2006. Phylogenetic analysis of turkey astroviruses reveals evidence of recombination. Virus Genes.32:187-192.
    • Pantin Jackwood, M.J., Spackman, E., Day, J.M. 2006. Molecular characterization of astroviruses and rotaviruses detected in turkey flocks from hatch to 12 weeks of age. In: Proceedings of the 55th Western Poultry Disease Conference, March 5-8, 2006, Sacramento, California. p.51-52
    • Day, J.M., Pantin Jackwood, M.J., Spackman, E. 2006. Molecular characterization of the S1 genome segment of turkey-origin reovirus. In: American Society for Microbiology Annual Meeting Scientific Program, May 21-25, 2006, Orlando, Florida. 2006 CDROM.
    • Spackman, E., Day, J.M., Pantin Jackwood, M.J. 2006. The pathogenesis of agents associated with runting-stunting syndrome of broilers in SPF turkeys. In: American Association of Avian Pathologists Annual Meeting Scientific Program, July 16-19, 2006, Honolulu, HI. 2006 CDROM.
    • Day, J.M., Pantin Jackwood, M.J., Spackman, E. 2006. Evidence of immune system dysfunction in poults infected with turkey-origin reoviruses. In: American Association of Avian Pathologists Annual Meeting Scienfitic Program, July 16-19, 2006, Honolulu, HI. 2006 CDROM.


