Progress 06/23/01 to 02/02/06
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? Animal health drugs have a remarkable history of safe use in the United States. With the exception of a few allergic reactions to penicillin or other antibiotics, there have been no documented examples of human toxicities as a result of animal drugs in meat products within the United States. Such is not the case in Europe and Asia, however, where humans have been poisoned by the ingestion of meats containing illegally used animal drugs. Phenylbutazone is an analgesic drug with a long history of use in humans and animals. Because phenylbutazone causes aplastic anemia in a small percentage of individuals, it is rarely used in human medicine and is banned for use in food animals. Nevertheless, a recent FDA/FSIS survey indicated that phenylbutazone residues were present in a surprisingly
large percentage of samples obtained from an abattoir processing cattle. We will determine the absorption, distribution, metabolism, and excretion (ADME) of phenylbutazone in cattle to learn how long it takes for phenylbutazone and its metabolites to deplete from treated animals. In addition, we will determine whether commercial immunologically-based assays are capable of detecting phenylbutazone and(or) its metabolites after prolonged withdrawal periods. The illegal, off-label, and inappropriate use of animal health drugs is discouraged by the US FDA and the USDA FSIS through nationwide surveillance and monitoring programs. A major tool for authorities overseeing these programs is immunologically based screening assays in which hundreds to thousands of samples are screened for specific drug residues. Samples that are positive for the drug residue are then analyzed by confirmatory methods designed to unequivocally identify the drug residue in question. Immunologically based
detection methods are rapid, sensitive, specific, inexpensive, and easy to perform. In addition, immunoassays may be field based and offer flexibility not encountered with other analytical assay formats. Once validated, the assays may be used by regulatory scientists, officials at packing plants, farmers, or other individuals who have a vested interest in whether an animal has been treated with a drug. We have developed immunoassays towards ractopamine HCl and other compounds of food safety interest and are showing their potential to detect drug residues in the excreta and tissues of treated animals. Cattle that graze N. coenophialum infected tall fescue are susceptible to a number of maladies that decrease their productive capabilities. The maladies, which are termed fescue foot or fescue toxicosis, are hypothesized to be caused by ergopeptine alkaloids produced by the endophytic fungus N. coenophialum. The most abundant ergopeptine alkaloid present in affected plants,
ergovaline, is thought to be the causative agent of fescue toxicosis, but the syndrome has not been replicated using purified materials. In order for toxicity to occur, toxins must be absorbed and must reach target tissues. Establishment of the rate of toxin elimination through metabolism and excretion -or accumulation due to slow elimination- is fundamental to understanding the development of clinical signs of toxicosis. Thus, an understanding of the metabolism and disposition of the major ergopeptine alkaloids such as ergovaline is fundamental to an understanding of fescue toxicity. Unfortunately, no data exist on the absorption of the major alkaloids present in infected tall fescue; in addition, once absorbed it is not known whether the major ergopeptine alkaloids bind to specific tissues, are excreted rapidly, or are biotransformed to more toxic metabolites. We plan to investigate whole animal absorption and biotransformation of ergopeptine alkaloids and cellular transport
mechanism using ergovaline as a model compound. 2. List by year the currently approved milestones (indicators of research progress) Objective 1. Determine the absorption, distribution, and metabolism of ergovaline in intact or surgically modified rodents. Year 1 (2001): Complete synthesis of [14C]ergovaline, complete live-phase of balance- excretion studies in rodents. Year 2 (2002): Complete metabolite identification including biliary metabolites of ergovaline in rodents; develop and validate residue methods for edible tissues. Year 3 (2003): No milestone for objective. Year 4 (2004): No milestone for objective. Year 5 (2005) No milestone for objective. Objective 2. Determine residue depletion characteristics of ergovaline and metabolites in cattle. Year 1. (2001): No milestone for objective. Year 2. (2002): No milestone for objective. Year 3 (2003): Conduct live phase of [14C]ergopeptine metabolism study in cattle. Measure total residues, initiate metabolite identification
in urine/bile. Initiate ruminal degradation studies. Year 4 (2004): Complete identification of ruminal ergovaline metabolites, complete tissue analysis of residue study. Initiate isolation of ruminal microbes that degrade ergovaline. Year 5 (2005): Initiate residue depletion study of ergovaline in cattle and determine tissue concentrations of residues. Objective 3. Determine the absorption, distribution, metabolism, and excretion of phenylbutazone in cattle. Determine the marker compound and target tissues for regulatory assays; determine whether available immunological techniques accurately detect phenylbutazone use. Year 1 (2001): Synthesize [14C]phenylbutazone; dose cattle for balance excretion study; complete total residue measurements. Year 2 (2002): Complete metabolite identification; determine immunoassay cross reactivity with metabolites. Year 3 (2003): Validate tissue residue methods for parent phenylbutazone. Year 4 (2004): Initiate residue depletion study in
cattle; complete tissue total residue analysis; determine marker compound; determine target tissue; conduct immunoassay analysis of edible tissues. Year 5 (2005): No objectives listed. Objective 4. Develop and validate rapid, sensitive, and simple immunological techniques for the detection of animal health products of potential food safety concern. Year 1 (2001): Design and synthesize haptens; complete conjugation of haptens to proteins. Year 2 (2002): Immunize animals; develop analysis for measuring antibody content. Year 3 (2003): Develop the antibody based analyses; explore matrix effects, variability. Initiate live phase metabolism study. Year 4 (2004): Complete validation of antibody properties; further optimize analysis if necessary; complete metabolism cross reactivity work. Year 5 (2005). Validation of immunoaffinity column technology. 4a List the single most significant research accomplishment during FY 2006. Development of Sodium Chlorate Feed Additive as a Food
Safety Tool. Studies were conducted in cattle to determine the identity of chemical residues in edible tissues after treatment with an investigational chlorate-based feed additive. It was demonstrated that residues of sodium chlorate in edible tissues of beef cattle will not limit the development of chlorate as a food safety tool. Chlorate is being developed as a feed additive because it inhibits the growth of pathogens such as E. coli O157:H7 and Salmonella in live animals. We determined that the residues fell below safe tissue concentrations estimated by the US FDA Center for Veterinary Medicine. This technology has the potential to be a safe and effective pre-harvest tool to reduce the incidence of pathogens in live animals prior to entering the human food chain. 4b List other significant research accomplishment(s), if any. Zilpaterol Residue Method Developed. A fellowship under the OECD co-operative Research Program: Biological Resource Management for Sustainable Agriculture
Systems has been completed. The fellowship allowed cooperative research between Department of Agriculture and Rural Development (Ireland) and Agricultural Research Service. A surface plasmon resonance biosensor method was developed to evaluate 9 zilpaterol antibodies. The sensor method showed excellent recovery and reproducibility for low ppb zilpaterol concentrations. A comparison of the zilpaterol biosensor method with ELISA demonstrated that both methods give equivalent results with a high correlation between them. This rapid analytical method is available for use to screen animal tissues for illegal residues. 4d Progress report. This report serves to document research conducted under a reimbursable agreement between ARS and the National Cattlmens Beef Association. Each year thousands of US consumers become ill because they have eaten food products that are contaminated with pathogenic bacteria. Intense efforts have been made to eliminate pathogenic organisms from beef
animals before they are slaughtered and to remove contamination after slaughter. A new pre-harvest food safety strategy has been developed that has been shown to greatly reduce, or even eliminate gram-negative pathogens from live cattle. Use of this new feed additive has not yet been approved by regulatory organizations because it is not known whether residues present in edible tissues of treated animals would represent a health risk. The purpose of this study was to quantify residues of a novel chlorate based feed additive in edible tissues of beef animals. For all of the doses tested, chlorate residues in liver, kidney, muscle, and fat fell well below amounts that the FDA have estimated to be safe. The major metabolite of chlorate was chloride, a nutrient already present in almost all human food sources. 5. Describe the major accomplishments to date and their predicted or actual impact. The research reported is conducted under National Program 108, Food Safety and directly
contributes to research Component 1.1, Pathogens, Toxins, and Chemical Contaminants Preharvest, and research Component 1.2, Pathogens, Toxins and Chemical Contaminants Postharvest. Research is further related to ARS strategic plan Objective 3.1: Provide Science Based Knowledge on the Safe Production, Processing, and Handling of Plant and Animal Products and on the Detection and Control of Toxin- producing and/or Pathogenic Bacteria and Fungi, Parasites, Chemical Contaminates, Mycotoxins, and Plant Toxins. A monoclonal antibody specific for ractopamine was generated and its usefulness at detecting ractopamine residues in urine and tissues of farm animals was developed. A U.S. patent for this technology was issued (no. 6,274,334; August 14, 2001); Testing Components Corporation and the Neogen Corporation have licensed the technology. Several other organizations have requested information regarding licensing agreements. The cell line has been deposited in American Type Culture
Collection (PTA- 2103) and Confidentiality and Material Transfer Agreements have been filed with several research groups. Details of the immunoassay has been requested by a variety of groups ranging from foreign government regulatory agencies (Germany, Northern Ireland, Hong Kong, South Korea), US government agencies (USDA-FSIS, FDA), State Agencies (Colorado), domestic and international industrial concerns, academia, and end users (State Fair Organizers, Horse Racing Officials). The development and use of this antibody will allow domestic and foreign regulatory agencies detect the off-label and(or) illegal use of ractopamine in target and non- target species. Detection of such use will increase the safety of domestic and foreign food supplies (Objective 4; Component 1.2)) A monoclonal antibody specific for the beta-agonist zilpaterol has been generated, its specificity determined, and its usefulness in detecting zilpaterol residues in urine and edible tissues of farm animals was
determined. This is the first antibody developed with application for the rapid detection of zilpaterols illegal use in food-animals. Material transfer agreements have been issued to several companies allowing for the evaluation of the antibody and one company has received a non-exclusive license for the technology. Rapid-screening assays for zilpaterol will be used by private industry and government laboratories for the determination of illegal residues of zilpaterol in edible tissues, and could be used by scientists to determine zilpaterol in other matrices (Objective 4; Component 1.2). The rates of ergovaline isomerization in solvents and cell culture media were measured. Ergovaline isomerizes extremely rapidly to ergovalinine in some cell culture media, emphasizing the need for researchers to store ergovaline under conditions of known stability and to dose ergovaline of known isomer composition. Two commercially available feed additives, marketed as products that reduce fescue
toxicosis, did not reduce cellular toxicity at the concentrations tested. Both ergovaline and its naturally occurring isomer, ergovalinine, readily crossed gastrointestinal. cells intact, and with similar kinetics. Because both isomers were transported, either isomer, or a combination of both, could be involved in the pathogenesis of fescue toxicosis at sites distal to the gastrointestinal tract. These studies advance the understanding of ergovalines involvement in fescue toxicity and will be used by scientists to further understand the overall mechanisms of fescue toxicosis (Objective 1; Component 1.1). The disposition and metabolism of orally and intravenously administered [14C]phenylbutazone was determined in beef cattle. In cattle, parent phenylbutazone is the major metabolite, whereas in other species, only small amounts of phenylbutazone are excreted intact. Data from these studies support the Food and Drug Administrations decision to monitor edible tissues for parent
