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? For livestock producers larkspurs (Delphinium spp.) are the most serious toxic plant problem on foothill and mountain rangeland in the western U.S. Cattle death losses average 2 to 5%, but may exceed 15% in areas where larkspurs are abundant. Total costs to the livestock industry from all larkspurs may exceed $20 million annually. In addition to dead livestock, significant amounts of forage are wasted, and management costs are increased as producers defer or avoid grazing larkspur-infested areas. Management costs also increase as producers purchase or raise replacement females and as calf crops are negatively impacted. The presence of larkspurs on ranges and pastures reduces productivity of animals and decreases the utilization of rangelands for grazing livestock. Research into larkspur toxicity and consumption by grazing livestock is leading to development of management strategies to reduce losses to larkspur. This research provides information to improve utilization of rangelands, promote biodiversity, and enhance livestock production. This research works closely with National Programs 205 Rangeland, Pastures, and Forages and 103 Animal Health to solve problems for rangeland and animal health. Unknown alkaloids will be isolated and characterized from larkspur species using large scale extraction followed by acid/base extraction to yield a crude alkaloid fraction. HPLC will be used to purify alkaloids for toxicity testing. Development of new analytical methods for larkspur alkaloids will continue. Immunogenic alkaloid-protein conjugates will be synthesized and immunized into animals. The resulting antibodies will be characterized, and if suitable, will be developed into ELISA detection systems. Immunogenic conjugates will be tested in animals as vaccines to determine if immunization alters larkspur toxicity. Rates of toxic metabolism and clearance will be determined using extracts and purified alkaloids. Diagnostic procedures will be enhanced. Treatment of intoxicated animals using cholinergic drugs will continue. Field and pen studies will evaluate plant, animal, and environmental factors influencing larkspur consumption and toxicity. Research will continue on management options such as sheep grazing larkspur, aversive conditioning and treatment of bloat. Objective 1: Isolate and identify individual alkaloids from various tall, low, and plains Delphinium species; test these alkaloids for toxicity; confirm structure-activity relationships for new alkaloids; determine seasonal toxicity of larkspur species. Objective 2: Develop analytical methods for larkspur alkaloids, including development of immunogenic alkaloid-protein conjugates and antibody-based detection methods and evaluate immunoconjugates as potential vaccines. Objective 3: Investigate mechanisms of intoxication, including molecular and pathophysiological alterations of poisoning, toxin receptor binding, metabolism and clearance, and treatment of intoxicated animals. Objective 4: Determine plant, animal, nutritional, or environmental factors influencing larkspur consumption of low and plains larkspurs, and continue development of management strategies to reduce losses for all classes of larkspur. Objective 5: Using molecular markers and cloning determine the genetic relatedness of toxic tall larkspurs. Compare genetic similarities with current taxonomic classification, and determine if new genetic taxonomic classifications better predict plant phenotype and toxicity. 2. List the milestones (indicators of progress) from your Project Plan. Milestone 1: Determined the toxic and relatively non-toxic alkaloids in larkspurs, particularly methyllycaconitine (MLA). Determined in vivo toxicity of purified alkaloids. Milestone 2: Developed the Fourier transform infrared spectroscopic method of analysis of toxic and total alkaloid levels in tall larkspur, thus allowing for rapid analysis of thousands of samples. Milestone 3: Determined mechanism of action of MLA and other toxic alkaloids, indicating that toxic alkaloids block nicotinic acetylcholine (nAch) receptors postsynaptically. Milestone 4: Developed grazing strategies to reduce losses to tall larkspurs including early- and late-season grazing management options. Milestone 5: Determined that cattle eat tall larkspur in a cyclic "toxification-detoxification" pattern. Milestone 6: Determined that adverse postingestive consequences regulate consumption of larkspur by cattle, not plant flavor (i.e., taste and smell). Milestone 7: Determined that mineral supplementation does not affect the amount of larkspur consumed by grazing cattle. Milestone 8: Developed aversive conditioning as a potentially viable tool for ranchers. Milestone 9: Developed herbicidal control recommendations for tall larkspurs. Milestone 10: Determined that sheep grazing before cattle can reduce tall larkspur availability and acceptability to cattle grazing afterward. 