    Progress 10/01/04 to 09/30/05

    Outputs
    1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Poult enteritis mortality syndrome (PEMS) is a highly infectious disease of young turkeys. PEMS was first reported in North Carolina in 1991. Since then, PEMS and similar disease conditions have been reported in most regions where turkeys are commercially produced including; the Southeastern United States, Texas, California, Arkansas, Missouri, and Israel. PEMS has been called one of the most devastating emergent disease to strike the poultry industry in recent years. Since its emergence in the early 1990s, outbreaks of PEMS have cost the turkey industry millions of dollars in losses annually. Although there are no detailed financial studies, its estimated that PEMS outbreaks cost the North Carolina turkey industry $34 million dollars in losses in 1995 alone, making it the most economically devastating disease of turkeys. Another recent outbreak occurred in 1999 when turkey flocks in Missouri were depopulated because of PEMS. PEMS represents an emerging disease. The major impact of PEMS is due to mortality and decreased production as turkeys are stunted and grow poorly when affected by the disease. Currently the agent or agents that cause PEMS are unknown. PEMS appears to be a complex disease, possibly involving multiple pathogens and some anecdotal evidence suggests that immunosuppression may be involved. Developing a better understanding of PEMS will be beneficial to the turkey industry. By understanding what agents are involved in causing PEMS, management practices may be modified to minimize the impact of the disease. There are three specific goals for this project: 1. Isolate and identify agents that are associated with PEMS. Novel molecular techniques will be used to sensitively and specifically identify these agents. 2. Develop rapid and accurate diagnostic tools and reagents for PEMS associated pathogens. 3. Evaluate the pathogenesis of PEMS associated agents for the development of control strategies. The goals of this project fall under National Program 103- Animal Health and addresses the following goals described in the National Programs Action Plan: Pathogen detection and diagnostics, microbial genomics, mechanism of disease and strategies to control infectious and non- infectious disease. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 FY 2002 Perform a field epidemiological study to help determine what agents involved with causing PEMS. Characterize a novel strain of turkey astrovirus Develop a standard RT-PCR test for turkey astrovirus detection in clinical samples Year 2 FY 2003 Design real-time reverse-transcriptase polymerase chain reaction (RT- PCR) primers and probes for three viruses, turkey astrovirus, turkey coronavirus and turkey reovirus, associated with PEMS Collect clinical specimens from commercial turkeys experiencing PEMS to isolate turkey astrovirus, turkey coronavirus and turkey reovirus. Year 3 FY 2004 Perform a bench validation of the real-time RT-PCR test for turkey astrovirus, turkey coronavirus and turkey reovirus and determine the optimal sample type for detection of each virus Evaluate the pathogenesis of turkey reovirus in turkeys and determine if these viruses have the potential to replicate and cause disease on chickens. Initiate development of a database of gene sequence information for viral agents associated with PEMS, focusing on turkey astrovirus and turkey reovirus Year 4 FY 2005 Evaluate the pathogenesis of simultaneous turkey astrovirus and turkey reovirus infection Evaluate the antigenic variation and pathogenesis of numerous turkey astrovirus isolates selected because of genetic variation from the reference isolate Continue development of a database of turkey astrovirus and turkey reovirus gene sequences and perform phylogenetic analysis on genes sequences from each virus Year 5 FY2006 Develop and bench validate an RT-PCR test for avian rotavirus Perform a nationwide survey of commercial poultry for enteric viruses Evaluate the pathogenesis of avian rotavirus in specific pathogen free and commercial-line turkeys. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Evaluate the antigenic variation and pathogenesis of numerous turkey astrovirus isolates selected because of genetic variation from the reference isolate. Milestone Not Met Redirection of Research focus due to change in priorities 2. Continue development of a database of turkey astrovirus and turkey reovirus gene sequences and perform phylogenetic analysis on genes sequences from each virus. Milestone Fully Met 3. Evaluate the pathogenesis of simultaneous turkey astrovirus and turkey reovirus infection. Milestone Not Met Redirection of Research focus due to change in priorities 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The Year 5 milestones are listed below. The entire project is scheduled to be completed during FY 2006 and a new project will be developed to undergo OSQR review, and subsequent implementation beginning FY 2007. Year 5 FY2006 Develop and bench validate an RT-PCR test for avian rotavirus Perform a nationwide survey of commercial poultry for enteric viruses Evaluate the pathogenesis of avian rotavirus in specific pathogen free and commercial-line turkeys. 4a What was the single most significant accomplishment this past year? An avian rotavirus was identified in specimens from turkeys with poult enteritis and broiler chickens with runting-stunting syndrome. Rotaviruses are well known enteric pathogens that have been minimally characterized in poultry. Initial pathogenesis studies were performed with clinical specimens containing rotavirus. Identification of the viral agents associated with poult enteritis and a new, similar condition in chickens, broiler runting-stunting syndrome, is critical to controlling the disease. 4b List other significant accomplishments, if any. A survey for enteric viruses was performed to determine which enteric viruses are present at what age. The age at which the birds are exposed to specific enteric viruses has not been determined. Samples were collected bi-weekly from each of six commercial turkey flocks through the life of each flock and screened by RT-PCR for astrovirus, rotavirus, coronavirus and reovirus. Viruses from positive samples were subsequently sequenced for a molecular epidemiological study. Since, age at exposure can affect the clinical manifestation of disease and may be controlled to some degree by management, understanding the age profiles of enteric virus infection in commercial turkeys will aid in mitigating disease impact. Gene sequences from turkey reoviruses, turkey astroviruses and avian rotaviruses, which have not previously been determined were obtained. Determination of markers for pathogenicity and development of diagnostic tests relies upon having sequence data from numerous virus strains. Selected genes, such as those expected to be associated with virulence, were sequenced and analyzed for, their relationships with previously characterized sequences, for molecular epidemiological analysis and to determine the target sites for molecular detection tests. The data obtained for the rotaviruses was used to develop a diagnostic test for avian rotavirus. Data from astroviruses and reoviruses was used for epidemiological purposes. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Accomplishments over the life of this project have included: A unique strain of astrovirus circulating in commercial turkey flocks with PEMS was isolated, identified and characterized. PEMS-like clinical disease could be reproduced by this virus in experimentally infected poults indicating that this virus may be involved in causing PEMS. Characterization of this virus revealed that the virus caused systemic infection and affected organs in the immune system suggesting that turkey astrovirus may cause immune dysfunction in turkeys. Addresses the action plan component: mechanism of disease. Cleaning and disinfection are often effective helping to control infectious agents, however turkey astrovirus is an unusually environmentally stable agent and most commercial disinfectants fail to inactivate it. Therefore use of these disinfectants would not effectively control the virus. Numerous disinfectants were evaluated for their ability to inactivate turkey astrovirus, two (formaldehyde and Virkon S) were found to be effective at inactivating the virus. Addresses the action plan component: strategies to control infectious and non- infectious disease. A standard RT-PCR test was developed and transferred to veterinary diagnostic laboratories to detect the avian astroviruses; and a multiplex standard RT-PCR test was developed and to detect astrovirus, reovirus, and turkey coronavirus from commercial turkey flocks using a single sample. Turkey astrovirus and turkey coronavirus are difficult to culture, therefore molecular detection methods are ideal for detection of these agents. Multiplexing reduces costs and time. Addresses the action plan component: Pathogen detection and diagnostics. A state-of-the-art diagnostic technique, multiplex real-time RT-PCR was developed to detect turkey astrovirus, turkey coronavirus and turkey reovirus. Real-time RT-PCR is a relatively new technology and offers numerous advantages over standard RT-PCR. This method is extremely rapid, highly specific and sensitive. Additionally, optimal samples types and times for collecting samples were determined for each virus. Historically, consistency of diagnostic results for these viruses has been problematic for the industry, this test should greatly improve this. The procedure for this test has been transferred to state veterinary diagnostic laboratories. Addresses the action plan component: pathogen detection and diagnostics. Turkey reoviruses collected from clinical cases of poult enteritis were evaluated for their ability to cause disease in poults in the absence of other viruses and for their ability to replicate and cause disease in chickens. Turkey reoviruses represent a previously un-described type of reovirus, which although related to chicken origin avian reoviruses are genetically distinct based on preliminary studies. Although, both chickens and turkey may be infected, clinical disease was only produced in turkeys, indicating that; 1) turkey reoviruses may not be a disease threat for the commercial chicken industry and 2) turkey reoviruses may be involved in causing PEMS. Importantly, it was shown that these reoviruses have the ability to cause immunosuppression in turkeys, therefore may contribute to disease as predisposing agents. This data will help the industry determine whether control measures for avian reoviruses need to be developed. Addresses the action plan component: mechanism of disease. A database of gene sequences from viruses associated with PEMS has been developed and will be maintained for the life of the project. Virus gene sequences from any viruses identified in PEMS specimens will be included. Currently the database contains sequence from turkey astrovirus, turkey reovirus, turkey coronavirus and avian rotavirus. Information from this database is being used for diagnostic test development which can be used directly by the industry, and molecular epidemiological studies. Sequence data will also be used for evaluating the mechanism of pathogenesis viruses associated with PEMS. Addresses the action plan components: microbial genomics. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The real-time RT-PCR test is available and has been transferred to veterinary diagnostic labs where it is currently available to the industry. An RT-PCR test for avian rotavirus should be available in early FY2006. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Pantin-Jackwood, M. and E. Spackman. Sequence analysis of turkey astroviruses Feed Info News Service Scientific Reviews. www.feedinfo.com, April 2005.

