Progress 11/15/01 to 11/14/06
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? Under National Program 103 (Animal Health), component 2 "Genetic and Biological Determinants of Disease Susceptibility (Avian Tumor Viruses)", we conduct research aimed at the epidemiology, diagnosis and control of avian leukosis virus (ALV) and reticuloendotheliosis virus (REV), the two most common naturally occurring retroviruses associated with neoplastic (cancer-like) diseases in poultry. In addition to causing tumors and other production problems, both ALV and REV are potential contaminants of live-virus vaccines of poultry. Control of retroviruses in poultry is complicated by lack of specific diagnostics and vaccines as well as a high frequency of antigenic and molecular variation among strains of virus. In late 1990s, the broiler breeder industry identified an ALV- induced disease
termed myeloid leukosis as one of its highest disease priorities. This disease had the potential to negatively influence the economic viability of the entire broiler industry. On the other hand, the principal economic concerns of REV infection are as contaminants of biologic products produced in chicken embryo cells or tissues or as a barrier to export of breeding stock to certain countries. However, sporadic outbreaks of REV-induced neoplastic diseases have been observed in turkeys and infected breeders can, in some cases, transmit the virus to progeny. Understanding the basic mechanisms involved in virus-host interaction, transmission of virus as well as the development of improved diagnostic technology for viruses and tumors are integral components of any successful program for control of retrovirus infection in poultry. The research emphasizes studies on influence of virus-, host-, and environment- related factors on retrovirus-induced diseases in poultry. Using current
information on molecular and antigenic characteristics of the virus, more sensitive and specific procedures for detection of virus and antibody will be developed. The research will provide the poultry industry with the necessary tools for better diagnosis and control of this economically important virus infection in poultry. 2. List by year the currently approved milestones (indicators of research progress) (14 month:)Studies of recombinant ALV, isolate and identify recombinant ALV from field cases; Role of strain of chickens in ALV infection and tumors; Interactions between MDV and ALV; Identify role B-haplotype in ALV infection and tumors; Screen fowlpox viruses for retrovirus (ALV) inserts; sequence env gene of newly isolated ALV; collaborate with industry to identify lines resisting selection for reduced ALV shedding. (32 month)Biological and molecular characterization of recombinant ALVs isolated from field cases; Identify role B-haplotype in ALV infection and tumors; compare
susceptibility of commercial breeders to ALV infection and tumors; Identify pathogen- and environment-related factors influencing ALV infection and tumors; Screen fowlpox viruses for REV inserts; sequence env genes of new isolates and develop PCR primers; develop monoclonal antibodies; Develop a cell line that is resistant to REV infection, clone REV env. (48 month) Biological and molecular characterization of recombinant ALVs isolated from field cases; Characterization of AlV isolated from commercial MD vaccines; Role of endogenous virus on ALV infection and tumors; Identify pathogen- and environment-related factors influencing ALV infection and tumors; Test REV cell line for resistance to REV; Test PCR primers for diagnosis of various subgroups of ALV, and provide breeders with sequence information. (60 month) Biological and molecular characterization of recombinant ALVs isolated from field cases, In vitro characterization REV isolated from fowlpox vaccines; Characterization of ALV
isolated from commercial MD vaccines; collaborate with commercial diagnostic companies to develop a test for detection of exogenous ALV using monoclonal antibodies and other reagents developed at our laboratory; Continue to communicate and collaborate with industry to develop effective programs for eradication of ALV; continue monitoring field cases of REV infection and tumors in chicken and turkey flocks. 4a List the single most significant research accomplishment during FY 2006. Under National Program 103, component 2 Genetic and Biological Determinants of Disease Susceptibility (Avian Tumor Viruses)", we were able to propagate and molecularly characterize the slow-growing subgroup A ALV that was isolated by USDA-Center for Veterinary Biologics (CVB), from commercial Mareks disease vaccines. Using chicken embryo cells obtained from a new chicken line developed at our laboratory named RFS for propagation of this contaminant ALV resulted in high titer virus stock, but only in cultures
treated for 24 hours with 10% chicken serum. This work demonstrated to USDA-CVB how to grow and obtain high titer stocks of the contaminant ALV for further characterization. Molecular analysis of this contaminant ALV suggested that the virus contains the envelope (gp85) of exogenous subgroup A ALV and the long terminal repeat (another part of virus genome) of endogenous subgroup E ALV. This finding explained the slow growing characteristics and low oncogenicity of this contaminant ALV. 4b List other significant research accomplishment(s), if any. Under National Program 103, component 2 "Genetic and Biological Determinants of Disease Susceptibility (Avian Tumor Viruses)", we studied development of viremia and antibody in infected chickens with ALV. It is not known why under certain circumstances, infection of susceptible chickens with ALV can lead to an infection category where chickens will develop viremia and in the mean have neutralizing antibodies (virus+ antibody+). Our results
show that 88% of ALV-infected chickens that are classed as V+A+ failed to neutralize the autologous virus, suggesting the emergence of the so called neutralizing antibody escape mutants. Under National Program 103, component 2 "Genetic and Biological Determinants of Disease Susceptibility (Avian Tumor Viruses)", we characterized recombinant ALV isolates from the field. Evaluation of seven more isolates of the naturally recombinant ALV-B/J revealed that the seven isolates are similar to the original isolate termed ADOL-AF-115- 4. DNA sequencing of two of the seven isolates, ADOL-AF 115-1 & 10, confirmed that they are recombinant ALV-B/J. ADOL-AF 115-1 was found to be more closely related to the original ALV-B/J (ADOL-AF 115-4) than ADOL- AF 115-10. 5. Describe the major accomplishments to date and their predicted or actual impact. Research conducted in this project is under National Program 103 (Animal Health), component 2 "Genetic and Biological Determinants of Disease Susceptibility