phenylbutazone rather than metabolites. Results indicated that liver or blood would be a more appropriate target tissue for regulatory measurement of phenylbutazone than the kidney, but with the sensitive analytical tools available to regulatory agencies, phenylbutazone residues are likely detectable in other tissues for extended periods of time. Results of this study will increase the efficiency with which regulatory agencies screen for illegal phenylbutazone use in food animals. (Objective 3; Component 1.2) The metabolism and residues of sodium [36Cl]chlorate were measured in beef cattle and the effect of sodium chlorate dose on chlorate residues in edible tissues was determined. Sodium chlorate is an investigational feed additive that is being developed for use in the cattle industry because of its specific bactericidal effects on pathogens of importance to the beef industry (E. Coli O157:H7). Studies indicated that chlorate is converted to chloride ion and that both chloride
and to a lesser extent chlorate ions are present in edible tissues. These results indicate that a large proportion of chlorate residues in edible tissues of cattle are present as natural products. Residues of chlorate in edible tissues of cattle fell below provisional safe tissue concentrations provided by the US FDA Center for Veterinary Medicine (Components 1.1 and 1.2). 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? Material Transfer Agreements for ractopamine monoclonal antibodies have been affected with Dr. Richard Sams, Ohio State University; Drs. Thomas Grace and Thomas Wida, Biacore, Inc., Dr. Fernando Rubio, ABRAXIS LLC, Dr. Richard Plange, Testing Components Corportation, Dr. Dwight Schreodt, Neogen Corporation, Dr. Deborah Morris,
International Diagnostics Systems Corporation and Dr. Karl Schmitt, R-Biopharm AG, Germany (2001 to present) . Shelver, W. L. and D. J. Smith. 2001. A monoclonal antibody, cell line and immunoassay for ractopamine. United States Patent 6,274,334 B1. An Individual Memorandum of Understanding Agreement (#58-5442-2M-631) was established with Biacore Inc. (New Jersey, headquarter Uppsala, Sweden);Use of the Surface Plasmon Resonance Technique to Determine Residue Levels of Agrochemicals in Animal Tissues and Excreta (October, 2001) Non-exclusive license for ractopamine antibody patent signed with Testing Components Corporation, October 2001; has been renewed annually to date. A specific cooperative agreement was signed between Xenosense Technologies (Belfast, Ireland) and the USDA ARS Biosciences Lab to conduct residue depletion studies with ractopamine and to test Biosensor technology for the detection of incurred ractopamine residues. Material Transfer Agreement signed with Neogen
Corporation, Lexington Kentucky, for ractopamine antibody and drug conjugation method. January 2002. Non-exclusive license for ractopamine antibody patent signed with Neogen Corporation, 2002; has been reviewed annually to date. A series of lectures on the dispositions and analysis of beta-adrenergic agonists in food animals was presented to the Korean National Veterinary Research and Quarantine Service, Veterinary Environmental Residues Division in October of 2002. Work on the development of a ractopamine immunoassay was presented to the Taiwan National Animal Industry Foundation and Taiwan Bureau of Animal and Plant Health Inspection and Quarantine (2002). The audience also included officers from the Taiwan Council of Agriculture and researchers from National Chung-Shing University. Trust Agreement with National Cattlmens Beef Association. 2004. Determination of a sodium chlorate dose that results in safe concentrations of tissue residues in beef cattle. $42,200. Trust Agreement
with the National Pork Board. 2005. Determination of a sodium chlorate dose that results in safe concentrations of tissue residues in swine. $20,000. Presentations to the Beef Safety Committee at the Annual Meeting of the Cattle Industry, San Antonio, TX (February, 2005) and at the Production Working group of the Beef Industry Safety Summit (Orlando, April 2005) on the safety of chlorate residues after chlorate use in beef cattle. Non-exclusive license for zilpaterol antibody issued to Testing Components Corporation, (June 2005). Material transfer agreements were signed with Euro-Diagnostica for zilpaterol and ractopamine antibodies (September, 2004), Beacon Analytical Systems for ractopamine antibodies (December 2004), and Immunalysis Corporation fro ractopamine antibodies (May 2005). Material transfer agreement with Envirologix, Inc and license agreement executed (August 2004) for thiamethoxam monoclonal antibodies. A Material transfer agreement with Envirologix, Inc. for purified
polyclonal antibody specific for sugerbeet pathogen Cercaspora was established August 2005, in collaboration with, ARS, Sidney, MT).
Impacts
(N/A)
Publications
- Shelver, W.L., Kim, H., Li, Q.X. 2005. Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the beta-adrenergic agonist zilpaterol. Journal of Agricultural and Food Chemistry 53:3273- 3280.
- Smith, D.J., Anderson, R.C., Ellig, D.A., Larsen, G.L. 2005. Tissue distribution, elimination, and metabolism of dietary sodium [36Cl]chlorate in beef cattle. Journal of Agricultural and Food Chemistry 53:4272-4280.
- Shelver, Weilin L. 2005. Generation and Testing of Immunoaffinity Columns IN: METHODS IN MOLECULAR BIOLOGY, Vol. 295: Immunochemical Protocols, Third Edition. Edited by R. Burns, Humana Press Inc., Totowa, NJ, pp: 269-280.
- Oliver, C.E., Smith, D.J., Anderson, R.C., Caton, J.S. 2005. Pharmacokinetics of intraruminally-dosed 36cl-labeled sodium chlorate in cattle. [abstract] American Chemical Society (ACS) Spring Meetings. March 14-17, 2005, San Diego, CA, Picogram No. 68, Abstract No. 105.
- Shappell, N.W., Smith, D.J. 2004. Ergovaline transport across human gastrointestinal cells (caco-2). [abstract] Joint Meeting of American Society of Animal Science, American Dairy Science Association, and Poultry Science Association, St. Louis, MO, July 25-29, 2004, Journal of Animal Science 82(Suppl 1):181, Abstract T71.
- Shappell, N.W., Billey, L.O. 2004. Assessment of tasco and ycwp on ergovaline toxicity in caco-2 cells. [abstract] Joint Meeting of American Society of Animal Science, American Dairy Science Association, and Poultry Science Association, St. Louis, MO, July 25-29, 2004, Journal of Animal Science 82(Suppl 1):181, Abstract T72.
- Smith, D.J., Huwe, J.K., Lorentzsen, M.K., Harrington, G.E. 2004. Biotransformation of [14c]-phenylbutazone after oral or intravenous administration to cattle. [abstract] American Chemical Society National Meeting, Philadelphia, PA, 8/22-26/2004, Picogram No. 67, Abstract No. 124.
- Smith, D.J., Anderson, R.C., Ellig, D.A., Larsen, G.L. 2004. Tissue distribution, elimination, and metabolism of dietary sodium [36cl]chlorate in beef cattle. [abstract] International Association for Food Protection 91st Annual Meeting, August 8-11, 2004, Phoenix, AZ. Abstract #T35.
- Smith, D.J., Oliver, C.E., Anderson, R.C., Caton, J.S. 2005. Residues and metabolism of 36cl-labeled sodium chlorate in cattle. [abstract] American Chemical Society (ACS) Spring Meeting, San Diego, CA, March 13-17, 2005, Picogram No. 68, Abstract No. 106.
- Kim, H., Shelver, W.L., Li, Q.X. 2004. Monoclonal antibody-based enzyme- linked immunosorbent assay for the insecticide imidacloprid. Analytica Chimica ACTA 509:111-118.
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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? Animal health drugs have a remarkable history of safe use in the United States. With the exception of a few allergic reactions to penicillin or other antibiotics, there have been no documented examples of human toxicities as a result of animal drugs in meat products within the United States. Such is not the case in Europe and Asia, however, where humans have been poisoned by the ingestion of meats containing illegally used animal drugs. Phenylbutazone is an analgesic drug with a long history of use in humans and animals. Because phenylbutazone causes aplastic anemia in a small percentage of individuals, it is rarely used in human medicine and is banned for use in food animals. Nevertheless, a recent FDA/FSIS survey indicated that phenylbutazone residues were present in a surprisingly large
percentage of samples obtained from an abattoir processing cattle. We will determine the absorption, distribution, metabolism, and excretion (ADME) of phenylbutazone in cattle to learn how long it takes for phenylbutazone and its metabolites to deplete from treated animals. In addition, we will determine whether commercial immunologically-based assays are capable of detecting phenylbutazone and(or) its metabolites after prolonged withdrawal periods. The illegal, off-label, and inappropriate use of animal health drugs is discouraged by the US FDA and the USDA FSIS through nationwide surveillance and monitoring programs. A major tool for authorities overseeing these programs is immunologically based screening assays in which hundreds to thousands of samples are screened for specific drug residues. Samples that are positive for the drug residue are then analyzed by confirmatory methods designed to unequivocally identify the drug residue in question. Immunologically based detection
methods are rapid, sensitive, specific, inexpensive, and easy to perform. In addition, immunoassays may be field based and offer flexibility not encountered with other analytical assay formats. Once validated, the assays may be used by regulatory scientists, officials at packing plants, farmers, or other individuals who have a vested interest in whether an animal has been treated with a drug. We have developed immunoassays towards ractopamine HCl and other compounds of food safety interest and are showing their potential to detect drug residues in the excreta and tissues of treated animals. Cattle that graze N. coenophialum infected tall fescue are susceptible to a number of maladies that decrease their productive capabilities. The maladies, which are termed fescue foot or fescue toxicosis, are hypothesized to be caused by ergopeptine alkaloids produced by the endophytic fungus N. coenophialum. The most abundant ergopeptine alkaloid present in affected plants, ergovaline, is
thought to be the causative agent of fescue toxicosis, but the syndrome has not been replicated using purified materials. In order for toxicity to occur, toxins must be absorbed and must reach target tissues. Establishment of the rate of toxin elimination through metabolism and excretion -or accumulation due to slow elimination- is fundamental to understanding the development of clinical signs of toxicosis. Thus, an understanding of the metabolism and disposition of the major ergopeptine alkaloids such as ergovaline is fundamental to an understanding of fescue toxicity. Unfortunately, no data exist on the absorption of the major alkaloids present in infected tall fescue; in addition, once absorbed it is not known whether the major ergopeptine alkaloids bind to specific tissues, are excreted rapidly, or are biotransformed to more toxic metabolites. We plan to investigate whole animal absorption and biotransformation of ergopeptine alkaloids and cellular transport mechanism using
ergovaline as a model compound. 2. List the milestones (indicators of progress) from your Project Plan. Objective 1. Determine the absorption, distribution, and metabolism of ergovaline in intact or surgically modified rodents. Year 1 (2001): Complete synthesis of [14C]ergovaline, complete live-phase of balance- excretion studies in rodents. Year 2 (2002): Complete metabolite identification including biliary metabolites of ergovaline in rodents; develop and validate residue methods for edible tissues. Year 3 (2003): No milestone for objective. Year 4 (2004): No milestone for objective. Year 5 (2005) No milestone for objective. Objective 2. Determine residue depletion characteristics of ergovaline and metabolites in cattle. Year 1. (2001): No milestone for objective. Year 2. (2002): No milestone for objective. Year 3 (2003): Conduct live phase of [14C]ergopeptine metabolism study in cattle. Measure total residues, initiate metabolite identification in urine/bile. Initiate
ruminal degradation studies. Year 4 (2004): Complete identification of ruminal ergovaline metabolites, complete tissue analysis of residue study. Initiate isolation of ruminal microbes that degrade ergovaline. Year 5 (2005): Initiate residue depletion study of ergovaline in cattle and determine tissue concentrations of residues. Objective 3. Determine the absorption, distribution, metabolism, and excretion of phenylbutazone in cattle. Determine the marker compound and target tissues for regulatory assays; determine whether available immunological techniques accurately detect phenylbutazone use. Year 1 (2001): Synthesize [14C]phenylbutazone; dose cattle for balance excretion study; complete total residue measurements. Year 2 (2002): Complete metabolite identification; determine immunoassay cross reactivity with metabolites. Year 3 (2003): Validate tissue residue methods for parent phenylbutazone. Year 4 (2004): Initiate residue depletion study in cattle; complete tissue