    Impacts
    (N/A)

    Publications

    • Spackman, E., Kapczynski, D.R., Sellers, H., 2005. Multiplex real-time rt- pcr for the detection of three viruses associated with poult enteritis complex: turkey astrovirus, turkey coronavirus, and turkey reovirus. Avian Diseases. 49:86-91.
    • Pantin Jackwood, M.J., Spackman, E. 2005. Molecular characterization of turkey astroviruses circulating in the united states [abstract]. American Veterinary Medical Association p.73.
    • Day, J.M., Pantin Jackwood, M.J., Spackman, E. 2005. Initial evaluation of the pathogenesis of novel turkey-origin reoviruses. In: American Society for Microbiology, June 8, 2005, Atlanta, Georgia. p.23.
    • Pantin Jackwood, M.J., Spackman, E., Day, J.M. 2005. Comparison of bursal histology lesions caused by different isolates of turkey reovirus. Proceedings of Western Poultry Disease Conference, p. 126.
    • Pantin Jackwood, M.J., Spackman, E. 2005. Sequence analysis of turkey astroviruses isolated from healthy and sick birds [abstract]. Southern Conference on Avian Diseases. p.22.
    • Spackman, E., Pantin Jackwood, M.J., Day, J.M., Sellers, H. 2005. The pathogenesis of turkey origin reoviruses in turkeys and chickens. Avian Pathology 34(4):291-296.