(Avian Tumor Viruses)". This project was initiated in fiscal year 2002 and was a continuation of work initiated under the previous project. During the life of this project infection with subgroup J ALV (ALV-J) and the tumors it induces have been a major concern, particularly to the broiler breeders industry. The disease was diagnosed in broiler breeder flocks in various regions of the U.S. Several new tools were developed that aided in the diagnosis of ALV-J: (1) a polymerase chain reaction (PCR) test (a DNA-based test); (2) a genetically engineered cell line that is resistant to infection with ALV- J (C/J); (3) monoclonal antibodies (specific reagents) for identification of ALV-J; and (4) a cloned envelope gene of ALV-J. Various industry and other diagnostic laboratories have successfully used these new tools in the diagnosis of ALV-J infection in field samples. Also, the envelope gene of the U.S. prototype of ALV-J termed ADOL-Hc1 was identified, sequenced and used as an excellent
source of antigen (protein) that is now being used in commercial diagnostic kits for detection of antibody to ALV-J. We were first to isolate natural recombinant ALVs with an envelope of subgroup B and LTR of subgroup J (ALV-B/J) from commercial layers flock suffering from myeloid leukosis. This confirmed that: a) natural recombination between two subgroups of ALV can occur, and b) a recombinant ALV-J was associated with an outbreak of myeloid leukosis in commercial layer flocks; commercial layers are thought to be relatively resistant to ALV-J-induced myeloid leukosis. Age at exposure of embryos and dose of virus may influence the development of ALV tolerant infection. Most recently, using biological and PCR assays, we were able to evaluate seven more isolates of naturally recombinant ALV-B/J. The results suggested that the seven isolates are similar to the original isolate termed ADOL-AF-115-4. DNA sequencing of two of the seven isolates, ADOL- AF 115-1 & 10, confirmed that they
are recombinant ALV-B/J. ADOL-AF 115-1 was found to be more closely related to the original ALV-B/J (ADOL-AF 115- 4) than ADOL-AF 115-10. This information is important and should assist scientists in academia and industry in their attempts to diagnose and eradicate ALV. Contamination of live-virus vaccines of poultry with ALV thought to be rare. WeNBwere requested to test Marek's disease (MD) vaccines produced by two manufacturers for possible contamination with ALV. Initially, samples tested positive by virus isolation for subgroup E (endogenous) ALV. However, upon re-passage, the vaccines also tested positive for exogenous (subgroup A) ALV, a virus that can cause cancer-like disease and other production problems in susceptible chickens.NBThis was a major finding of interest to various segments of the poultry industry, government and academia. We held a meeting on July 20, 2003 during the World Veterinary Poultry Congress in Denver, CO and presented our findings to approximately 125
persons representing various concerned parties including poultry breeders and growers, vaccine manufacturers, suppliers of specific-pathogen-free (SPF) eggs, and scientists from academia and government. This work indicated that: a) under certain conditions (to be determined yet), current tests used by vaccine manufacturers and USDA- APHIS-CVB to screen live-virus vaccines of poultry for contamination with ALV may fail to identify vaccine serials that are contaminated with ALV and b) there is a need for reevaluating current tests used by vaccine manufacturers and USDA-APHIS-CVB for detection of ALV in live-virus vaccines of poultry. In FY 2005, we demonstrated that this extraneous ALV- A that we isolated from commercial Mareks disease vaccines is of a very low oncogenicity, compared with that of RAV-1, the prototype strain of ALV-A. This finding, at least partially explained the lack of ALV-induced tumors in commercial flocks that were vaccinated with contaminated Mareks disease
vaccines. This year we were asked by USDA-APHIS-VS-CVB to propagate and molecularly characterize the contaminant ALV isolated by them from commercial MD vaccines, the so called official virus. Using CEFs from a new ADOL chicken line named RFS for propagation of this contaminant ALV resulted in high titer virus stock, but only in cultures treated for 24 hours with 10% chicken serum. DNA sequence analysis of the contaminant virus indicates that this slow-growing ALV-A contains gp85 of exogenous subgroup A ALV and the LTR of endogenous subgroup E ALV. This finding explained the slow growing characteristics and low oncogenicity of this contaminant ALV. As a collaborating laboratory(with the University of Georgia), we found truncated 3 LTR and envelope sequences of REV in 5 field isolates and in 1 vaccine strain of fowlpox virus. This study provided clear evidence that to differentiate fowlpox strains that carry intact REV provirus from those that carry only solo 5 LTR sequences, positive
PCR results with primers that amplify the 5 LTR should be confirmed with more specific PCR assays, such as the envelope, or the REV 3 LTR PCR. Our studies of effect of stress on ALV infection profile demonstrated that treatment of ALV seroconverted chickens with a hormone that is usually associated with stress(ACTH) resulted in reversion to ALV viremia and shedding of virus, but only in chickens infected with virus at hatch. This study suggested that stress may induce ALV seroconverted chickens to become viremic and shed virus. We also demonstrated influence of status of ALV viremia and antibody on tumor incidence and spectrum, distribution of viral antigen and proviral DNA. Lower incidence of tumors and limited tumor spectrum were noted in chickens that were able to develop antibodies to virus. Infection of meat-type chickens with ALV-J results in a high incidence of chickens with persistence viremia even in the presence of neutralizing antibodies against inoculated virus (V+A+).