total residue analysis; determine marker compound; determine target tissue; conduct immunoassay analysis of edible tissues. Year 5 (2005): No objectives listed. Objective 4. Develop and validate rapid, sensitive, and simple immunological techniques for the detection of animal health products of potential food safety concern. Year 1 (2001): Design and synthesize haptens; complete conjugation of haptens to proteins. Year 2 (2002): Immunize animals; develop analysis for measuring antibody content. Year 3 (2003): Develop the antibody based analyses; explore matrix effects, variability. Initiate live phase metabolism study. Year 4 (2004): Complete validation of antibody properties; further optimize analysis if necessary; complete metabolism cross reactivity work. Year 5 (2005). Validation of immunoaffinity column technology. 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. Determine the absorption, distribution, and metabolism of ergovaline in intact or surgically modified rodents. Milestone Not Met Redirection of Research focus due to change in priorities 2. Initiate residue depletion study of ergovaline in cattle and determine tissue concentrations of residues. Milestone Not Met Redirection of Research focus due to change in priorities 3. Initiate residue depletion study (of phenylbutazone) in cattle; complete tissue total residue analysis; determine marker compound; determine target tissue; conduct immunoassay analysis of edible tissues. Milestone Not Met Redirection of Research focus due to change in priorities 4. Complete validation of antibody properties; further optimize analysis if necessary; complete metabolism cross reactivity work. Milestone Substantially Met 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? This current CRIS project will expire in 2005 and a replacement project is currently in the review process. The new project plan will be implemented during the fall of 2005. Therefore, speculative fiscal year 2006, 2007, and 2008 objectives will not be included in this report. 4a What was the single most significant accomplishment this past year? Development of Sodium Chlorate Feed Additive as a Food Safety Tool. Studies were conducted in cattle to determine the identity of chemical residues in edible tissues after treatment with an investigational chlorate-based feed additive. It was demonstrated that residues of sodium chlorate in edible tissues of beef cattle will not limit the development of chlorate as a food safety tool. Chlorate is being developed as a feed additive because it inhibits the growth of pathogens such as E. coli O157:H7 and Salmonella in live animals. We determined that the residues fell below safe tissue concentrations estimated by the US
FDA Center for Veterinary Medicine. This technology has the potential to be a safe and effective pre-harvest tool to reduce the incidence of pathogens in live animals prior to entering the human food chain. 4b List other significant accomplishments, if any. Zilpaterol Residue Method Developed. A fellowship under the OECD co-operative Research Program: Biological Resource Management for Sustainable Agriculture Systems has been completed. The fellowship allowed cooperative research between Department of Agriculture and Rural Development (Ireland) and Agricultural Research Service. A surface plasmon resonance biosensor method was developed to evaluate 9 zilpaterol antibodies. The sensor method showed excellent recovery and reproducibility for low ppb zilpaterol concentrations. A comparison of the zilpaterol biosensor method with ELISA demonstrated that both methods give equivalent results with a high correlation between them. This rapid analytical method is available for use to
screen animal tissues for illegal residues. 4d Progress report. This report serves to document research conducted under a reimbursable agreement between ARS and the National Cattlmens Beef Association. Each year thousands of US consumers become ill because they have eaten food products that are contaminated with pathogenic bacteria. Intense efforts have been made to eliminate pathogenic organisms from beef animals before they are slaughtered and to remove contamination after slaughter. A new pre-harvest food safety strategy has been developed that has been shown to greatly reduce, or even eliminate gram-negative pathogens from live cattle. Use of this new feed additive has not yet been approved by regulatory organizations because it is not known whether residues present in edible tissues of treated animals would represent a health risk. The purpose of this study was to quantify residues of a novel chlorate based feed additive in edible tissues of beef animals. For all of the doses
tested, chlorate residues in liver, kidney, muscle, and fat fell well below amounts that the FDA have estimated to be safe. The major metabolite of chlorate was chloride, a nutrient already present in almost all human food sources. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The research reported is conducted under National Program 108, Food Safety and directly contributes to research Component 1.1, Pathogens, Toxins, and Chemical Contaminants Preharvest, and research Component 1.2, Pathogens, Toxins and Chemical Contaminants Postharvest. Research is further related to ARS strategic plan Objective 3.1: Provide Science Based Knowledge on the Safe Production, Processing, and Handling of Plant and Animal Products and on the Detection and Control of Toxin- producing and/or Pathogenic Bacteria and Fungi, Parasites, Chemical Contaminates, Mycotoxins, and Plant Toxins. A monoclonal antibody specific for ractopamine was
generated and its usefulness at detecting ractopamine residues in urine and tissues of farm animals was developed. A U.S. patent for this technology was issued (no. 6,274,334; August 14, 2001); Testing Components Corporation and the Neogen Corporation have licensed the technology. Several other organizations have requested information regarding licensing agreements. The cell line has been deposited in American Type Culture Collection (PTA- 2103) and Confidentiality and Material Transfer Agreements have been filed with several research groups. Details of the immunoassay has been requested by a variety of groups ranging from foreign government regulatory agencies (Germany, Northern Ireland, Hong Kong, South Korea), US government agencies (USDA-FSIS, FDA), State Agencies (Colorado), domestic and international industrial concerns, academia, and end users (State Fair Organizers, Horse Racing Officials). The development and use of this antibody will allow domestic and foreign regulatory
agencies detect the off-label and(or) illegal use of ractopamine in target and non- target species. Detection of such use will increase the safety of domestic and foreign food supplies (Objective 4; Component 1.2)) A monoclonal antibody specific for the beta-agonist zilpaterol has been generated, its specificity determined, and its usefulness in detecting zilpaterol residues in urine and edible tissues of farm animals was determined. This is the first antibody developed with application for the rapid detection of zilpaterols illegal use in food-animals. Material transfer agreements have been issued to several companies allowing for the evaluation of the antibody and one company has received a non-exclusive license for the technology. Rapid-screening assays for zilpaterol will be used by private industry and government laboratories for the determination of illegal residues of zilpaterol in edible tissues, and could be used by scientists to determine zilpaterol in other matrices
(Objective 4; Component 1.2). The rates of ergovaline isomerization in solvents and cell culture media were measured. Ergovaline isomerizes extremely rapidly to ergovalinine in some cell culture media, emphasizing the need for researchers to store ergovaline under conditions of known stability and to dose ergovaline of known isomer composition. Two commercially available feed additives, marketed as products that reduce fescue toxicosis, did not reduce cellular toxicity at the concentrations tested. Both ergovaline and its naturally occurring isomer, ergovalinine, readily crossed gastrointestinal. cells intact, and with similar kinetics. Because both isomers were transported, either isomer, or a combination of both, could be involved in the pathogenesis of fescue toxicosis at sites distal to the gastrointestinal tract. These studies advance the understanding of ergovalines involvement in fescue toxicity and will be used by scientists to further understand the overall mechanisms of
fescue toxicosis (Objective 1; Component 1.1). The disposition and metabolism of orally and intravenously administered [14C]phenylbutazone was determined in beef cattle. In cattle, parent phenylbutazone is the major metabolite, whereas in other species, only small amounts of phenylbutazone are excreted intact. Data from these studies support the Food and Drug Administrations decision to monitor edible tissues for parent phenylbutazone rather than metabolites. Results indicated that liver or blood would be a more appropriate target tissue for regulatory measurement of phenylbutazone than the kidney, but with the sensitive analytical tools available to regulatory agencies, phenylbutazone residues are likely detectable in other tissues for extended periods of time. Results of this study will increase the efficiency with which regulatory agencies screen for illegal phenylbutazone use in food animals. (Objective 3; Component 1.2) The metabolism and residues of sodium [36Cl]chlorate were
measured in beef cattle and the effect of sodium chlorate dose on chlorate residues in edible tissues was determined. Sodium chlorate is an investigational feed additive that is being developed for use in the cattle industry because of its specific bactericidal effects on pathogens of importance to the beef industry (E. Coli O157:H7). Studies indicated that chlorate is converted to chloride ion and that both chloride and to a lesser extent chlorate ions are present in edible tissues. These results indicate that a large proportion of chlorate residues in edible tissues of cattle are present as natural products. Residues of chlorate in edible tissues of cattle fell below provisional safe tissue concentrations provided by the US FDA Center for Veterinary Medicine (Components 1.1 and 1.2). 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? Material Transfer Agreements for ractopamine monoclonal antibodies have been affected with Dr. Richard Sams, Ohio State University; Drs. Thomas Grace and Thomas Wida, Biacore, Inc., Dr. Fernando Rubio, ABRAXIS LLC, Dr. Richard Plange, Testing Components Corportation, Dr. Dwight Schreodt, Neogen Corporation, Dr. Deborah Morris, International Diagnostics Systems Corporation and Dr. Karl Schmitt, R-Biopharm AG, Germany (2001 to present) . Shelver, W. L. and D. J. Smith. 2001. A monoclonal antibody, cell line and immunoassay for ractopamine. United States Patent 6,274,334 B1. An Individual Memorandum of Understanding Agreement (#58-5442-2M-631) was established with Biacore Inc. (New Jersey, headquarter Uppsala, Sweden);Use of the Surface Plasmon Resonance Technique to Determine Residue Levels of Agrochemicals in Animal Tissues and Excreta (October, 2001) Non-exclusive license for ractopamine
antibody patent signed with Testing Components Corporation, October 2001; has been renewed annually to date. A specific cooperative agreement was signed between Xenosense Technologies (Belfast, Ireland) and the USDA ARS Biosciences Lab to conduct residue depletion studies with ractopamine and to test Biosensor technology for the detection of incurred ractopamine residues. Material Transfer Agreement signed with Neogen Corporation, Lexington Kentucky, for ractopamine antibody and drug conjugation method. January 2002. Non-exclusive license for ractopamine antibody patent signed with Neogen Corporation, 2002; has been reviewed annually to date. A series of lectures on the dispositions and analysis of ?-adrenergic agonists in food animals was presented to the Korean National Veterinary Research and Quarantine Service, Veterinary Environmental Residues Division in October of 2002. Work on the development of a ractopamine immunoassay was presented to the Taiwan National Animal Industry
Foundation and Taiwan Bureau of Animal and Plant Health Inspection and Quarantine (2002). The audience also included officers from the Taiwan Council of Agriculture and researchers from National Chung-Shing University. Trust Agreement with National Cattlmens Beef Association. 2004. Determination of a sodium chlorate dose that results in safe concentrations of tissue residues in beef cattle. $42,200. Trust Agreement with the National Pork Board. 2005. Determination of a sodium chlorate dose that results in safe concentrations of tissue residues in swine. $20,000. Presentations to the Beef Safety Committee at the Annual Meeting of the Cattle Industry, San Antonio, TX (February, 2005) and at the Production Working group of the Beef Industry Safety Summit (Orlando, April 2005) on the safety of chlorate residues after chlorate use in beef cattle. Non-exclusive license for zilpaterol antibody issued to Testing Components Corporation, (June 2005). Material transfer agreements were signed
with Euro-Diagnostica for zilpaterol and ractopamine antibodies (September, 2004), Beacon Analytical Systems for ractopamine antibodies (December 2004), and Immunalysis Corporation fro ractopamine antibodies (May 2005). Material transfer agreement with Envirologix, Inc and license agreement executed (August 2004) for thiamethoxam monoclonal antibodies. A Material transfer agreement with Envirologix, Inc. for purified polyclonal antibody specific for sugerbeet pathogen Cercaspora was established August 2005, in collaboration with, ARS, Sidney, MT).
Impacts
(N/A)
Publications
- Shappell, N.W., Billey, L.O. 2004. Assessment of tasco and ycwp on ergovaline toxicity in caco-2 cells. [abstract] Joint Meeting of American Society of Animal Science, American Dairy Science Association, and Poultry Science Association, St. Louis, MO, July 25-29, 2004, Journal of Animal Science 82(Suppl 1):181, Abstract T72.