    Progress 10/01/03 to 09/30/04

    Outputs
    1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Poult enteritis mortality syndrome (PEMS) is a highly infectious disease of young turkeys. PEMS was first reported in North Carolina in 1991. Since then, PEMS and similar disease conditions have been reported in most regions where turkeys are commercially produced including; the Southeastern United States, Texas, California, Arkansas, Missouri, and Israel. PEMS has been called one of the most devastating emergent disease to strike the poultry industry in recent years. Since its emergence in the early 1990's, outbreaks of PEMS have cost the turkey industry millions of dollars in losses annually. Although there are no detailed financial studies, it's estimated that PEMS outbreaks cost the North Carolina turkey industry $34 million dollars in losses in 1995 alone, making it the most economically devastating disease of turkeys. Another ercent outbreak occurred in 1999 when turkey flocks in Missouri were depopulated because of PEMS. PEMS represents an emerging disease. The major impact of PEMS is due to mortality and decreased production as turkeys are stunted and grow poorly when affected by the disease. Currently the agent or agents that cause PEMS are unknown. PEMS appears to be a complex disease, possibly involving multiple pathogens and some anecdotal evidence suggests that immunosuppression may be involved. Developing a better understanding of PEMS will be beneficial to the turkey industry. By understanding what agents are involved in causing PEMS, management practices may be modified to minimize the impact of the disease. There are three specific goals for this project: 1. Isolate and identify agents that are associated with PEMS. Novel molecular techniques will be used to sensitively and specifically identify these agents. 2. Develop rapid and accurate diagnostic tools and reagents for PEMS associated pathogens. 3. Evaluate the pathogenesis of PEMS associated agents for the development of control strategies. The goals of this project fall under National Program 103- Animal Health and addresses the following goals described in the National Programs Action Plan: Pathogen detection and diagnostics, microbial genomics, mechanism of disease and strategies to control infectious and non- infectious disease. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 FY 2002 Perform a field epidemiological study to help determine what agents involved with causing PEMS. Characterize a novel strain of turkey astrovirus. Develop a standard RT-PCR test for turkey astrovirus detection in clinical samples. Year 2 FY 2003 Design real-time reverse-transcriptase polymerase chain reaction (RT-PCR) primers and probes for three viruses, turkey astrovirus, turkey coronavirus and turkey reovirus, associated with PEMS. Collect clinical specimens from commercial turkeys experiencing PEMS to isolate turkey astrovirus, turkey coronavirus and turkey reovirus. Year 3 FY 2004 Perform a bench validation of the real-time RT-PCR test for turkey astrovirus, turkey coronavirus and turkey reovirus and determine the optimal sample type for detection of each virus. Evaluate the pathogenesis of turkey reovirus in turkeys and determine if these viruses have the potential to replicate and cause disease on chickens. Initiate development of a database of gene sequence information for viral agents associated with PEMS, focusing on turkey astrovirus and turkey reovirus. Year 4 FY 2005 Evaluate the pathogenesis of simultaneous turkey astrovirus and turkey reovirus infection. Evaluate the antigenic variation and pathogenesis of numerous turkey astrovirus isolates selected because of genetic variation from the reference isolate. Continue development of a database of turkey astrovirus and turkey reovirus gene sequences and perform phylogenetic analysis on genes sequences from each virus. Year 5 FY 2006 Determine if turkey reoviruses or turkey astroviruses induce immune dysfunction in turkeys. Develop a vaccine for turkey astroviruses and/or turkey reoviruses. Continue development of a database of turkey astrovirus and turkey reovirus gene sequences and perform phylogenetic analysis on genes sequences from each virus. 3. Milestones: The milestones listed below were scheduled to be completed during FY 2004. All milestones were completed. Perform a bench validation of the real-time RT-PCR test for turkey astrovirus, turkey coronavirus and turkey reovirus and determine the optimal sample type for detection of each virus. Evaluate the pathogenesis of turkey reovirus in turkeys and determine if these viruses have the potential to replicate and cause disease on chickens. Initiate development of a database of gene sequence information for viral agents associated with PEMS, focusing on turkey astrovirus and turkey reovirus. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The Year 4 and 5 milestones are listed below. The entire project is scheduled to be completed during FY 2006 and a new project will be developed to undergo OSQR review, and subsequent implementation beginning FY 2007. Year 4 FY 2005 Evaluate the pathogenesis of simultaneous turkey astrovirus and turkey reovirus infection. Evaluate the antigenic variation and pathogenesis of numerous turkey astrovirus isolates selected because of genetic variation from the reference isolate. Continue development of a database of turkey astrovirus and turkey reovirus gene sequences and perform phylogenetic analysis on genes sequences from each virus. Year 5 FY2006 Determine if turkey reoviruses or turkey astroviruses induce immune dysfunction in turkeys. Develop a vaccine for turkey astroviruses and/or turkey reoviruses. Continue development of a database of turkey astrovirus and turkey reovirus gene sequences and perform phylogenetic analysis on genes sequences from each virus. Year 6 FY 2007 (assuming continuation of the project) Continue characterization of viruses associated with PEMS, including mechanisms of pathogenesis. Evaluate the efficacy of vaccine(s) developed in FY2006 and develop vaccination strategies. Utilize molecular techniques to evaluate clinical specimens for previously undescribed viruses. 