Most recently, our results show that 88% of chickens that are classed as V+A+ failed to neutralize the autologous virus, suggesting the emergence of the so called neutralizing antibody escape mutants. 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? Several MTAs were issued to transfer specific reagents such as monoclonal antibodies, PCR primers and cloned env gene to scientists in industry and academia. These technologies are being used by end-users to help them in the diagnosis of ALV infection.
Impacts
(N/A)
Publications
- Fadly, A.M. 2006. Current methods for detection of extraneous avian retroviruses in live virus vaccines of poultry. In: Proceedings of the 7th Scientific Conference of the Egyptian Veterinary Poultry Association, March 6-9, 2006, Sharm El-Sheikh, Egypt. p. 54-62.
- Fadly, A.M., Garcia, M. 2005. Detection of reticuloendotheliosis virus in live virus vaccines of poultry [abstract]. New Diagnostic Technology: Applications in Animal Health and Biologics Controls. p.65.
- Malkinson, M., Banet-Noach, C., Davidson, I., Fadly, A.M., Witter, R.L. 2004. Comparison of serological and virological findings from subgroup J avian leukosis virus-infected neoplastic and non-neoplastic flocks in Israel. Avian Pathology. p. 281-287.
- Witter, R.L. 2006. Prevention and control of reticuloendotheliosis virus infection: rationale and strategies. American Association of Avian Pathologists. In: A.A.A.P. Symposium Program, "Impact of Subclinical Immunosuppression on Poultry Production", July 2006, Honolulu, Hawaii. p. 81-89.
- Fadly, A.M. 2006. Epidemiology of reticuloendotheliosis virus. American Association of Avian Pathologists. In: A.A.A.P. Symposium Program, "Impact of Subclinical Immunosuppression on Poultry Production", July 2006, Honolulu, Hawaii. p. 67-69.
- Pandiri, A., Reed, W., Silva, R.F., Fadly, A.M. 2006. Emergence of subgroup J avian leukosis virus neutralizing antibody escape variants in meat-type chickens infected with virus at hatch [abstract]. American Association of Avian Pathologists and American Veterinary Medical Association Scientific Program. p. 73.
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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? Avian leukosis virus (ALV) and reticuloendotheliosis virus (REV) are the most common naturally occurring retroviruses associated with neoplastic (cancer-like) diseases in poultry. In addition to causing tumors and other production problems, both ALV and REV are potential contaminants of live-virus vaccines of poultry. Control of retroviruses in poultry is complicated by lack of specific diagnostics and vaccines as well as a high frequency of antigenic and molecular variation among strains of virus. In the late 1990s, the broiler breeder industry identified an ALV- induced disease termed myeloid leukosis as one of its highest disease priorities. This disease had the potential to negatively influence the economic viability of the entire broiler industry. On the other hand, the principal economic concerns
of REV infection are as contaminants of biologic products produced in chicken embryo cells or tissues or as a barrier to export of breeding stock to certain countries. However, sporadic outbreaks of REV-induced neoplastic diseases have been observed in turkeys and infected breeders can sometimes transmit the virus to progeny. Understanding the basic mechanisms involved in virus-host interaction and virus transmission, along with improved diagnostic technology, are integral components for controlling retrovirus infection in poultry. The research emphasizes studies on the influence of virus-, host-, and environment-related factors on retrovirus-induced diseases in poultry. Using current information on molecular and antigenic characteristics of the virus, more sensitive and specific procedures for detection of virus and antibody will be developed. Currently available technology will also be explored to develop vaccines for use as an adjunct to eradication programs, the principal means
for controlling retrovirus infection in poultry. The research will provide the poultry industry with the necessary tools for better diagnosis and control of this economically important virus infection in poultry. 2. List the milestones (indicators of progress) from your Project Plan. Significant milestones for this project include: (1) biological and molecular characterization of recombinant ALVs isolated from field cases; (2) identify role of host-related factors such as B-haplotype and presence of endogenous viral genes on ALV infection and tumors; (3) identify pathogen- and environment-related factors influencing ALV infection and tumors; (4) determine role of virulent and vaccine strains of Mareks disease virus on ALV infection and tumors; (5) identify inserts of REV field and vaccine strains of fowlpox virus; (6) development of specific reagents including monoclonal antibodies, cell lines and PCR primers to improve diagnosis of avian retrovirus infection and tumors; (7) study ALV
infection profile in commercial lines resisting selection for reduced shedding of ALV; and 8) evaluate programs for control of retrovirus infection including vaccines. 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. Characterization of recombinant ALV/challenge studies. Milestone Fully Met 2. Role of strain of chickens and identify pathogen/environment-related factors influencing ALV infection and tumors. Milestone Fully Met 3. Molecular assays/develop PCR primers and test in field. Milestone Fully Met 4. Utilize REV-resistant cell line to remove REV from test material. Milestone Not Met Other 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? During FY 06, we plan to address the following milestones listed in question
#2: (1) continue characterization of recombinant ALVs isolated from field flocks; (2)conduct laboratory trials to test the influence of REV inserts on pathogenicity of fowlpox viruses; (3) compare pathogenicity of REV isolates obtained from field and vaccine strains of fowlpox viruses with reference laboratory strains of REV; (4)continue working with commercial breeders of chickens to identify lines resisting selection for reduced shedding of ALV and develop program for control of infection in such lines; (4) collaborate with commercial diagnostic companies to develop a test for detection of exogenous ALV using monoclonal antibodies developed at our laboratory; and (5) continue monitoring field cases of REV infection and tumors in chicken and turkey flocks. During FY-07, this project is schedule to terminate on November 16, 2006, within the first two months of FY-07. 4a What was the single most significant accomplishment this past year? Our studies of factors that influence avian