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. A study determined the toxicokinetics (aborption and elimination profiles) of larkspur alkaloids in blood, serum, and feces in cattle. These results provide a physiological basis for the difference in toxicity of various alkaloids with different structural characteristics. Milestone Substantially Met 2. Grazing studies have been conducted in Colorado and Idaho to determine if sheep will consume substantial amounts of low larkspur flowers. During the 2004 study, sheep ate almost no larkspur flowers. It does not appear that sheep grazing will diminish risk for cattle producers with dense low larkspur populations. Milestone Substantially Met 3. A study was conducted to determine consumption patterns and toxicity of tall larkspur (D. occidentale) in western Montana. Cattle consumed little tall larkspur before the plants flowered. Milestone Substantially Met 4. Research is continuing on the relationship between low larkspur population densities and environmental conditions (e.g., precipitation). Low larkspur populations fluctuate from year-to-year, and this study seeks to determine the relationship between plant populations and climate variables. 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? FY 2006: Determine the genetic basis for receptor binding of toxic alkaloids in different breeds of cattle. Determine the synergistic or antagonistic effects of methyllycoconitine and deltaline, two predominant alkaloid toxins in larkspurs, in a mouse bioassay. Continue studies into alkaloid absorption and serum elimination rate in cattle fed tall larkspur. Begin studies to determine the interaction of bloat and larkspur toxicity in cattle (if an animal physiologist is hired). Continue studies on consumption of tall larkspur by cattle on timothy- dominated rangelands in Montana. Continue support of alkaloid analyses for rangeland or toxicity studies. Continue immunization and toxicokinetic studies with mice and sheep. Continue to study relationship between precipitation and abundance of low larkspur populations. Complete comparative toxicity study of MLA and Deltaline in mice, rats, sheep and calves. FY 2007: Continue study to determine the genetic basis for receptor binding of toxic alkaloids in different breeds of cattle. Continue studies on consumption of tall larkspur on timothy-dominated rangelands in Montana. Continue physiology studies examining interaction between bloat and larkspur toxicity. Continue ELISA and immunization studies. Conclude study of relationships between precipitation and abundance of low larkspur populations Evaluate the synergistic and/or antagonistic effects of various MDL larkspur alkaloids on the more toxic MSAL alkaloids in a mouse bioassay. FY 2008: Continue study to determine the genetic basis for receptor binding of toxic alkaloids in different breeds of cattle. Continue studies on consumption of tall larkspur on timothy-dominated rangelands. Continue physiology studies examining interaction between bloat and larkspur toxicity. Continue ELISA and immunization studies. 4a What was the single most significant accomplishment this past year? Larkspur kinetics in cattle: A study determined the toxicokinetics (absorption and elimination profiles) of larkspur alkaloids in blood, serum, and feces in cattle. Cattle are the major livestock species poisoned by larkspur, and this study will provide valuable information on toxicokinetics. The stress of repeated bleeding reduced the effective dose substantially, supporting previous work that affected animals should be not be stressed. These results provide a physiological basis for the difference in toxicity of various alkaloids with different structural characteristics. 4b List other significant accomplishments, if any. Sheep grazing larkspur: Sheep may be an important tool to reduce cattle deaths to larkspurs because sheep are relatively resistant to the toxic alkaloids. Anecdotal accounts indicate that if sheep eat the flowering heads of low larkspurs (e.g., D. nuttallianum), the plants senesce quickly, and toxicity and acceptability to cattle decrease. Grazing studies have been conducted in Colorado and Idaho to determine if sheep will consume substantial amounts of low larkspur flowers. During the 2004 study, sheep ate almost no larkspur flowers, instead consuming other forbs and green grasses. This study is currently being repeated at the Idaho location with a high density of low larkspur. Even so, it does not appear that sheep grazing will diminish risk for livestock producers with dense low larkspur populations. Cattle consumption of larkspur in Montana: A study was conducted to determine consumption patterns and toxicity of tall larkspur (D. occidentale) in western Montana. On these timothy- dominated rangelands, larkspur is reportedly eaten during the vegetative and bud stages by cattle because timothy is slow to grow, and other forbs are lacking. The tall larkspur in this area has been found to be very toxic. Although this study is ongoing, it does not appear that cattle select tall larkspur before the plant flowers, as we have noted in past studies in other locations. Further work will be required to confirm this finding. Low larkspur density: Research is continuing on the relationship between low larkspur population densities and environmental conditions (e.g., precipitation). Low larkspur populations fluctuate from year-to-year, and this study seeks to determine the relationship between plant populations and climate variables. This is a multi-state and multi-year project. Because of recent losses to D. andersonii in various locations, including California, we have added the Bodie Hills area of California to our sampling regime. 