4. What were the most significant accomplishments this past year? Real-time reverse transcriptase polymerase chain reaction (RRT-PCR), a new molecular diagnostic technique, which is rapid, highly specific and highly sensitive (superior to standard RT-PCR) was developed and applied to the detection of three viruses commonly associated with Poult Enteritis Mortality Syndrome (PEMS); turkey astrovirus, turkey coronavirus and turkey reovirus. Previously, no rapid or specific diagnostic tools are available for the detection of viral agents which are involved in causing PEMS. In FY 2004, a laboratory validation for this test with clinical samples from experimental cases was successfully completed with optimization of for sample types and times were determined for each of the three viruses. This test is superior to previous test and will accurately identify infected turkeys. B. Other Significant Accomplishment(s), if any. Turkey reoviruses collected from clinical cases of poult enteritis were evaluated for their ability to cause disease in poults in the absence of other viruses and for their ability to replicate and cause disease in chickens. Turkey reoviruses represent a previously undescribed type of reovirus, which although related to chicken origin avian reoviruses are genetically distinct based on preliminary studies. Because these are novel viruses their ability to cause disease in commercial poultry needs to be determined. Although, both chickens and turkey may be infected, clinical disease was only produced in turkeys, indicating that; 1) turkey reoviruses may not be a disease threat for the commercial chicken industry and 2) turkey reoviruses may be involved in causing PEMS. A database of gene sequences from viruses associated with PEMS was initiated. Virus gene sequences from both turkey astroviruses and turkey reovirus isolates collected from commercial turkeys in 2003 were obtained. A higher level of genetic variation was observed among turkey astrovirus than expected which may have implications for the specificity of current diagnostics tests. Sequence information was obtained from previously unrecognized turkey reoviruses. Information from this database will be used for: aiding vaccine development, future diagnostic test development, aiding in understanding the pathogenesis of PEMS; and molecular epidemiology. C. Significant Accomplishments/Activities that support special target populations: None. D. Progress Report opportunity to submit additional programmatic information to your Area Office and NPS (optional for all in-house ("D") projects and the projects listed in Appendix A; mandatory for all other subordinate projects). None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Accomplishments over the life of this project have included: A unique strain of astrovirus circulating in commercial turkey flocks with PEMS was isolated, identified and characterized. PEMS-like clinical disease could be reproduced by this virus in experimentally infected poults indicating that this virus may be involved in causing PEMS. Characterization of this virus revealed that the virus caused systemic infection and affected organs in the immune system suggesting that turkey astrovirus may cause immune dysfunction in turkeys. Addresses the action plan component: mechanism of disease. Cleaning and disinfection are often effective helping to control infectious agents, however turkey astrovirus is an unusually environmentally stable agent and most commercial disinfectants fail to inactivate it. Therefore use of these disinfectants would not effectively control the virus. Numerous disinfectants were evaluated for their ability to inactivate turkey astrovirus, two (formaldehyde and Virkon S) were found to be effective at inactivating the virus. Addresses the action plan component: strategies to control infectious and non- infectious disease. A standard RT-PCR test was developed and transferred to veterinary diagnostic laboratories to detect the avian astroviruses; and a multiplex standard RT-PCR test was developed and to detect astrovirus, reovirus, and turkey coronavirus from commercial turkey flocks using a single sample. Turkey astrovirus and turkey coronavirus are difficult to culture, therefore molecular detection methods are ideal for detection of these agents. Multiplexing reduces costs and time. Addresses the action plan component: Pathogen detection and diagnostics. A state-of-the-art diagnostic technique, multiplex real-time RT-PCR was developed to detect turkey astrovirus, turkey coronavirus and turkey reovirus. Real-time RT-PCR is a relatively new technology and offers numerous advantages over standard RT-PCR. This method is extremely rapid, highly specific and sensitive. Additionally, optimal samples types and times for collecting samples were determined for each virus. Historically, consistency of diagnostic results for these viruses has been problematic for the industry, this test should greatly improve this. Addresses the action plan component: pathogen detection and diagnostics. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The real-time RT-PCR test will be available in early FY 2005 to be transferred to veterinary diagnostic labs. Although now common in most diagnostic labs, the primary constraint to adoption of this technology may be availability of the necessary equipment, in which case the previously described standard RT-PCR test may be used. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Spackman, E. "Multiplex Real-time RT-PCR for Turkey Coronavirus, Turkey Astrovirus and Avian Reovirus" Presented at the American Association of Avian Pathologists Annual Meeting Philadelphia, PA, July 24-27, 2004.