leukosis virus (ALV) infection profile demonstrated that treatment of ALV seroconverted chickens (immune chickens that already developed antibody to ALV), with adrenocorticotrophic hormone (ACTH), resulted in reversion to ALV viremia and shedding, but only in chickens infected with virus at hatch. Chickens infected with virus as adults were not influenced by ACTH treatment. These results suggested that stress may induce seroconverted chickens that had been exposed to ALV at hatch to become viremic and shed virus. The information is of value to chicken breeders and growers in their attempts to control this economically important virus infection. 4b List other significant accomplishments, if any. Last year we reported on isolation and identification of an extraneous avian leukosis virus subgroup A (ALV-A) as a contaminant in commercial Mareks disease vaccines. These vaccines are routinely used by chicken breeders and growers to protect against Mareks disease. This year, we demonstrated
that the extraneous ALV-A isolated from commercial Mareks disease vaccines is of a very low oncogenicity (ability to induce cancer- like disease), compared with that of standard prototype strain of ALV named RAV-1. This explained the lack of ALV-induced tumors in commercial flocks that were vaccinated with contaminated Mareks disease vaccines. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project was initiated in fiscal year 2002 and was a continuation of work initiated under the previous project. During the life of this project infection with subgroup J ALV (ALV-J) and the tumors it induces has been a major concern, particularly to the broiler breeder industry. The disease was diagnosed in broiler breeder flocks in various regions of the U.S. Several new tools were developed that aided in the diagnosis of ALV-J: (1) a polymerase chain reaction (PCR) test (a DNA- based test); (2) a genetically engineered cell line
that is resistant to infection with ALV- J (C/J); (3) monoclonal antibodies (specific reagents) for identification of ALV-J; and (4) a cloned envelope gene of ALV-J. Various industry and other diagnostic laboratories have successfully used these new tools in the diagnosis of ALV-J infection in field samples. Also, the envelope gene of the U.S. prototype of ALV-J termed ADOL-Hcl was identified, sequenced and used as an excellent source of antigen (protein) that is now being used in commercial diagnostic kits for detection of antibody to ALV-J (Milestone #6; NP-103 Action plan 2, Pathogen Detection and Diagnostics). We were first to isolate natural recombinant ALVs with an envelope of subgroup B and LTR of subgroup J (ALV-B/J) from commercial layers suffering from myeloid leukosis. This confirmed that: a) natural recombination between two subgroups of ALV can occur, and b) a recombinant ALV-J was associated with an outbreak of myeloid leukosis in commercial layer flocks; commercial
layers are thought to be relatively resistant to ALV-J-induced myeloid leukosis. Age at exposure of embryos and dose of virus may influence the development of ALV tolerant infection (Milestones 2 and 3; NP-103, Action Plan 7, Epidemiology of Disease). Contamination of live-virus vaccines of poultry with ALV is thought to be rare. We were requested to test Marek's disease (MD) vaccines produced by two manufacturers for possible contamination with ALV. Initially, samples tested positive by virus isolation for subgroup E (endogenous) ALV. However, upon re- passage, the vaccines also tested positive for exogenous (subgroup A) ALV, a virus that can cause cancer-like disease and other production problems in susceptible chickens. This was a major finding of interest to various segments of the poultry industry, government and academia. We held a meeting on July 20, 2003 during the World Veterinary Poultry Congress in Denver, CO and presented our findings to various concerned parties including
poultry breeders and growers, vaccine manufacturers, suppliers of specific-pathogen-free (SPF) eggs, and scientists from academia and government. This work indicated that improved tests are needed to screen live-virus vaccines of poultry. In FY 2005, we demonstrated that this extraneous ALV-A that we isolated from commercial Mareks disease vaccines is of a very low oncogenicity, compared with that of RAV-1, the prototype strain of ALV-A. This finding, at least partially explained the lack of ALV-induced tumors in commercial flocks that were vaccinated with contaminated Mareks disease vaccines. (NP-103, Action Plan 2, Pathogen Detection and Diagnostics). As a collaborating laboratory (with the University of Georgia), we found truncated 3 LTR and envelope sequences of REV in 5 field isolates and in 1 vaccine strain of fowlpox virus. This study provided clear evidence that to differentiate fowlpox strains that carry intact REV provirus from those that carry only solo 5 LTR sequences,
positive PCR results with primers that amplify the 5 LTR should be confirmed with more specific PCR assays, such as the envelope, or the REV 3 LTR PCR (Milestone # 5; NP-103 Action Plan 3, Microbial Genomics). Our studies of effect of stress on ALV infection profile demonstrated that treatment of ALV seroconverted chickens with a hormone that is usually associated with stress (ACTH) resulted in reversion to ALV viremia and shedding of virus, but only in chickens infected with virus at hatch. This study suggested that stress may induce ALV seroconverted chickens to become viremic and shed virus. We also demonstrated influence of status of ALV viremia and antibody on tumor incidence and spectrum, distribution of viral antigen and proviral DNA. Lower incidence of tumors and limited tumor spectrum were noted in chickens that were able to develop antibodies to virus. (Milestone #2 and 3; NP-103 Action Plan 4, Mechanism of Diseases). 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? Scientists at ADOL continued to provide their counterparts in industry, government and academia with protocols and reference reagents used for detection of ALV in vaccines and other samples. Material Transfer Agreements were used to transfer PCR primers that detect ALVs to a major breeder company. Most recently, and based on research conducted by ADOL Scientists in this CRIS, USDA-APHIS-CVB has revised their prescribed procedures for detection of ALVs in live-virus vaccines of poultry. The revised procedures, namely Supplemental Assay Method (SAM-412)for Detecting Extraneous Avian Leukosis Virus in Biologic Products was issued on August 10, 2005.