- Smith, D.J., Huwe, J.K., Lorentzsen, M.K., Harrington, G.E. 2004. Biotransformation of [14c]-phenylbutazone after oral or intravenous administration to cattle. [abstract] American Chemical Society National Meeting, Philadelphia, PA, 8/22-26/2004, Picogram No. 67, Abstract No. 124.
- Smith, D.J., Anderson, R.C., Ellig, D.A., Larsen, G.L. 2004. Tissue distribution, elimination, and metabolism of dietary sodium [36cl]chlorate in beef cattle. [abstract] International Association for Food Protection 91st Annual Meeting, August 8-11, 2004, Phoenix, AZ. Abstract #T35.
- Smith, D.J., Oliver, C.E., Anderson, R.C., Caton, J.S. 2005. Residues and metabolism of 36cl-labeled sodium chlorate in cattle. [abstract] American Chemical Society (ACS) Spring Meeting, San Diego, CA, March 13-17, 2005, Picogram No. 68, Abstract No. 106.
- Kim, H., Shelver, W.L., Li, Q.X. 2004. Monoclonal antibody-based enzyme- linked immunosorbent assay for the insecticide imidacloprid. Analytica Chimica ACTA 509:111-118.
- Shelver, W.L., Kim, H., Li, Q.X. 2005. Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the beta-adrenergic agonist zilpaterol. Journal of Agricultural and Food Chemistry 53:3273- 3280.
- Smith, D.J., Anderson, R.C., Ellig, D.A., Larsen, G.L. 2005. Tissue distribution, elimination, and metabolism of dietary sodium [36Cl]chlorate in beef cattle. Journal of Agricultural and Food Chemistry 53:4272-4280.
- Shelver, Weilin L. 2005. Generation and Testing of Immunoaffinity Columns IN: METHODS IN MOLECULAR BIOLOGY, Vol. 295: Immunochemical Protocols, Third Edition. Edited by R. Burns, Humana Press Inc., Totowa, NJ, pp: 269-280.
- Oliver, C.E., Smith, D.J., Anderson, R.C., Caton, J.S. 2005. Pharmacokinetics of intraruminally-dosed 36cl-labeled sodium chlorate in cattle. [abstract] American Chemical Society (ACS) Spring Meetings. March 14-17, 2005, San Diego, CA, Picogram No. 68, Abstract No. 105.
- Shappell, N.W., Smith, D.J. 2004. Ergovaline transport across human gastrointestinal cells (caco-2). [abstract] Joint Meeting of American Society of Animal Science, American Dairy Science Association, and Poultry Science Association, St. Louis, MO, July 25-29, 2004, Journal of Animal Science 82(Suppl 1):181, Abstract T71.
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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? Animal health drugs have a remarkable history of safe use in the United States. With the exception of a few allergic reactions to penicillin or other antibiotics, there have been no documented examples of human toxicities as a result of animal drugs in meat products within the United States. Such is not the case in Europe and Asia, however, where humans have been poisoned by the ingestion of meats containing illegally used animal drugs. Phenylbutazone is an analgesic drug with a long history of use in humans and animals. Because phenylbutazone causes aplastic anemia in a small percentage of individuals, it is rarely used in human medicine and is banned for use in food animals. Nevertheless, a recent FDA/FSIS survey indicated that phenylbutazone residues were present in a surprisingly large
percentage of samples obtained from an abattoir processing cattle. We will determine the absorption, distribution, metabolism, and excretion (ADME) of phenylbutazone in cattle to learn how long it takes for phenylbutazone and its metabolites to deplete from treated animals. In addition, we will determine whether commercial immunologically-based assays are capable of detecting phenylbutazone and(or) its metabolites after prolonged withdrawal periods. The illegal, off-label, and inappropriate use of animal health drugs is discouraged by the US FDA and the USDA FSIS through nationwide surveillance and monitoring programs. A major tool for authorities overseeing these programs is immunologically based screening assays in which hundreds to thousands of samples are screened for specific drug residues. Samples that are positive for the drug residue are then analyzed by confirmatory methods designed to unequivocally identify the drug residue in question. Immunologically based detection
methods are rapid, sensitive, specific, inexpensive, and easy to perform. In addition, immunoassays may be field based and offer flexibility not encountered with other analytical assay formats. Once validated, the assays may be used by regulatory scientists, officials at packing plants, farmers, or other individuals who have a vested interest in whether an animal has been treated with a drug. We have developed immunoassays towards ractopamine HCl and other compounds of food safety interest and are showing their potential to detect drug residues in the excreta and tissues of treated animals. Cattle that graze N. coenophialum infected tall fescue are susceptible to a number of maladies that decrease their productive capabilities. The maladies, which are termed "fescue foot" or "fescue toxicosis", are hypothesized to be caused by ergopeptine alkaloids produced by the endophytic fungus N. coenophialum. The most abundant ergopeptine alkaloid present in affected plants, ergovaline, is
thought to be the causative agent of fescue toxicosis, but the syndrome has not been replicated using purified materials. In order for toxicity to occur, toxins must be absorbed and must reach target tissues. Establishment of the rate of toxin elimination through metabolism and excretion -or accumulation due to slow elimination- is fundamental to understanding the development of clinical signs of toxicosis. Thus, an understanding of the metabolism and disposition of the major ergopeptine alkaloids such as ergovaline is fundamental to an understanding of fescue toxicity. Unfortunately, no data exist on the absorption of the major alkaloids present in infected tall fescue; in addition, once absorbed it is not known whether the major ergopeptine alkaloids bind to specific tissues, are excreted rapidly, or are biotransformed to more toxic metabolites. We plan to investigate whole animal absorption and biotransformation of ergopeptine alkaloids and cellular transport mechanism using
ergovaline as a model compound. 2. List the milestones (indicators of progress) from your Project Plan. Objective 1. Determine the absorption, distribution, and metabolism of ergovaline in intact or surgically modified rodents. Year 1 (2001): Complete synthesis of [14C]ergovaline, complete live-phase of balance- excretion studies in rodents. Year 2 (2002): Complete metabolite identification including biliary metabolites of ergovaline in rodents; develop and validate residue methods for edible tissues. Year 3 (2003): No milestone for objective. Year 4 (2004): No milestone for objective. Year 5 (2005) No milestone for objective. Objective 2. Determine residue depletion characteristics of ergovaline and metabolites in cattle. Year 1. (2001): No milestone for objective. Year 2. (2002): No milestone for objective. Year 3 (2003): Conduct live phase of [14C]ergopeptine metabolism study in cattle. Measure total residues, initiate metabolite identification in urine/bile. Initiate
ruminal degradation studies. Year 4 (2004): Complete identification of ruminal ergovaline metabolites, complete tissue analysis of residue study. Initiate isolation of ruminal microbes that degrade ergovaline. Year 5 (2005): Initiate residue depletion study of ergovaline in cattle and determine tissue concentrations of residues. Objective 3. Determine the absorption, distribution, metabolism, and excretion of phenylbutazone in cattle. Determine the marker compound and target tissues for regulatory assays; determine whether available immunological techniques accurately detect phenylbutazone use. Year 1 (2001): Synthesize [14C]phenylbutazone; dose cattle for balance excretion study; complete total residue measurements. Year 2 (2002): Complete metabolite identification; determine immunoassay cross reactivity with metabolites. Year 3 (2003): Validate tissue residue methods for parent phenylbutazone. Year 4 (2004): Initiate residue depletion study in cattle; complete tissue
total residue analysis; determine marker compound; determine target tissue; conduct immunoassay analysis of edible tissues. Year 5 (2005): No objectives listed. Objective 4. Develop and validate rapid, sensitive, and simple immunological techniques for the detection of animal health products of potential food safety concern. Year 1 (2001): Design and synthesize haptens; complete conjugation of haptens to proteins. Year 2 (2002): Immunize animals; develop analysis for measuring antibody content. Year 3 (2003): Develop the antibody based analyses; explore matrix effects, variability. Initiate live phase metabolism study. Year 4 (2004): Complete validation of antibody properties; further optimize analysis if necessary; complete metabolism cross reactivity work. Year 5 (2005). Validation of immunoaffinity column technology. 3. Milestones: Objective 1. Determine the absorption, distribution, and metabolism of ergovaline in intact or surgically modified rodents. No specific
experiments were planned under this objective for fiscal 2004 because radiolabeled ergovaline was not procured. The chemical synthesis of radiolabeled ergovaline was not successful; therefore this objective was not met. No metabolism studies with radiolabeled ergovaline have been conducted or are currently being planned. Objective 2. Determine residue depletion characteristics of ergovaline and metabolites in cattle. The chemical synthesis of radiolabeled ergovaline was not successful; therefore this objective was not met. Because radiolabeled ergovaline was not available during the life of the project, a series of other studies have superseded studies outlined with radiolabeled ergovaline. In 2004, the transport of ergovaline and its diastereoisomer, ergovalanine, across an in vitro intestinal cell model was completed. An additional study was conducted that measured the effectiveness of a commercial seaweed extract and a yeast extract at preventing the toxicity of ergovaline in
an intestinal cell model. Objective 3. Initiate residue depletion study in cattle; complete tissue total residue analysis; determine marker compound; determine target tissue; conduct immunoassay analysis of edible tissues. Studies on the identification of phenylbutazone metabolites were not sufficiently completed to initiate a phenylbutazone residue depletion trial. The identification and characterization of the major urinary metabolites of phenylbutazone was completed in fiscal 2004, as was the identification of the major tissue residue. Because absorption, metabolism, and excretion data for phenylbutazone are no longer critical to the FDA and USDA-FSIS regulatory needs, this objective has been superseded with an different objective: to measure the absorption, distribution, metabolism, and excretion of sodium chlorate in beef cattle. Sodium chlorate is specifically toxic to pathogens such as E. Coli O157:H7 and Salmonella in live animals, and its commercial use is dependent upon
the residues of chlorate being determined in food animals. To this end, two studies investigating the fate and metabolism of sodium [36Cl]chlorate in 8 beef cattle were conducted. Objective 4. Complete validation of antibody properties; further optimize analysis if necessary; complete metabolism cross reactivity work. Monoclonal antibodies for the feed additive zilpaterol and for the insecticides imidacloprid and thiamethoxam were developed. A feeding trial was conducted in order to generate excreta and tissues containing incurred zilpaterol residues; these tissues were used to validate the zilpaterol immunoassay that was developed. A series of experiments were initiated to develop a zilpaterol biosensor assay, which uses the monoclonal antibody as the basis of zilpaterol detection. B. List the milestones that you expect to address over the next 3 years (FY2005, 2006, and 2007). What do you expect to accomplish, year-by-year, over then next 3 years under each milestone? This CRIS
project will expire after 2005 and a new project will be written within the next year. FY2005. Objectives 1 and 2: Because the chemical synthesis of radiolabeled ergovaline was unsuccessful and because the resources are not available to produce radiolabeled lysergic acid via fermentation, focus will continue on the development of sodium chlorate as a pre- harvest intervention method for the elimination of human pathogens. To this end, a titration study of sodium chlorate dose on tissue residues has been initiated in beef cattle and will be completed in FY2005; its goal is to determine the dose of sodium chlorate that will result in safe chlorate concentrations in tissues. A pharmacokinetic study of sodium chlorate in cattle has been initiated and will be completed in 2005. Investigate the fate and transport of excreted sodium chlorate in soils. Investigate ruminal conditions that influence the ruminal biotransformation of sodium chlorate in cattle. Investigate the intestinal
transport of sodium chlorate using cell models. Objective 3. The cross reactivity of identified phenylbutazone metabolites to a commercially available phenylbutazone immunoassay will be determined. Determine the biotransformation of zilpaterol in sheep using LC/MS/MS techniques. Objective 4: Conduct animal feeding trials for the validation of the immunoassays for neonicotinoid insecticides. FY2006. Objectives 1 and 2. Determine the metabolic fate and residues of sodium chlorate in poultry and(or) swine. Develop a determinative analytical method for chlorate in animal tissues. Determine the sites and mechanisms of sodium chlorate absorption in ruminants. Determine whether mammalian tissues have the capability of biotransforming sodium chlorate. Objective 3. It is not anticipated that further work with phenylbutazone will be pursued. Objective 4: Studies will continue supporting the development of the zilpaterol antibody as well as other new antibodies to target compounds that