4d Progress report. Characterization of toxic larkspur species: Toxic larkspur species which have not been previously chemically characterized were collected and screened for toxic alkaloids using electrospray mass spectrometry (ESMS) and/or liquid chromatography mass spectrometry. Collected plant material were freeze-dried, ground, extracted with methanol, diluted and analyzed by ESMS. The presence of known or unknown toxic alkaloids was determined. Newly isolated alkaloids were tested for mammalian toxicity using a mouse bioassay. Chemotaxonomy and genetic basis of larkspurs: Chemotaxonomic and genotaxonomic approaches were used to define tall larkspur taxonomic divisions. Approximately 25 different accessions of Delphinium occidentale, D. glaucum, and D. barbeyi, were collected from the Rocky Mountain regions of Utah, Idaho, Wyoming, California, and Colorado based on past knowledge of plant locations and association with livestock poisonings. Genetic evaluation of tall larkspurs classified D. occidentale, D. barbeyi and D. glaucum as distinct species; chemotaxonomic studies of larkspurs using RAPD (randomly amplified polymorphic DNA) markers and chemical analyses supported this view. Results were integrated to show how knowledge of taxonomy, alkaloid concentration, and other factors can be used to make informed management decisions. ELISA diagnosis techniques: Rapid, sensitive and specific diagnostic techniques are important to identify poisoned animals and to determine when and what plants are likely to poison livestock. Enzyme-linked immunosobent assays have been developed as altermatives to traditional analytical techniques for the detection of natural products. Competitive inhibition enzyme-linked immunosorbent assays (CI-ELISA) for toxic larkspur alkaloids were developed. Absorption and excretion of larkspur alkaloids: Small ruminant models (i.e., sheep) and rodent models (i.e., mice) were compared to cows in the role of toxicity of MLA and deltaline. Toxicity of purified alkaloids from larkspur including MLA and deltaline were compared in cattle, sheep and mice. Newly identified alkaloids and alkaloid analogs (synthesized fragments of larkspur alkaloids thought to be important in toxicity) were tested in mice for toxicity. Using a sheep model, studies determined the toxicokinetics (absorption and elimination profiles) of larkspur alkaloids in blood. The elimination profile of the dominant alkaloid methyllycaconitine (MLA) was slower than the other less-toxic alkaloids. These results provide a physiological basis for the difference in toxicity of various alkaloids with different structural characteristics. Studies with cattle are ongoing. Alkaloid concentrations and predicting toxicity: Knowledge of the toxic alkaloid concentrations in larkspur plants, and when and why these concentrations change, is necessary to reduce the risk of poisoning. The pools of toxic alkaloids (biomass x concentration) were determined in larkspur plants and plant parts over the growing season. Results suggest that alkaloids are synthesized in the roots during the early growth and translocated throughout the above-ground growth. Concentrations are diluted as the biomass increases during the growing season, and mature larkspur translocates alkaloids back into roots before senescence. Cattle and sheep grazing of larkspurs: Sheep have been shown to periodically graze tall larkspurs and sometimes reduce the availabilaity and toxicity to cattle grazing thereafter. This type of information is not available for low larkspurs. Grazing studies were conducted with sheep and cattle in mountain areas with high densities (>4plants/m2 of low larkspur (D. nuttallianum). Sheep may be an important tool to reduce cattle deaths to larkspurs because sheep are relatively resistant to the toxic alkaloids. Anecdotal accounts indicate that if sheep eat the flowering heads of low larkspurs (e.g., D. nuttallianum), the plants senesce quickly, and toxicity and acceptability to cattle decrease. Grazing studies have been conducted in Colorado and Idaho to determine if sheep will consume substantial amounts of low larkspur flowers. Sheep ate almost no larkspur flowers, instead consuming other forbs and green grasses. It does not appear that sheep grazing will diminish risk for livestock producers with dense low larkspur populations. Other grazing studies have provided information on plant/animal interactions, including concentrations of alkaloids, probability of consumption by livestock, and risk assessment. 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 103 Animal Health directly contributing to research component VII Strategies to Control Infectious and Non-Infectious Disease, and National Program 205 Rangeland, Pasture, and Forages directly contributing to research component V Integrated Management of Weeds and Other Pests, and the Poisonous Plant problem area. Research activities are further related to ARS Strategic Plan Goal 3.2.1.1 Animal Disease Diagnosis, Develop specific, efficient tests for detecting and identifying diseases of animals to facilitate effective diagnosis, control, and prevention; develop protocols for movement of postnatal animals, sperm, and embryos with minimum disease risk to support a national animal genetic resources management program and ARS Strategic Plans Goal 3.2.3.2 Natural and Synthetic Chemicals - Animals, Develop knowledge about ways of controlling the toxic effects of natural and synthetic chemicals in animals to reduce losses and illegal product residues. Knowledge of