Impacts
(N/A)
Publications
- Fadly, A.M. 2004. Neoplastic diseases of chickens: Marek's disease and avian leukosis. In: Proceedings of International Symposium on Avian Immunology. ANECA, November 18-20, 2004, Queretaro, Mexico. 2004 CDROM. p. 57-59.
- Mays, J.K., Bacon, L.D., Pandiri, A.P., Fadly, A.M. 2005. Response of white leghorn chickens of various B haplotypes to infection at hatch with subgroup J avian leukosis virus. Avian Diseases. 49(2):214-219.
- Fadly, A.M., Witter, R.L., Crespo, R., Davidson, I., Hafez, H.M. 2005. Virus-induced neoplastic diseases of turkeys: an update. In: Proceedings of the German Branch of the World Veterinary Poultry Association. 5th International Symposium on Turkey Diseases. p. 64-67.
- Pandiri, A., Reed, W., Fadly, A.M. 2003. Factors affecting subgroup J avian leukosis virus tolerance in commercial chickens [abstract]. American Veterinary Medical Association. p. 99.
- Williams, S.M., Reed, W.M., Bacon, L.D., Fadly, A.M. 2004. Response of white leghorn chickens of various genetic lines to infection with avian leukosis virus subgroup J. Avian Diseases. 48:61-67.
- Williams, S.M., Fitzgerald, S.D., Reed, W.M., Lee, L.F., Fadly, A.M. 2004. Tissue tropism and bursal transformation ability of subgroup J avian leukosis virus in white leghorn chickens. Avian Diseases. 48:921-927.
- Xu, B., Dong, W., Yu, C., He, Z., Lv, Y., Sun, Y., Feng, X., Li, N., Lee, L.F. 2004. Occurrence of avian leukosis virus subgroup J in commercial layer flocks in china. Avian Pathology. 33(1):13-17.
- Xu, B., Dong, W., He, Z., Lv, Y., Yu, C., Lee, L.F., Li, M. 2003. Diagnosis of avian leukosis virus subgroup J in commercial egg-type chickens in china [abstract]. American Veterinary Medical Association. p. 142-143.
- Fadly, A.M. 2003. Current and future strategies for control of virus- induced neoplastic diseases of poultry. Proceedings of North Central Avian Disease Conference, September 21-23, 2003, Ohio State University. p. 11.
- Pandiri, A.R., Reed, W.M., Fadly, A.M. 2005. Effect of viremia and antibody status on tumor incidence, tumor spectrum, and viral distribution in different tissues of ALV-J infected meat type chickens [abstract]. American Association of Avian Pathologists. p. 57.
- Davis, C.A., Mays, J.K., Pandiri, A., Fadly, A.M. 2005. Comparative evaluation of the pathogenicity of an extraneous subgroup A avian leukosis virus isolated from commercial Marek's disease vaccines [abstract]. American Veterinary Medical Association. p. 58.
- Mays, J.K., Hunt, H.D., Pandiri, A., Bacon, L.D., Fadly, A.M. 2005. Evaluation of response of a new experimental line of chickens (line 0-1) to avian leukosis virus infection [abstract]. American Veterinary Medical Association. p. 58.
- Fadly, A.M. 2005. Neoplasms-poultry. In: Kahn, C., editor. Whitehouse Station, New Jersey: Merck & Company, Inc. Merck Veterinary Manual. p. 2248-2255.
- Fadly, A.M. 2005. Neoplastic diseases of poultry. 13th International Moscow Veterinary Congress of Poultry, April 18-22, 2005, Moscow, Russia. p. 152-157.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Avian leukosis virus (ALV) and reticuloendotheliosis virus (REV) are the most common naturally occurring retroviruses associated with neoplastic (cancer-like) diseases in poultry. In addition to causing tumors and other production problems, both ALV and REV are potential contaminants of live-virus vaccines of poultry. Control of retroviruses in poultry is complicated by lack of specific diagnostics and vaccines as well as a high frequency of antigenic and molecular variation among strains of virus. In recent years, the broiler breeder industry has identified an ALV-induced disease termed myeloid leukosis as its highest disease priority. This disease has the potential to negatively influence the economic viability of the entire broiler industry. On the other hand, the principal economic concerns of REV infection are as contaminants of biologic products produced in chicken embryo cells or
tissues or as a barrier to export of breeding stock to certain countries. However, sporadic outbreaks of neoplastic disease have been observed in turkeys and infected breeders can, in some cases, transmit the virus to progeny. Understanding the basic mechanisms involved in virus-host interaction, transmission of virus as well as the development of improved diagnostic technology for viruses and tumors are integral components of any successful program for control of retrovirus infection in poultry. The research emphasizes studies on influence of virus-, host-, and environment- related factors on retrovirus-induced diseases in poultry. Using current information on molecular and antigenic characteristics of the virus, more sensitive and specific procedures for detection of virus and antibody will be developed. Currently available technology will also be explored to develop vaccines for use as an adjunct to eradication programs, the principal means for controlling retrovirus infection in
poultry. The research will provide the necessary tools for better diagnosis and control of this economically important virus infection of retrovirus infection in poultry. 2. How serious is the problem? Why does it matter? In addition to economic losses from tumor mortality, infection with such viruses is known to be associated with decreased productivity and poor performance in both chickens and turkeys. The presence of ALV or REV in poultry breeding stocks can also have a significant negative impact on breeding companies involved in exporting poultry to some countries. Improved methods for control of these economically important viruses in chickens and turkeys will benefit poultry breeders and growers as well as the American consumer 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 Health (100%) The research supports National Program 103 and its components identified as Pathogen Detection,
Microbial Genomics, Epidemiology of Disease, Host/Pathogen Interactions, Genetic Resistance to Disease, Disease Control Strategies. Research to improve control of avian tumor virus infection and the tumors they induce is also conducted through extramural collaboration with Michigan State University and the University of Georgia. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2003: Contamination of live-virus vaccines of poultry with ALV thought to be rare. Recently, scientists at USDA-ARS Avian Disease and Oncology Laboratory (ADOL), East Lansing, MI, were requested to test Marek's disease (MD) vaccines for possible contamination with ALV. Samples of MD vaccines manufactured by two different companies were received from a breeder company; samples were also received directly from one of the two vaccine companies. Initially, samples tested positive by virus isolation for subgroup E (endogenous) ALV.