may have been developed during fiscal 2004 and 2005. FY2007. Objectives 1 and 2. Continue residue, mechanistic, and analytical studies to support the use of sodium chlorate as a safe pre- harvest intervention strategy to eliminate human pathogens in live animals. Objective 4. Support the development of new rapid screening assays and the development of new immunologically-based technologies to improve the safety of animal-derived food products. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment. A monoclonal antibody specific for the beta-agonist zilpaterol has been generated, its specificity determined, and its usefulness in detecting zilpaterol residues in urine and edible tissues of farm animals was determined. Beta-adrenergic agonists have a sordid history of illegal use across the globe and rapid screening assays for all classes of beta- agonists are in high demand. Dr. Shelver developed the first antibody with
application for the rapid detection of zilpaterol's illegal use in food-animals and other species. The importance of Dr. Shelver's work is illustrated by her invitation to work in the Laboratory of Dr. Chris Elliot, Belfast, Ireland, to develop a the antibody into a rapid, high- throughput assay using state of the art Biosensor technology. Once developed and validated, the assay has the potential to be widely used by European, Asian, and US regulatory agencies. B. Other significant accomplishments. The metabolism and residues of sodium [36Cl]chlorate were measured in beef cattle. Sodium chlorate is an investigational drug that is being developed for use in the cattle industry because of its specific bactericidal effects on pathogens of importance to the beef industry (E. Coli O157:H7). Studies indicated that chlorate is converted to a non toxic metabolite (chloride ion) and that both chloride and, to a lesser extent, chlorate ions are present in edible tissues. These results
indicate that a large proportion of chlorate residues in edible tissues of cattle are present as natural products. The study had an immediate impact on leaders of the beef industry; after reviewing results of the initial preliminary study, the National Cattlemen's Beef Association provided support for a second residue study that is now in progress. C. Significant Accomplishments/Activities that Support Special Target Populations. None. D. Progress Report. (Optional) 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A monoclonal antibody specific for ractopamine was generated and its usefulness at detecting ractopamine residues in urine and tissues of farm animals was developed. A U.S. patent for this technology was issued (no. 6,274,334; August 14, 2001); Testing Components Corporation and the Neogen Corporation have licensed the technology. Several other organizations have requested information regarding licensing
agreements. The cell line has been deposited in American Type Culture Collection (PTA- 2103) and Confidentiality and Material Transfer Agreements have been filed with several research groups. Details of the immunoassay has been requested by a variety of groups ranging from foreign government regulatory agencies (Germany, Northern Ireland, Hong Kong, South Korea), US government agencies (USDA-FSIS, FDA), State Agencies (Colorado), domestic and international industrial concerns, academia, and end users (State Fair Organizers, Horse Racing Officials). The development and use of this antibody will allow domestic and foreign regulatory agencies detect the off-label and(or) illegal use of ractopamine in target and non- target species. Detection of such use will increase the safety of domestic and foreign food supplies (Objective 4)) A monoclonal antibody specific for the -agonist zilpaterol has been generated, its specificity determined, and its usefulness in detecting zilpaterol
residues in urine and edible tissues of farm animals was determined. This is the first antibody developed with application for the rapid detection of zilpaterol's illegal use in food-animals. The use of a rapid-screening assay for zilpaterol will increase the safety of domestic and foreign food supplies (Objective 4). The rates of ergovaline isomerization in solvents and cell culture media were measured. Ergovaline isomerizes extremely rapidly to ergovalinine in some cell culture media, emphasizing the need for researchers to store ergovaline under conditions of known stability and to dose ergovaline of known isomer composition. Two commercially available feed additives, marketed as products that reduce fescue toxicosis, did not reduce cellular toxicity at the concentrations tested. Both ergovaline and its naturally occurring isomer, ergovalinine, readily crossed gastrointestinal. cells intact, and with similar kinetics. Because both isomers were transported, either isomer, or a
combination of both, could be involved in the pathogenesis of fescue toxicosis at sites distal to the gastrointestinal tract. These studies advance the understanding of ergovaline's involvement in fescue toxicity (Objective 1). The disposition and metabolism of orally and intravenously administered [14C]phenylbutazone was determined in beef cattle. In cattle, parent phenylbutazone is the major metabolite, whereas in other species, only small amounts of phenylbutazone are excreted intact. Because of these studies, the Food Safety and Inspection Service. Results indicated that liver or blood would be a more appropriate target tissue for regulatory measurement of phenylbutazone than the kidney. Results of this study will increase the efficiency with which regulatory agencies screen for illegal phenylbutazone use in food animals (Objective 3). The metabolism and residues of sodium [36Cl]chlorate were measured in beef cattle. Sodium chlorate is an investigational drug that is being
developed for use in the cattle industry because of its specific bactericidal effects on pathogens of importance to the beef industry (E. Coli O157:H7). Studies indicated that chlorate is converted to chloride ion and that both chloride and to a lesser extent chlorate ions are present in edible tissues. These results indicate that a large proportion of chlorate residues in edible tissues of cattle are present as natural products. These studies are being supported by the National Cattlemen's Beef Association and by EKA Chemicals. 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? Material Transfer Agreements for ractopamine monoclonal antibodies have been affected with Dr. Richard Sams, Ohio State University; Drs. Thomas Grace and Thomas Wida, Biacore,
Inc., Dr. Fernando Rubio, ABRAXIS LLC, Dr. Richard Plange, Testing Components Corportation, Dr. Dwight Schreodt, Neogen Corporation, Dr. Deborah Morris, International Diagnostics Systems Corporation and Dr. Karl Schmitt, R-Biopharm AG, Germany (2001 to present) . Shelver, W. L. and D. J. Smith. 2001. A monoclonal antibody, cell line and immunoassay for ractopamine. United States Patent 6,274,334 B1. An Individual Memorandum of Understanding Agreement (#58-5442-2M-631) was established with Biacore Inc. (New Jersey, headquarter Uppsala, Sweden);"Use of the Surface Plasmon Resonance Technique to Determine Residue Levels of Agrochemicals in Animal Tissues and Excreta" (October, 2001) Non-exclusive license for ractopamine antibody patent signed with Testing Components Corporation, October 2001; has been renewed annually to date. A specific cooperative agreement was signed between Xenosense Technologies (Belfast, Ireland) and the USDA ARS Biosciences Lab (W. Shelver) to conduct residue
depletion studies with ractopamine and to test Biosensor technology for the detection of incurred ractopamine residues. Material Transfer Agreement signed with Neogen Corporation, Lexington Kentucky, for ractopamine antibody and drug conjugation method. January 2002. Non-exclusive license for ractopamine antibody patent signed with Neogen Corporation, 2002; has been reviewed annually to date. Application package for Non-exclusive license was requested by International Diagnostics Systems Corporation and was sent February 2004. Trust Agreement with National Cattlmen's Beef Association. 2004. Determination of a sodium chlorate dose that results in safe concentrations of tissue residues in beef cattle. $42,200. A series of lectures on the dispositions and analysis of -adrenergic agonists in food animals was presented to the Korean National Veterinary Research and Quarantine Service, Veterinary Environmental Residues Division in October of 2002 (D. J. Smith). Work on the development of
a ractopamine immunoassay was presented to the Taiwan National Animal Industry Foundation and Taiwan Bureau of Animal and Plant Health Inspection and Quarantine. The audience also included officers from the Taiwan Council of Agriculture and researchers from National Chung-Shing University (W. Shelver). 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Suszkiw, J. 2004. New Tests Detect Growth Promoter in Livestock, Agricultural Research, 52:20.
Impacts
(N/A)
Publications
- Shelver, W.L., Smith, D.J. 2003. Determination of ractopamine in cattle and sheep urine using biosensor. VII International Conference on Agri-Food Antibodies, September 10-13, 2003, Uppsala, Sweden.
- Shields, T.H., Smith, T.R., Althen, T., Smith, D.J. The effect of ractopamine stereoisomers on lipolysis of bovine subcutaneous and intramuscular adipose tissue. [abstract]. Federation of American Societies for Experimental Biology Annual Meeting, April 17-24, 2004, Washington, DC. Abstract #8086.
- Shelver, Weilin L. and Smith, David J. 2004. Enzyme-linked immunosorbent assay development for the beta-adrenergic agonist zilpaterol. Journal of Agricultural and Food Chemistry. 52:2159-2166.
- Shelver, W.L., Smith, D.J. 2004. Development of an enzyme-linked immunosorbent assay for beta-agonist zilpaterol. EuroResidue V, May 10-12, 2004, Noordwijkerhout, The Netherlands.
- Shaikh, B., Rummel, N., Smith, D.J. 2004. Determination of 14C residue in eggs of laying hens administered orally with [14C]sulfaquinoxaline. Journal of Food Additives & Contaminants. 21:545-554.
- Shappell, N.W. 2003. Ergovaline toxicity on Caco-2 cells as assessed by MTT, alamarBlue, and DNA assay. In Vitro Cellular and Developmental Biology-Animal. 39:329-335.
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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? Animal health drugs have a remarkable history of safe use in the United States. With the exception of a few allergic reactions to penicillin or other antibiotics, there have been no documented examples of human toxicities as a result of animal drugs in meat products within the United States. Such is not the case in Europe and Asia, however, where humans have been poisoned by the ingestion of meats containing illegally used animal drugs. Phenylbutazone is an analgesic drug with a long history of use in humans and animals. Because phenylbutazone causes aplastic anemia in a small percentage of individuals, it is rarely used in human medicine and is banned for use in food animals. Nevertheless, a recent FDA/FSIS survey indicated that phenylbutazone residues were present in a surprisingly large percentage of samples obtained from an abattoir processing cattle. We will determine the
absorption, distribution, metabolism, and excretion (ADME) of phenylbutazone in cattle to learn how long it takes for phenylbutazone and its metabolites to deplete from treated animals. In addition, we will determine whether commercial immunologically-based assays are capable of detecting phenylbutazone and(or) its metabolites after prolonged withdrawal periods. The illegal, off-label, and inappropriate use of animal health drugs is discouraged by the US FDA and the USDA FSIS through nationwide surveillance and monitoring programs. A major tool for authorities overseeing these programs is immunologically based screening assays in which hundreds to thousands of samples are screened for specific drug residues. Samples that are positive for the drug residue are then analyzed by confirmatory methods designed to unequivocally identify the drug residue in question. Immunologically based detection methods are rapid, sensitive, specific, inexpensive, and easy to perform. In addition,
immunoassays may be field based and offer flexibility not encountered with other analytical assay formats. Once validated, the assays may be used by regulatory scientists, officials at packing plants, farmers, or other individuals who have a vested interest in whether an animal has been treated with a drug. We have developed immunoassays towards ractopamine HCl and other compounds of food safety interest and are showing their potential to detect drug residues in the excreta and tissues of treated animals. Cattle that graze N. coenophialum infected tall fescue are susceptible to a number of maladies that decrease their productive capabilities. The maladies, which are termed 'fescue foot' or 'fescue toxicosis', are hypothesized to be caused by ergopeptine alkaloids produced by the endophytic fungus N. coenophialum. The most abundant ergopeptine alkaloid present in affected plants, ergovaline, is thought to be the causative agent of fescue toxicosis, but the syndrome has not been
replicated using purified materials. In order for toxicity to occur, toxins must be absorbed and must reach target tissues. Establishment of the rate of toxin elimination through metabolism and excretion -or accumulation due to slow elimination- is fundamental to understanding the development of clinical signs of toxicosis. Thus, an understanding of the metabolism and disposition of the major ergopeptine alkaloids such as ergovaline is fundamental to an understanding of fescue toxicity. Unfortunately, no data exist on the absorption of the major alkaloids present in infected tall fescue; in addition, once absorbed it is not known whether the major ergopeptine alkaloids bind to specific tissues, are excreted rapidly, or are biotransformed to more toxic metabolites. We plan to investigate whole animal absorption and biotransformation of ergopeptine alkaloids and cellular transport mechanism using ergovaline as a model compound. 2. How serious is the problem? Why does it matter?