    Impacts
    (N/A)

    Publications

    • Kapczynski, D.R., Sellers, H.S., Simmons, V., Schultz-Cherry, S. Sequence analysis of the S3 gene from a turkey reovirus. Virus Genes. 2002. v.25. p. 95-100.


    Progress 10/01/02 to 09/30/03

    Outputs
    1. What major problem or issue is being resolved and how are you resolving it? Poult enteritis mortality syndrome (PEMS) is a highly infectious disease of young turkeys. PEMS was first reported in North Carolina in 1991. Since then, PEMS has been reported throughout the Southeastern United States and Texas. There are also reports of a PEMS-like disease in California, Arkansas, Missouri, and Israel. PEMS has been called the most devastating emergent disease to strike the poultry industries since infectious bursal disease virus. In spite of years of research, the causative agent of PEMS remains unknown. The research under this CRIS seeks to identify the PEMS agent(s) using novel molecular techniques, develop diagnostic reagents for rapid identification, and understand the pathogenesis of the disease. 2. How serious is the problem? Why does it matter? Since its emergence in the early 1990's, outbreaks of PEMS have cost the turkey industry millions of dollars in losses annually. Although there are no detailed financial studies, it's estimated that PEMS outbreaks cost the North Carolina turkey industry $34 million dollars in losses in 1995 alone, making it the most economically devastating disease of turkeys. In 1999, turkey flocks in regions of Missouri were depopulated because of PEMS. PEMS represents an emerging disease. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program 103, Animal Diseases (100%) This research allows scientists to identify factors involved in PEMS and to determine the causative agent(s). This information is necessary for diagnostic agencies, regulatory agencies, and industry specialists who need to detect, control and minimize the impact of PEMS in commercial poultry. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY2003 year: Currently no rapid or sensitive laboratory tests are available to identify the viruses involved in PEMS production. A rapid and sensitive real-time reverse transcriptase polymerase chain reaction (RT-PCR) test was developed to detect turkey astrovirus type-2, one of the major viruses involved in production of PEMS in turkeys. This test was shown to be very accurate in detecting the astrovirus in experimentally infected turkeys. This test provides more accurate diagnosis of PEMS in turkeys. B. Other significant accomplishment(s): C.Significant Accomplishments/Activities that support special target populations: 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Accomplishments over the life of this project have included: 1) isolated, identified and characterized of a unique strain of astrovirus in turkeys with PEMS; 2) reproduced of a PEMS-like clinical disease in turkeys with the astrovirus; 3) reproduction of immune deficiencies in turkeys with the astrovirus which reduces the ability of turkeys to fight off other pathogens; 4) determined that most commercial disinfectants fail to kill the astrovirus, but formaldehyde and Virkon S were effective at killing the virus; 4) a specific and sensitive standard RT-PCR test was developed, validated and transferred to veterinary diagnostic laboratories to detect the avian astroviruses; and 5) an RT-PCR test was developed to detect and differentiate astrovirus, reovirus, and turkey coronavirus in a single sample from PEMS affected turkeys. 6. What do you expect to accomplish, year by year, over the next 3 years? FY2004: Develop and improve diagnostic assays specific for viral agents associated with PEMS including a multiplex real-time RT-PCR test for turkey astrovirus type-2, turkey reoviruses and turkey coronavirus and an antibody detection assay for turkey astrovirus. FY2005: Perform field surveillance and an epidemiological study utilizing improved diagnostic and detection methods to determine the prevalence of the primary viruses associated with PEMS. FY2006: Evaluate the biology and pathogenesis the most prevalent PEMS associated viruses with the focus of improving disease control by vaccination or management practices.

    Impacts
    (N/A)

    Publications

    • Kapczynski, D.R., Sellers, H.S., Simmons, V., Schultz-Cherry, S. Sequence analysis of the S3 gene from a turkey reovirus. Virus Genes. 2002. v.25. p. 95-100.