However, upon re-passage, the vaccines also tested positive for exogenous (subgroup A) ALV, a virus that can cause cancer-like disease and other production problems in susceptible chickens. polymerase chain reaction (PCR) and DNA sequencing of the envelope of ALVs isolated from vaccines confirmed the results obtained from virus isolation assays that in addition to endogenous subgroup E ALV, an exogenous subgroup A ALV was also present in the vaccines tested. ADOL scientists held a meeting on July 20, 2003 during the World Veterinary Poultry Congress in Denver, CO, and presented their findings to approximately 125 persons representing various concerned parties including poultry breeders and growers, vaccine manufacturers, suppliers of specific-pathogen-free (SPF) eggs, and scientists from academia and government. This work indicated that: a) under certain conditions (to be determined yet), current tests used by vaccine manufacturers and USDA- Animal and Plant Health Inspection
Service (APHIS), Center for Veterinary Biologics (CVB) to screen live-virus vaccines of poultry for contamination with ALV may fail to identify vaccine serials that are contaminated with ALV; and b) there is a need for reevaluating current tests used by vaccine manufacturers and USDA-APHIS-CVB for detection of ALV in live-virus vaccines of poultry. B. Other Significant Accomplishment(s), if any: Screening for retroviral insertions in the genome of infectious laryngotracheitis virus(ILTV) was performed using several amplification procedures to specifically amplify regions of the ILTV genome that expand from the internal and terminal repeats into the unique long and unique short regions. After amplification products obtained from field isolates and vaccine strains were sequenced, sequences were compared to each other, and to the NCBI data bank to search for any retroviral sequence homology. ILTV sequences obtained did not present any significant homology with avian retroviral sequences.
Therefore, unlike Marek's disease virus (another avian herpesvirus) where retroviral insertions have been commonly found in the sequence junctions between the unique short and the terminal repeat of the genome, no retroviral sequences were detected within the ILTV genome of strains analyzed in the present study. Examining the role of the B haplotype on pathogenesis of ALV-J in white leghorn chickens revealed no influence of B haplotype on incidence of ALV- J-induced viremia and tumors. C. Significant Activities that Support Special Target Populations: None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project was initiated in fiscal year 2002. During the last five years (the life of the previous project), infection with subgroup J ALV (ALV-J) and tumors previously thought to be not present in flocks in the U.S. were diagnosed in broiler breeder flocks in various regions of the U. S. Two new tools were developed
that aided in the diagnosis of ALV-J: 1) a PCR test (a DNA-based test); and 2) a genetically engineered cell line that is resistant to infection with ALV-J (C/J). Various industry and other diagnostic laboratories have successfully used these new tools in the diagnosis of ALV-J infection in field samples. Also, the envelope gene of the U.S. prototype of ALV-J termed ADOL-Hc1 was identified, sequenced and used as an excellent source of antigen (protein) that is now being used in commercial diagnostic kits for detection of antibody to ALV-J. Scientists at ADOL were first to isolate natural recombinant ALVs with an envelope of subgroup B and LTR of subgroup J (ALV-B/J) from commercial layers flocks suffering from myeloid leukosis. This confirmed that: a) natural recombination between two subgroups of ALV can occur, and b) a recombinant ALV-J was associated with an outbreak of myeloid leukosis in commercial layer flocks; commercial layers are thought to be relatively resistant to
ALV-J-induced myeloid leukosis. Preliminary data suggest an influence of age at exposure of embryos and dose of virus on the development of ALV tolerant infection (consistent viremia and lack of antibody). 6. What do you expect to accomplish, year by year, over the next 3 years? In FY 04, we plan to: a) determine role of route of exposure and dose of virus on ALV-J-induced tumor and infection, b) continue biological and molecular characterization of new recombinant ALV with components from ALV subgroups B and J, and c) develop a cell line that is resistant to REV to be used in the diagnosis of REV infection. In FY 05, we plan to: a) compare pathogenicity of new recombinant ALV (ALV-B/J) with that of ALV-J, and b) use monoclonal antibodies to develop an ELISA test for detection of ALV-J specific antigen. In FY 06, we plan to: a) test efficacy of recombinant vaccines using commercially available new adjuvants to boost immune system of chickens, and b) work with Commercial breeders to
identify lines resisting selection for reduced shedding of ALV. Monitor field cases of REV infection and tumors. 7. 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? Scientists at ADOL transferred to their counterparts at USDA-APHIS-CVB and industry the protocol used at ADOL for detection of ALV in vaccines and other samples.