Illegal use of beta-adrenergic agonist drugs for enhancing the leanness of food animals has been documented to occur in Belgium, Canada, China, France, Germany, Hong Kong, Ireland, Italy, Mexico, Portugal, Spain, The Netherlands, and the United States. Consumption of liver, muscle, and in one case soup prepared from lungs, harvested from animals illegally treated with clenbuterol has been documented to cause human intoxication. In the United States, the FDA has prosecuted at least 4 cases of illegal beta-adrenergic agonist use. Although clenbuterol has been widely studied, residue studies of several beta-agonists that are widely available have not been conducted. The extent to which the anti- inflammatory agent phenylbutazone is being used illegally is not known for certain, but recent surveys have indicated that a large proportion of large-animal veterinarians use phenylbutazone in food animals. Data generated by FDA and FSIS suggest that as many as 5% of cattle from one
slaughterhouse had phenylbutazone residues. Knowledge of the ADME of these compounds will enhance the understanding of human risks from residues, and will help determine which chemical moieties need to be detected for monitoring purposes. Rapid screening methodology does not exist for many animal-health compounds that are used in an off-label or illegal manner. Development of these assays will aid local, state, and federal regulatory agencies in the detection of treated animals or tainted carcasses. It has been estimated that nearly 8.5 million cattle and over 1 million horses are annually managed on pastures seeded primarily with tall fescue. The continued use of tall fescue as a forage crop, even when toxicity problems sometimes occur, testifies to its value to animal producers. Tall fescue's value rests in its outstanding heartiness, durability, nutrient composition, and drought-tolerance. However, the annual cost of fescue related toxicity to the livestock industry exceeds 600
million dollars. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? This research was conducted under National Program 108, Food Safety (Animal Products) and is designed to detect, quantify, and characterize chemicals and their metabolites, of potential toxicological concern present in edible tissues of food animals. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment. In order to satisfy export requirements, a great need has arisen for inexpensive, rapid, and accurate analytical methods for the detection of ractopamine residues in food animals. Immunochemical isolation and detection studies conducted by Shelver and Smith at the Biosciences Research Laboratory have established the utility of a ractopamine monoclonal antibody as a viable tool for a rapid preparation and screening method for determining the presence or absence of ractopamine residues in live animals
or animal carcasses. The studies have shown specifically that the ractopamine antibody, developed at the Biosciences Research Laboratory, may be used as an immunoaffinity column for the purification of ractopamine residues from animal tissues and that biosensor technology can serve as the basis for ractopamine screening programs. These purification and detection techniques may be used by local state, federal, or international regulatory officials for the determination of ractopamine residues in live animals or in animal carcasses. B. Other significant accomplishments. Studies investigating the absorption and fate of toxic alkaloids present in endophyte-infected tall fescue have been difficult to conduct because: 1) naturally exposed animals receive extremely small doses that are difficult to measure after absorption; and 2) attempts to synthesize radiolabeled materials (used to track the alkaloid during and after absorption) have been unsuccessful. Studies performed by Shappell, of
the Biosciences Research Laboratory, demonstrated that a tall fescue alkaloid, ergovaline, readily crosses through intestinal cells. Thus, limitations in ergovaline bioavailability at the level of the intestinal cell is not likely to occur. Dr. Shelver has successfully developed polyclonal antibodies towards the b-agonist zilpaterol, which has been approved by some countries for use as a production enhancing agent in food animals. The antibody is both sensitive and specific and shows great promise for use in screening assays. C. Significant Accomplishments/Activities that Support Special Target Populations. None. D. Progress Report. (Optional) This report serves to document research conducted under a reimbursable agreement between the ARS and Auburn University. Additional details of research can be found in the report for the parent project 5442-32000-008- 00 D. The goal of the subordinate CRIS is to procure [14C]ergovaline by synthetic techniques. The synthetic strategy is to
incorporate [14C]proline into the tricyclic peptide portion of ergovaline; due to the nature of radiolabel incorporation a novel synthetic strategy had to be developed. A synthetic strategy has been completed using 'cold' starting materials, but attempts to incorporate [14C]proline in a small scale synthesis were not successful. Currently efforts are being made to determine the cause of the poor incorporation of [14C]proline. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A monoclonal antibody to ractopamine HCl has been developed, characterized, and its utility described in a number of applications including enzyme-linked immunosorbent assay, immunoaffinity chromatography, and biosensor analysis in excreta and urine from the major food animal species. The excretion, depletion, and(or) tissue residues of ractopamine and its major metabolites have been measured in four species of food animals. These studies have
demonstrated the utility and characterized the application limits of immunologically based detection methods for ractopamine screening methods in biological systems. Two companies have licensed the technology and several other groups have signed Material Transfer Agreements in order to evaluate it for possible licensing. The toxicity of ergovaline to CACO-2 cells has been described and the cell model is being used in transport studies. Studies investigating the stereochemical stability of ergovaline in protic and aprotic solvents have established appropriate storage and experimental handling procedures of the toxin. A disposition study with phenylbutazone has established that cattle exposed to the drug tend to concentrate the drug in liver rather than in kidney. Results suggest that in efforts to monitor illegal or off-label phenylbutazone use, analysts should utilize liver rather than kidney. 6. What do you expect to accomplish, year by year, over the next 3 years? Year 1. With
the aid of a new liquid chromatograph with quadrapole time- of-flight mass spectrometer, phenylbutazone metabolism and ractopamine depletion projects will be completed and submitted for publication. Characterization and validation of a polyclonal-based zilpaterol immunoassay will continue. Animals will be dosed with zilpaterol to provide incurred tissue samples. A monoclonal antibody for zilpaterol will be developed and characterized. The absorption and transport of ergovaline by CACO-2 cells will be described. Studies on the metabolism and disposition of sodium [36Cl]chlorate in cattle and poultry will commence. Year 2. We will pursue the biochemical mechanisms of phenylbutazone binding to liver tissues and will characterize individual metabolites in edible tissues of animals treated with phenylbutazone. If radiolabeled ergovaline is not available from chemical synthesis, we plan to initiate studies to produce radiolabeled lysergic acid from culture and to use it as a starting
material for the production of [14C]ergovaline. If that is not possible, studies investigating the importance of peramine, quantitatively the most important alkaloid in endophyte-infected tall fescue will be initiated. These studies will include synthesis of [14C]labeled peramine and investigation of peramine's ability to induce cytochrome P450. Studies will be continued which characterize the recently developed zilpaterol polyclonal and monoclonal antibodies as applied to animal-derived samples. Results from antibody-based methods will be quantitatively compared to results from LC-MS methods. Studies investigating the fate of [36Cl]-labeled sodium chlorate will continue in potential target animals (swine, poultry, or sheep). The ruminal biotransformation of sodium chlorate will be described. Year 3. Radiolabeled ergovaline(if available) or peramine will be dosed to cattle (or other appropriate species, depending upon the mass of label available) and its absorption,
distribution, metabolism, and excretion will be determined. Studies will continue supporting the development of the zilpaterol antibody as well as other new antibodies that may have been developed during year two. It is anticipated that sodium chlorate disposition studies conducted in years 1 and 2 will lead to further studies investigating the mechanisms of clearance in food animals. Studies in year 3 will determine the rate of sodium chlorate clearance and clearance mechanisms. 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? A licensing agreement was signed between the USDA ARS technology transfer office and Testing Components, Inc. for the monoclonal antibody developed against ractopamine and patented by Shelver and Smith. The licensing
agreement was signed in fiscal 2001, 2002, and renewed for 2003. A similar licensing agreement was established between Neogen, Inc. and the USDA ARS technology transfer office in fiscal 2002 and 2003. Material Transfer Agreements were established with International Diagnostics Systems Corporation, St. Joseph, MI and R-Biopharm AG, Germany in order for them to evaluate the previously mentioned ractopamine antibody. Availability of commercial products derived from the licensing agreements, will depend upon development schedules of the licensing companies. David Smith presented a series of lectures on the dispositions and analysis of b-adrenergic agonists in food animals to the Korean National Veterinary Research and Quarantine Service, Veterinary Environmental Residues Division in October of 2002. Dr. Weilin Shelver presented her work on the development of a ractopamine immunoassay to the Taiwan National Animal Industry Foundation and Taiwan Bureau of Animal and Plant Health
Inspection and Quarantine. The audience also included officers from the Taiwan Council of Agriculture and researchers from National Chung-Shing University. Dr. Shelver's talk was entitled, 'The development of drug residue screening methods: ractopamine as a case study.' 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). No popular publications or presentations.
Impacts
(N/A)
Publications
- Mills, S. E., Kissel, J., Bidwell, C.A., Smith, D.J. Stereoselectivity of porcine beta-adrenergic receptors for ractopamine isomers. Journal of Animal Science. 2003. v. 81. p. 122-129.
- KLOTZ, K.L., FINGER, F.L., SHELVER, W.L. CHARACTERIZATION OF TWO SUCROSE SYNTHASE ISOFORMS IN SUGARBEET ROOT. PLANT PHYSIOLOGY AND BIOCHEMISTRY. 2003. V. 41 P. 107-115.
- Mills, S.E., Spurlock, M.E., Smith, D.J. Beta-adrenergic receptor subtypes that mediate ractopamine stimulation of lipolysis. Journal of Animal Science. 2003. v. 81. p. 662-668.
- Shelver, Weilin L., Smith, David J. Application of an immunoaffinity column as a sample cleanup method for the beta-adrenergic agonist ractopamine and its metabolites. Journal of Association of Official Analytical Chemists International. 2002. v. 85(6). p. 1302-1307.
- Shelver, Weilin L., Shan, Guomin, Gee, Shirley J., Stanker, Larry H., Hammock, Bruce D. Comparison of immunoaffinity column retention patterns of PCDDs/PCDFs on columns generated with different monoclonal antibody clones and polyclonal antibodies produced using different hapten conjugates. 2002. Analytica Chimica ACTA. v.457. p. 199-209.
- Shelver, Weilin L., Smith, David J. Immunologically based assays for pesticide/veterinary medicine residues in animal products. Philip W. Lee, ed. John Wiley Sons, Ltd. Handbook of Residue Analytical Methods for Agrochemicals, Vol. 2. 2003. p. 680-710.
- Shelver, Weilin L., Smith, David J. Determination of ractopamine in cattle and sheep urine samples using an optical biosensor analysis: comparative study with HPLC and ELISA. Journal of Agriculture and Food Chemistry. 2003. v. 51. p. 3715-3721.
- Smith D.J., Ehrenfreid, K. Dalidowicz, J.D., Turberg, M.P. The in vivo binding of ractopamine HCl to ocular tissues of cattle and turkeys and to melanin in vitro. Journal of Animal Science. 2002. v. 80. p. 2931-2941.