    Progress 10/01/01 to 09/30/02

    Outputs
    1. What major problem or issue is being resolved and how are you resolving it? Poult enteritis mortality syndrome (PEMS) is a highly infectious disease of young turkeys. PEMS was first reported in North Carolina in 1991. Since then, PEMS has been reported throughout the Southeastern United States and Texas. There are also reports of a PEMS-like disease in California, Arkansas, Missouri, and Israel. PEMS has been called the most devastating emergent disease to strike the poultry industries since infectious bursal disease virus. In spite of years of research, the causative agent of PEMS remains unknown. The research under this CRIS seeks to identify the PEMS agent(s) using novel molecular techniques, develop diagnostic reagents for rapid identification, and understand the pathogenesis of the disease. 2. How serious is the problem? Why does it matter? Since its emergence in the early 1990's, outbreaks of PEMS have cost the turkey industry millions of dollars in losses annually. Although there are no detailed financial studies, it's estimated that PEMS outbreaks cost the North Carolina turkey industry $34 million dollars in losses in 1995 alone, making it the most economically devastating disease of turkeys. In 1999, turkey flocks in regions of Missouri were depopulated because of PEMS. PEMS represents an emerging disease. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? National Program 103, Animal Diseases (100%) This research allows scientists to pinpoint factors involved in PEMS and determine the causative agents(s). Such data will be useful to diagnostic and regulatory agencies, and industry specialists who need to be able to detect PEMS infections in poultry. 4. What was your most significant accomplishment this past year? A. Single most significant accomplishment during FY2002 year: Various viruses and bacteria have been identified in poults affected with PEMS, but the cause is unknown. Southeast Poultry Research Laboratory in collaboration with North Carolina State University, Perdue University and Ohio State University conducted a field epidemiological study on the agents involved with PEMS. The collaborative study demonstrated a statistically-significant relationship of the PEMS-associated astrovirus (TAstV-2) with field outbreaks of PEMS. This information emphasizes the central role of astrovirus in the PEMS and need to concentrate control efforts on astrovirus. B. Other significant accomplishment(s): Most enteric viruses replicate only in the intestines and have limited effect on non-intestinal organs. Research at Southeast Poultry Research Laboratory conducted research to understand why infected turkeys are immune depressed. During FY2002, research demonstrated that TAstV-2 caused viremia in exposed poults and infectious virus was present throughout the bird including the breast muscle. This information indicates the new astrovirus produces a systemic infection that may be responsible for effects outside the intestines including growth depression and immune dysfunction. C.Significant Accomplishments/Activities that support special target populations: None. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? This is a new CRIS project that replaces a previous CRIS project on PEMS, 6612-32000-032-00D. Accomplishments over the life of the previous project included: 1) a unique strain of astrovirus circulating in commercial turkey flocks with PEMS was isolated, identified and characterized; 2) in experimental studies, the virus reproduced a PEMS-like clinical disease and induced changes to the immune system that reduced the ability to fight off secondary disease problems; 3) most commercial disinfectants fail to inactivate the astrovirus, but formaldehyde and Virkon S were effective at inactivating the virus; 4) a specific and sensitive RT-PCR test was developed, validated and transferred to veterinary diagnostic laboratories to detect the avian astroviruses; and 5) a multiplex RT-PCR test was developed to detect astrovirus, reovirus, and turkey coronavirus from commercial turkey flocks using a single sample. 6. What do you expect to accomplish, year by year, over the next 3 years? FY2003: Determine the role of the macrophage in PEMS related disease FY2004: Develop molecular diagnostic (RT-PCR) tests to other enteric viruses including enterovirus, parvovirus and calicivirus. FY2005: Preliminary vaccine studies on astrovirus immunization for protection against PEMS. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? The molecular diagnostic (RT-PCR) test for the astrovirus was transferred to the University of Guelph diagnostic laboratory and is currently being used to screen commercial turkey flocks for PEMS. 8. List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) "Update on PEMS research" National Turkey Federation. 2002.

    Impacts
    (N/A)

    Publications

    • Kapczynski, D.R., Sellers, H.S., Simmons, V., Schultz-Cherry, S. Sequence analysis of the S3 gene from a turkey reovirus. Virus Genes. 2002. v.25. p. 95-100.