Impacts
(N/A)
Publications
- KULKARNI, G., FADLY, A. SEROLOGICAL EVIDENCE OF CHICKEN ANEMIA VIRUS INFECTION IN SPECIFIC PATHOGEN-FREE CHICKENS. POULTRY SCIENCE MEETING. 2003. V. 82 (SUPPLEMENT 1). ABSTRACT P. 42.
- YONDEM, B., REED, W., FADLY, A. EFFECTS OF VIRULENT AND VACCINE STRAINS OF MAREK'S DISEASE VIRUS ON SUBGROUP J AVIAN LEUKOSIS VIRUS INFECTION IN MEAT- TYPE CHICKENS. WORKSHOP ON MOLECULAR PATHOGENESIS OF MAREK'S DISEASE AND AVIAN IMMUNOLOGY. 2002. ABSTRACT P. 81.
- Lupiani, B., Williams, S.M., Silva, R.F., Hunt, H.D., Fadly, A.M. 2003. Pathogenicity of two recombinant avian leukosis viruses. Avian Diseases. 47:425-432.
- MAYS, J., BACON, L., PANDIRI, A., FADLY, A. RESPONSE OF WHITE LEGHORN CHICKENS OF VARIOUS B HAPLOTYPE TO INFECTION WITH SUBGROUP J AVIAN LEUKOSIS VIRUS. XIII CONGRESS OF WORLD VETERINARY POULTRY ASSOCIATION. 2003. ABSTRACT P. 99.
- FADLY, A. 2003. NEOPLASTIC DISEASES OF POULTRY: INTRODUCTION. SAIF, M., BARNES, J., FADLY, A., GLISSON, J., MCDOUGAL, L., SWAYNE, D., EDITORS. IOWA STATE UNIVERSITY PRESS, AMES, IA. DISEASES OF POULTRY. P. 405-407.
- FADLY, A., PAYNE, L. LEUKOSIS AND SARCOMA GROUP. SAIF, M., BARNES, J., FADLY, A., GLISSON, J., MCDOUGAL, L., SWAYNE, D., EDITORS. IOWA STATE UNIVERSITY PRESS, AMES, IA. DISEASES OF POULTRY. 2003. P. 465-516.
- WITTER, R.L., FADLY, A.M. RETICULOENDOTHELIOSIS. SAIF, M., BARNES, J., FADLY, A., GLISSON, J., MCDOUGAL, L., SWAYNE, D., EDITORS. IOWA STATE UNIVERSITY PRESS, AMES, IA. DISEASES OF POULTRY. 2003. P. 517-536.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Avian leukosis virus (ALV) and reticuloendotheliosis virus (REV) are the most common naturally occurring retroviruses associated with neoplastic (cancer-like) diseases in poultry. In addition to causing tumors and other production problems, both ALV and REV are potential contaminants of live-virus vaccines of poultry. Control of retroviruses in poultry is complicated by lack of specific diagnostics and vaccines as well as a high frequency of antigenic and molecular variation among strains of virus. In recent years, the broiler breeder industry has identified an ALV-induced disease termed myeloid leukosis as its highest disease priority. This disease has the potential to negatively influence the economic viability of the entire broiler industry. On the other hand, the principal economic concerns of REV infection are as contaminants of biologic products produced in chicken embryo cells or
tissues or as a barrier to export of breeding stock to certain countries. However, sporadic outbreaks of neoplastic disease have been observed in turkeys and infected breeders can, in some cases, transmit the virus to progeny. Understanding the basic mechanisms involved in virus-host interaction, transmission of virus as well as the development of improved diagnostic technology for viruses and tumors are integral components of any successful program for control of retrovirus infection in poultry. The research emphasizes studies on influence of virus-, host-, and environment- related factors on retrovirus-induced diseases in poultry. Using current information on molecular and antigenic characteristics of the virus, more sensitive and specific procedures for detection of virus and antibody will be developed. Currently available technology will also be explored to develop vaccines for use as an adjunct to eradication programs, the principal means for controlling retrovirus infection in
poultry. The research will provide the necessary tools for better diagnosis and control of this economically important retrovirus infection in poultry. 2. How serious is the problem? Why does it matter? In addition to economic losses from tumor mortality, infection with such viruses is known to be associated with decreased productivity and poor performance in both chickens and turkeys. The presence of ALV or REV in poultry breeding stocks can also have a significant negative impact on breeding companies involved in exporting poultry to some countries. Improved methods for control of these economically important viruses in chickens and turkeys will benefit poultry breeders and growers as well as the American consumer. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? The research supports National Program 103 ARS Animal Health and its components identified as Pathogen Detection, Microbial Genomics, Epidemiology of
Disease, Host/Pathogen Interactions, Genetic Resistance to Disease, Disease Control Strategies. Research to improve control of avian tumor virus infection and the tumors they induce is also conducted through extramural collaboration with Michigan State University and the University of Georgia. 4. What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment during FY 2002: Field outbreaks of ALV-J-induced myeloid leukosis in commercial layers are thought to be rare. For the first time, scientists at the ADOL showed that the etiologic agent of an outbreak of myeloid leukosis in commercial layers was a recombinant ALV with genetic materials from two different subgroups (B and J). This finding indicated that under certain conditions: a) recombination between ALV-J and ALV-B can occur in the field and b) commercial layers are as vulnerable to ALV-induced outbreaks of myeloid leukosis as meat-type chickens. B. Other Significant Accomplishment(s),