- Smith D.J., Stehly, G.S., Turberg, M.T. Procedures and practices for conducting residue studies of animal health compounds in food animals. P.W. Lee, ed. John Wiley Sons, Ltd. Handbook of Residue Analytical Methods for Agrochemicals, Vol. 1. 2003. p. 248-299.
- Shelver, Weilin L., Smith, David J. Application of a monoclonal antibody based ELISA for the determination of ractopamine in incurred samples from food animals. American Chemical Society. 2002. Abstract p. 37.
- Shelver, Weilin, L., Shan, Guomin, Gee, Shirley J., Stanker, Larry H., Hammock, Bruce D. Comparison of retention patterns of PCDDs/PCDFs on immunoaffinity columns generated with different monoclonal antibody clones and polyclonal antibodies produced using different hapten conjugates. American Chemical Society. 2002. Abstract p. 38.
|
Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Animal health drugs have a remarkable history of safe use in the United States. With the exception of a few allergic reactions to penicillin or other antibiotics, there have been no documented examples of human toxicities as a result of animal drugs in meat products within the United States. Such is not the case in Europe and Asia, however, where humans have been poisoned by the ingestion of meats containing illegally used animal drugs. Phenylbutazone is an analgesic drug with a long history of use in humans and animals. Because phenylbutazone causes aplastic anemia in a small percentage of individuals, it is rarely used in human medicine and is banned for use in food animals. Nevertheless, a recent FDA/FSIS survey indicated that phenylbutazone residues were present in a surprisingly large percentage of samples obtained from abattoir processing cattle. We will determine the
absorption, distribution, metabolism, and excretion (ADME) of phenylbutazone in cattle to learn how long it takes for phenylbutazone and its metabolites to deplete from treated animals. In addition, we will determine whether commercial immunologically-based assays are capable of detecting phenylbutazone and(or) its metabolites after prolonged withdrawal periods. The illegal, off-label, and inappropriate use of animal health drugs is discouraged by the US FDA and the USDA FSIS through nationwide surveillance and monitoring programs. A major tool for authorities overseeing these programs is immunologically based screening assays in which hundreds to thousands of samples are screened for specific drug residues. Samples that are positive for the drug residue are then analyzed by confirmatory methods designed to unequivocally identify the drug residue in question. Immunologically based detection methods are rapid, sensitive, specific, inexpensive, and easy to perform. In addition,
immunoassays may be field based and offer flexibility not encountered with other analytical assay formats. Once validated, the assays may be used by regulatory scientists, officials at packing plants, farmers, or other individuals who have a vested interest in whether an animal has been treated with a drug. We have developed immunoassays towards ractopamine HCl and have shown its potential to detect the drug and its metabolites in the excreta and tissues of treated animals. Cattle that graze Neotyphodium coenophialum infected tall fescue are susceptible to a number of maladies that decrease their productive capabilities. The maladies, which are termed "fescue foot" or "fescue toxicosis", are caused by ergopeptine alkaloids produced by the endophytic fungus N. coenophialum. The most abundant alkaloid present in affected plants, ergovaline, has been hypothesized to be the causative agent of fescue toxicosis, but the syndrome has not been replicated using purified materials. In
order for toxicity to occur, toxins must be absorbed and must reach target tissues. The rate of toxin elimination through metabolism and excretion -or accumulation due to slow elimination- is fundamental to the understanding of the developments of clinical signs of toxicosis. Thus an understanding of the metabolism and disposition of the major ergopeptine alkaloids such as ergovaline is fundamental to an understanding of fescue toxicity. Unfortunately, no data exist on the absorption of the major alkaloids present in infected tall fescue; in addition, once absorbed it is not known whether the major ergopeptine alkaloids bind to specific tissues, are excreted rapidly, or biotransformed to more toxic metabolites. We plan to investigate whole animal absorption and biotransformation of ergopeptine alkaloids and cellular transport mechanism using ergovaline as a model compound. 2. How serious is the problem? Why does it matter? Illegal use of beta-adrenergic agonists for enhancing
the leanness of food animals has been documented to occur in Belgium, Canada, China, France, Germany, Hong Kong, Ireland, Italy, Mexico, Portugal, Spain, The Netherlands, and the United States. Consumption of liver, muscle, and in one case soup prepared from lungs, harvested from animals illegally treated with clenbuterol has been documented to cause human intoxication. In the United States, the FDA has prosecuted at least 4 cases of illegal beta-adrenergic agonist use. Although clenbuterol has been widely studied, residue studies of several beta-agonists that are widely available have not been conducted. The extent to which phenylbutazone is being used illegally is not known for certain, but recent surveys have indicated that a large proportion of large-animal veterinarians use phenylbutazone in food animals. Data generated by FDA and FSIS suggest that as many as 5% of cattle from one slaughterhouse had phenylbutazone residues. Knowledge of the ADME of these compounds will
enhance the understanding of human risks from residues, and will help determine which chemical moieties need to be detected for monitoring purposes. It has been estimated that nearly 8.5 million cattle and over 1 million horses are annually managed on pastures seeded primarily with tall fescue. The continued use of tall fescue as a forage crop, even when toxicity problems sometimes occur, testifies to its value to animal producers. Tall fescue's value rests in its outstanding heartiness, durability, nutrient composition, and drought-tolerance. The annual cost of fescue related toxicity to the livestock industry exceeds 600 million dollars, however. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? This research was conducted under National Program 108, Food Safety (Animal Products) and is designed to detect, quantify, and characterize chemicals and their metabolites, of potential toxicological concern present in edible
tissues of food animals. 4. What was your most significant accomplishment this past year? A. Single most significant accomplishment. The distribution of phenylbutazone residues was studied in cattle in order to determine appropriate tissues for the development of residue screening programs. To this end, cattle were dosed intravenously or orally with radioactive phenylbutazone for 5 consecutive days and then slaughtered after a 5-day withdrawal period by scientists at the Biosciences Research Laboratory, Fargo, ND. Residues of phenylbutazone and metabolites were greatest in liver and blood, and parent phenylbutazone represented a major urinary excretory product. These results indicate that the current target tissue for residue screening (kidney) is likely not the best choice for a residue monitoring program and that urinary phenylbutazone residues could be measured when assessing live-animal exposure to phenylbutazone. B. Other significant accomplishments. Ractopamine HCl is a feed
additive approved for use in the United States, but European regulators have banned the importation of animal carcasses having residues of ractopamine. Therefore an immunoaffinity column was developed in order to simplify the time-consuming and multi-step analysis of ractopamine in food-animals. The immunoaffinity column worked well for the isolation of residual ractopamine from a variety of tissue types and results of tissue assays using the immunoaffintiy columns compared favorably with results using conventional assays. Use of the immunoaffintiy column would be applicable to determinative or confirmatory regulatory assays of ractopamine. Ergovaline is an ergopeptine alkaloid toxic to cattle after it is absorbed from the gastrointestinal tract. In order study absorption using a cellular model, the toxicity of ergovaline to the cells had to be evaluated. Studies at the USDA ARS Biosciences Research Laboratory showed that ergovaline is toxic to differentiated CACO-2 cells only at
high concentrations. These findings establish that the cell line will be viable tool for study of the transport of ergopeptines across gastrointestinal cells. 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 CRIS project was initiated in February 2000. Accomplishments of this project are itemized under number 4, above. For the previous CRIS project "Disposition of Beta-Agonists in Farm Animals", project number 5442-32000-006-00D, we described the tissue residues of ractopamine HCl and clenbuterol hydrochloride in several species. These studies also included descriptions of the stereochemical compositions of tissue residues and biotransformation products. We also demonstrated for the first time that the leanness enhancing activity of a phenolic phenethanolamine beta-agonist resides with specific stereoisomers. This
work was conducted with in vitro and in vivo studies. Of additional interest was the demonstration that the beta-agonist clenbuterol caused a redistribution of dioxins away from muscle and fat towards the liver of treated rats when clenbuterol was included in diets of dioxin- contaminated animals. We also were able to successfully generate polyclonal and monoclonal antibodies towards phenolic phenethanolamine beta agonists and have developed and validated immunologically based assays and sample preparation techniques from these antibodies. The stability of ergopeptine alkaloids in protic and aprotic solvents has been described and the toxicity of ergovaline to CACO-2 cells has been described. 6. What do you expect to accomplish, year by year, over the next 3 years? Year 1 (FY 2003). Tissue metabolites of phenylbutazone will be identified and a large-scale phenylbutazone residue depletion study in cattle will be initiated. The goal of this study will be to determine the relationship
of parent phenylbutazone with total metabolites at various stages of a withdrawal period. Commercial immunoassays will be evaluated for their abilities to predict tissue levels of phenylbutazone and for their ability to detect low levels of parent drug or metabolites. CACO-2 cells will be used to determine whether ergopeptine alkaloids are readily transported across the mucosal layer of the gastrointestinal tract. We will also continue the development and validation of rapid detection methods of ractopamine by determining the depletion of ractopamine residues from swine tissues using chromatographic, immunoassay, and biosensor techniques. Year 2 (FY 2004). Work will continue on the analysis of tissues from phenylbutazone depletion studies. We will characterize individual metabolites in edible tissues of animals treated with phenylbutazone. If radiolabeled ergovaline is not available from chemical synthesis, we plan to produce radiolabeled lysergic acid from culture and to use it
as a starting material for the production of [14C]ergovaline. Finally, we plan to continue to develop and validate rapid immunoassays for animal health drugs that have the potential for illicit use; rapid screening assays may be used for regulatory purposes in the detection illegal drug residues. Year 3 (FY 2005). Radiolabeled ergovaline will be dosed to cattle (or other appropriate species, depending upon the mass of ergovaline available) and its absorption, distribution, metabolism, and excretion will be determined. Excreta and tissues will be used for metabolite identification. 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? A licensing agreement was signed between the USDA ARS technology transfer office and Testing Components, Inc. for the monoclonal antibody developed and patented by
Shelver and Smith. The licensing agreement was signed in fiscal 2001 and renewed for fiscal 2002. A confidentiality agreement was signed between Xenosense Technologies (Belfast, Ireland) and the USDA ARS Biosciences Lab (W. Shelver) to conduct residue depletion studies with ractopamine and to test Biosensor technology for the detection of incurred ractopamine residues. 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) Dr. David J. Smith, presented a talk entitled, "Relative safety of clenbuterol and ractopamine residues in edible tissues of hogs" at the International Pig Veterinarian Society Meeting in Ames, Iowa, June 2-5, 2002.
Impacts
(N/A)
Publications
- Shelver, W.L., Smith, D.J. A monoclonal antibody, cell line, and immunoassay for ractopamine. 2001. U.S. Patent 6,274,334 B1.
- Smith, D.J., Shelver, W.L. Tissue residues of ractopamine and urinary excretion of ractopamine and metabolites in animals treated for 7 days with dietary ractopamine. Journal of Animal Science. 2002. v. 80. p. 1240- 1249.
- Shelver, W., Smith, David J. Application of a monoclonal antibody-based enzyme linked immunosorbent assay for the determination of ractopamine in incurred samples from food animals. Journal of Agricultural and Food Chemistry. 2002. v. 50. p. 2742-2747.
- Shappell, N.W. Toxicity of ergovaline on Caco2 cells as assessed by MTT, alamarBlue, and DNA analysis. Journal of Animal Science. 2002. v. 80(Suppl. 1). p. 30. Abstract 118.
- Shelver, W.L., Smith, D.J. The use of immunoaffinity columns for the isolation of ractopamine from edible tissues of food animals. Journal of Animal Science. 2002. v. 80(Suppl.1). p. 260. Abstract 1041.
- Smith, D.J., Shappell, N.W. Technical Note: Epimerization of ergopeptine alkaloids in organic and aqueous solvents. Journal of Animal Science. 2002. v. 80. p. 1616-1622.
- Lehotay, S.J., Lightfield, A.R., Anastassiades, M., Smith, D.J. Analysis of veterinary growth promoting drugs in animal tissues by LC/MS-MS and fluorescence. American Chemical Society. 2002. Picogram No. 62. Abstract No. 30.
- Lehotay, S.J., Lightfield, A.R., Anastassiades, M., Smith, D.J. Simultaneous analysis of beta-agonists and thyreostats in animal tissues by LC/MS and in-line fluorescence. Proceedings of the Fourth International Symposium on Hormone and Veterinary Drug Residue Analysis. 2002.
- Churchwell, M.I., Holder, C.L., Little, D., Preece, S., Smith, D.J., Doerge, D.R. Liquid chromatography/electrospray tandem mass spectrometric analysis of incurred ractopamine residues in livestock. Rapid Communication Mass Spectrometry. 2002. v. 16. p. 1261-1265.
- Shelver W.L., Smith, D.J. ELISA and immunoaffinity column development for beta adrenergic agonist ractopamine. 224th American Chemical Society National Meeting. 2002. Picogram No. 63:Abstract No. 20.
- Smith, D.J., Huwe, J.K. Disposition of orally and intravenously administered phenylbutazone in cattle. 224th American Chemical Society National Meeting. 2002. Picogram No. 63:Abstract No. 7.
|
Progress 10/01/00 to 09/30/01
Outputs
1. What major problem or issue is being resolved and how are you resolving it?
Animal health drugs have a remarkable history of safe use in the United States. With the exception of a few allergic reactions to penicillin or other antibiotics, there have been no documented examples of human toxicities as a result of animal drugs in meat products within the United States. Such is not the case in Europe and Asia, however, where humans have been poisoned by the ingestion of meats containing illegally used animal drugs. Phenylbutazone is an analgesic drug with a long history of use in humans and animals. Because phenylbutazone causes aplastic anemia in a small percentage of individuals, it is rarely used in human medicine and is banned for use in food animals. Nevertheless, a recent FDA/FSIS survey indicated that phenylbutazone residues were present in a surprisingly large percentage of samples obtained from an abattoir processing cattle. We will determine the absorption, distribution, metabolism, and excretion (ADME) of phenylbutazone in cattle to learn how
long it takes for phenylbutazone and its metabolites to deplete from treated animals. In addition, we will determine whether commercial immunologically-based assays are capable of detecting phenylbutazone and(or) its metabolites after prolonged withdrawal periods. The illegal, off-label, and inappropriate use of animal health drugs is discouraged by the US FDA and the USDA FSIS through nationwide surveillance and monitoring programs. A major tool for authorities overseeing these programs is immunologically based screening assays in which hundreds to thousands of samples are screened for specific drug residues. Samples that are positive for the drug residue are then analyzed by confirmatory methods designed to unequivocally identify the drug residue in question. Immunologically based detection methods are rapid, sensitive, specific, inexpensive, and easy to perform. In addition, immunoassays may be field based and offer flexibility not encountered with other analytical assay
formats. Once validated, the assays may be used by regulatory scientists, officials at packing plants, farmers, or other individuals who have a vested interest in whether an animal has been treated with a drug. We have developed immunoassays towards ractopamine HCl and have shown its potential to detect the drug and its metabolites in the excreta and tissues of treated animals. Cattle that graze N. coenophialum infected tall fescue are susceptible to a number of maladies that decrease their productive capabilities. The maladies, which are termed "fescue foot" or "fescue toxicosis", are caused by ergopeptine alkaloids produced by the endophytic fungus N. coenophialum. The most abundant alkaloid present in affected plants, ergovaline, has been hypothesized to be the causative agent of fescue toxicosis, but the syndrome has not been replicated using purified materials. In order for toxicity to occur, toxins must be absorbed and must reach target tissues. The rate of toxin
elimination through metabolism and excretion -or accumulation due to slow elimination- is fundamental to the understanding of the developments of clinical signs of toxicosis. Thus an understanding of the metabolism and disposition of the major ergopeptine alkaloids such as ergovaline is fundamental to an understanding of fescue toxicity. Unfortunately, no data exist on the absorption of the major alkaloids present in infected tall fescue; in addition, once absorbed it is not known whether the major ergopeptine alkaloids bind to specific tissues, are excreted rapidly, or biotransformed to more toxic metabolites. We plan to investigate whole animal absorption and biotransformation of ergopeptine alkaloids and cellular transport mechanism using ergovaline as a model compound.
2. How serious is the problem? Why does it matter?
Illegal use of beta-adrenergic agonists for enhancing the leanness of food animals has been documented to occur in Belgium, Canada, China, France, Germany, Hong Kong, Ireland, Italy, Mexico, Portugal, Spain, The Netherlands, and the United States. Consumption of liver, muscle, and in one case soup prepared from lungs, harvested from animals illegally treated with clenbuterol has been documented to cause human intoxication. In the United States, the FDA has prosecuted at least 4 cases of illegal beta-adrenergic agonist use. Although clenbuterol has been widely studied, residue studies of several beta-agonists that are widely available have not been conducted. The extent to which phenylbutazone is being used illegally is not known for certain, but recent surveys have indicated that a large proportion of large-animal veterinarians use phenylbutazone in food animals. Data generated by FDA and FSIS suggest that as many as 5% of cattle from one slaughterhouse had phenylbutazone
residues. Knowledge of the ADME of these compounds will enhance the understanding of human risks from residues, and will help determine which chemical moieties need to be detected for monitoring purposes. It has been estimated that nearly 8.5 million cattle and over 1 million horses are annually managed on pastures seeded primarily with tall fescue. The continued use of tall fescue as a forage crop, even when toxicity problems sometimes occur, testifies to its value to animal producers. Tall fescue's value rests in its outstanding heartiness, durability, nutrient composition, and drought-tolerance. The annual cost of fescue related toxicity to the livestock industry exceeds 600 million dollars, however.
3. How does it relate to the National Program(s) and National Component(s)?
This research was conducted under National Program 108, Food Safety (Animal Products) and is designed to detect, quantify, and characterize chemicals and their metabolites, of potential toxicological concern present in edible tissues of food animals.
4. What were the most significant accomplishments this past year?
A. Single Most Significant Accomplishment during FY 2000 year: A monoclonal antibody based ELISA towards ractopamine HCl was developed at the Animal Metabolism-Agricultural Chemicals Research Unit. The performance of this ELISA was validated using tissues and excreta from animals treated with Paylean and correlated (>90%) extremely well with analytical results obtained by HPLC for both tissues and excreta from sheep and cattle. The results suggest that the monoclonal based ELISA could be a very useful tool for screening tissue and/or excreta samples from animals with possible exposure to ractopamine, and such a rapid screening assay is needed for markets both nationally and internationally. B. Other significant accomplishments: Cattle and sheep were treated with dietary ractopamine and the urinary excretion of ractopamine and its conjugated metabolites was measured during a seven-day withdrawal period from the feed additive. Concentrations of parent ractopamine in sheep urine
were less than 20 ppb during the seven-day feeding period and fell below the limit of quantitation within 48 hours of being withdrawn from dietary ractopamine. When urine samples were pretreated with glucuronidase, ractopamine was easily detected in sheep urine seven days after the last dietary exposure. Ractopamine was readily detected in cattle urine during the ractopamine feeding period and was detectable by HPLC through about 3 days of withdrawal. After hydrolysis of ractopamine conjugates, ractopamine was detectable in urine samples 6 days after withdrawal. Analysis of the same sample hydrolysates using a sensitive ractopamine ELISA indicated that ractopamine could be detected in cattle and sheep urine samples at least 7 days after withdrawal. These results indicate that the off-label use of ractopamine can easily be detected in excreta of sheep and cattle at least 1 week after removal from ractopamine containing feed. Toxicity of ergovaline in gastrointestinal cells found to
depend on the differentiation state of the cells and the exposure period. Undifferentiated cells were more sensitive to the effects of ergovaline than were differentiated cells. This may, in part, explain the lack of thriftiness in animals exposed to ergovaline in contaminated fescue. If gastrointestinal tract replacement cells are preferentially sensitive to erogvaline, then the integrity of digestive system would be compromised, and perhaps absorptive capacity decreased. Stability studies were conducted with the ergopeptine alkaloids ergocryptine and ergovaline. Isomerization rates, as assessed by high performance liquid chromatography, of both ergocryptine and ergovaline varied with temperature, solvent, and pH. In general, rates of isomerization were greatest in protic solvents having a pH of 7 or greater. Addition of 10% fetal bovine serum to water adjusted to pH 3, rapidly increased the rate of isomerization. Solutions of ergocryptine stored at -40 degrees C. in
acetonitrile, water:methanol (70:30), acetone, chloroform, or methanol were stable. Ergocryptine and ergocryptinine reached complete equilibrium in methanol and water:methanol (70:30) within about 500 and 250 hours, respectively. Ergovaline and ergovalinine reached complete equilibrium within 7 hours when stored in buffered phosphate (pH 9 or 7.5; 37 degrees C.) and when stored in cell culture media or water fortified with 10% FBS. These results indicate that isomerization of ergot alkaloids occurs rapidly under conditions used for cell culture, and therefore the proportion of the active isomer remaining in solution during in vitro experiments will not be constant. The chemical synthesis of [14C]phenylbutazone, to be used in metabolism and depletion studies in cattle, was completed. C. Significant Accomplishments/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 CRIS project was initiated in June of 2001.
6. What do you expect to accomplish, year by year, over the next 3 years?
Year 1: We plan to describe the absorption, disposition, metabolism, and excretion (ADME) of the anti-inflammatory agent phenylbutazone in cattle. Immunoassays developed for detecting phenylbutazone will also be evaluated for cross-reactivity to phenylbutazone and phenylbutazone metabolites. We will continue efforts to generate radiolabeled ergovaline for use in ADME studies; this effort will include the development of screening assays to evaluate strains of Claviceps paspali for their propensity to produce lysergic and paspalic acids. These acids are used as starting materials during the chemical synthesis of ergopeptine alkaloids. We will continue to validate a ractopamine immunoassay and ractopamine immunoaffinity column developed at this laboratory. We also plan to validate previous studies determining whether dietary administration of beta-agonists allows remediation of persistent environmental contaminants. Year 2: Phenyl- butazone residue depletion studies will be
conducted in cattle using intravenous and oral administration. Tissues will be analyzed for parent phenylbutazone and(or) major phenylbutazone metabolites. Commercial immunassays will be evaluated for their abilities to predict tissue levels of phenylbutazone and for their ability to detect low levels of parent drug or metabolites. If radiolabeled ergovaline is not available from chemical synthesis, we plan to produce radiolabeled lysergic acid from culture and to use it as a starting material for the production of [14C]ergovaline. Finally, we plan to continue to develop and validate rapid immunoassays for animal health drugs that have the potential for illicit use; rapid screening assays may be used for regulatory purposes in the detection illegal drug residues. Year 3: Radiolabeled ergovaline will be dosed to cattle (or other appropriate species, depending upon the mass of ergovaline available) and its absorption, distribution, metabolism, and excretion will be determined.
Excreta and tissues will be used for metabolite identification.
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 & durability of the technology product?
None
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
No popular publications or presentations.
Impacts
(N/A)
Publications
- Kissel, J., Smith, D.J., Mills, S.E. Stereoselectivity of porcine -adrenergic receptors for ractopamine isomers. Journal of Animal Science. 2001. v. 79(Suppl. 1). p. 429.
- Smith, D. J., Shelver, W.L. Ractopamine residues in edible tissues and excreta of farm animals. American Chemical Society Agrochemicals Division. 2001. Abstract No. 36.
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