if any: Primary poultry breeders monitor their breeding flocks for infection with ALV using antibody tests. Analysis of various tests including two commercial kits used to monitor serum antibody to ALV-J indicated that a test termed flocytometry was most reliable. This finding suggests to scientists in academia and industry that flocytometry can be used in cases where other tests fail to give a firm diagnosis. In collaboration with one of the major primary broiler breeders, we examined a flock that had been identified by the breeder to have a relatively high incidence (20% - 60%) of ALV-J infection. Unexpectedly, scientists at ADOL found that only 0.6% of chicks tested from this flock were positive for ALV-J, but 56% of chicks tested positive for a closely related, nonpathogenic (does not cause disease) endogenous virus. This information indicated to the commercial primary breeder that methods used to identify infected breeders must be optimized. Scientists at ADOL suggested minor
modifications to the testing protocol that minimized effects of endogenous virus on test results and consequently reduced the frequency of false positive tests. This research resulted in significant savings to the primary breeder, as the practice of discarding these false positive chickens was significantly reduced or eliminated. 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? The current research activity follows from the previous project, 3635- 32000-011-00D, entitled "Epidemiology, Diagnosis and control of retrovirus infection in poultry." This is a new project initiated on 11/15/2001. During the life of the previous project, infection with subgroup J ALV (ALV J) and tumors previously thought to be not present in flocks in the U.S. were diagnosed in broiler breeder flocks in various regions of the U.S. Two new tools were developed
that aided in the diagnosis of ALV-J: 1) a polymerase chain reaction (PCR) test (a DNA based test) and 2) a genetically engineered cell line that is resistant to infection with ALV-J (C/J). Various industry and other diagnostic laboratories have successfully used these new tools in the diagnosis of ALV-J infection in field samples. Also, the envelope gene of the U.S. prototype of ALV J termed ADOL Hc1 was identified, sequenced and used as an excellent source of antigen (protein) that is now being used in commercial diagnostic kits for detection of antibody to ALV J. 6. What do you expect to accomplish, year by year, over the next 3 years? In FY 03, we plan to determine role of route of exposure and dose of virus on ALV-J-induced infection and tumors. Also, biological and molecular characterization of new recombinant ALV with genetic components from subgroup B and J will be completed. In FY 04, we plan to determine the role of endogenous virus and role of other genetic constitution of
host (B-haplotype) on ALV-J infection and tumors. Develop an ELISA test for detection of ALV-J antigen. Also, we plan to complete research to investigate insertion of REV in various vaccine and field strains of fowlpox virus. In FY 05, we plan to test efficacy of recombinant ALV-J vaccines using commercially available new adjuvants to enhance the immune system of host. We plan to work closely with Commercial breeders to identify lines resisting selection for reduced shedding of ALV. Also, we will continue to monitor field flocks for REV tumors in chickens and turkeys. 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? Scientists at ADOL expressed genetic components (envelope) of the U.S. prototype of ALV-J, ADOL-Hc1, and made them available to two new manufacturers of avian diagnostics to develop
kits for antibody to ALV-J. Scientists transferred to two primary poultry breeder companies the protocol used at ADOL in conducting virus isolation assays for ALV-J. Implementing such protocol reduced or eliminated false positive reactors and consequently the companies avoided significant economic losses due to the practice of discarding false positive breeders.
Impacts
(N/A)
Publications
- Lupiani, B., Pandiri, A.R., Hunt, H.D., Reed, W.M., Fadly, A.M. Molecular and biological characterization of naturally occurring recombinant avian leukosis (ALV) isolated form egg-type chickens suffering from myeloid leukosis. American Veterinary Medical Association/American Association Avian Pathologists. 2002. Abstract p. 20.
- Miles, A.M., Pittman, J.S., Barnes, J.H., Gimeno, I.M., Fadly, A.M., Witter, R.L., Zavala, G. Determining the etiology of unusual tumors in chickens. American Veterinary Medical Association/American Association Avian Pathologists. 2002. Abstract p. 46
- Pandiri, A.R., Lupiani, B., Reed, W.M., Fadly, A.M. Real time quantitation of viral and proviral levels of subgroup J avian leukosis virus. American Veterinary Medical Association/American Association Avian Pathologists. 2002. Abstract p. 21.
- Gingerich, E., Porter, R.E., Lupiani, B., Fadly, A.M. Diagnosis of myeloid leukosis induced by a recombinant avian leukosis virus in commercial white leghorn egg laying flocks. Avian Diseases. 2002. v. 46. p. 745-748.
- Sung, H.W., Reddy, S.M., Fadly, A.M. High virus titer in feather pulp of chickens infected with subgroup J avian leukosis virus. Avian Diseases. 2002. v. 46. p. 281-286.
- Fadly, A.M., Dybing, J.K., Pandiri, A.R. Influence of Marek's disease vaccines on the response of commercial broiler breeder chickens to infection with subgroup J avian leukosis virus. Western Poultry Disease Conference. 2002. Abstract p. 129.
- Fadly, A.M., Mays, J.K., Pandiri, A.R. Response of chickens from three commercial broiler breeders and two experimental lines to infection with a field strain of subgroup J avian leukosis virus. American Veterinary Medical Association/American Association Avian Pathologists. 2002. Abstract p. 21.
- Mays, J.K., Pandiri, A.R., Bacon, L.D., Fadly, A.M. Studies of subgroup J avian leukosis virus infection and tumors in a naturally infected commercial broiler breeder flock. American Veterinary Medical Association/American Association Avian Pathologists. 2002. Abstract p. 21.
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