the toxic alkaloid concentrations in larkspur plants, and when and why these concentrations change, is necessary to reduce the risk of poisoning. In the past, the concentrations of the toxic alkaloid, methyllycaconitine (MLA) were typically used alone to determine toxicity. Improved analytical procedures now allow for simultaneous measurement of 4 other MSAL-type toxic alkaloids, significantly improving the ability to determine the concentrations of all the toxic alkaloids in the plants. Development continues on three competitive inhibition enzyme-linked immunosorbent assays (CI-ELISA) for toxic larkspur alkaloids. One assay is class specific toward the N-(methylsuccinimido)anthranoyllycoctonine (MSAL) alkaloids, while two assays are specific for individual alkaloids. With additional development these techniques are likely to be excellent tools to diagnose poisoned animals and identify highly toxic plants. Developed the Fourier transform infrared spectroscopic method of analysis of toxic and total alkaloid levels in tall larkspur, thus allowing for rapid analysis of thousands of samples. Elucidated the comparative toxicity of 24 alkaloids from larkspurs and structure activity relationships. Understanding toxicity has important implications for developing rational treatments to reduce losses. Developed grazing strategies to reduce losses to tall larkspurs. Continued implementation of early- and late-season grazing strategies has resulted in reduced cattle losses for ranchers grazing on tall larkspur- infested pastures. Numerous grazing studies in Colorado, Idaho, Utah, and now Montana (this year) have confirmed that cattle eat most tall larkspur after the plant has flowered (i.e., elongated flowering racemes) thus allowing a low risk grazing window early in the season, and another later in the season after pods have shattered. Determined that cattle eat tall larkspur in a cyclic toxification- detoxification pattern after ingesting a daily threshold of 14-18 mg toxic alkaloid/kg b.w. This research helps to understand patterns of consumption and how much larkspur is necessary to poison cattle. Determined that when cattle eat larkspur, adverse postingestive consequences regulate consumption, not plant flavor (i.e., taste and smell). Increases in the toxic alkaloid concentration deter consumption by sheep, but have little impact on short-term consumption by cattle. This research provides fundamental information about when and why cattle eat larkspurs. Determined that mineral supplementation does not affect the amount of larkspur consumed by grazing cattle, indicating that producers can save money by providing mineral supplements as necessary for nutritional needs, rather than using minerals to alter larkspur consumption. Developed aversive conditioning as a potentially viable tool for ranchers. This practice could potentially greatly reduce the risk of cattle losses for those livestock producers that implement aversive conditioning. Developed herbicidal control recommendations for tall larkspurs. Ranchers with dense populations of larkspur can greatly reduce losses and enhance economic returns through the use of herbicides. Verified that sheep grazing before cattle can reduce larkspur availability and acceptability to cattle grazing afterward. Collaborative work with Colorado State University investigated the mechanism of action of MLA, nudicauline, 12-deacetylnudicauline, barbanine, and deltaline. It was determined that the alkaloids block nicotinic acetylcholine (nAch) receptors postsynaptically. The potency of deltaline is much lower, indicating that deltaline does not bind as tightly as do the other alkaloids at nAch receptors. 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? Toxic larkspur alkaloid analyses done on a number of plant, blood, and rumen samples for ranchers (samples from Idaho, Montana, Colorado), veterinarians (e.g., USU Vet. Diagnostic Lab.), and extension personnel. This is a particularly useful tool for ranchers to help determine if they can begin grazing with low risk, or continue to graze, a particular pasture or allotment. The management concepts referred to as primary toxic window and early season grazing have been developed and transmitted to numerous ranchers and USFS personnel. These concepts are based on plant chemistry and cattle diet selection patterns, and are being used to reduce losses on summer-grazed Forest Service allotments where tall larkspur is problematic. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Toured larkspur-infested areas and discussed management options with Shoshone-Bannock Tribe (Ft. Hall, Idaho) extension personnel (May, 2005). Gardner, D.R. and Pfister. 2005. Toxic alkaloid profiles in low and plains larkspur of the Western U.S., 7th International Symposium on Poisonous Plants, Logan, Utah.

Impacts
(N/A)

Publications

• Ralphs, M.H., Jones, W.A. 2000. Population cycles of the larkspur mirid heteroptera: miridae. Journal of Entomological Science.
• Ralphs, M.H., Gardner, D.R., Pfister, J.A. 2000. A functional explanation for patterns of norditerpenoid alkaloid levels in tall larkspur (delphinium barbeyi). Journal of Chemical Ecology.
• Ralphs, M.H., Gardner, D.R. 2001. Alkaloid levels in duncecap (delphinium occidentale) and tall larkspur (d. barbeyi)grown in reciprocal gardens: separating genetic from environmental influences. Biochemical Systematics and Ecology.
• GARDNER, D.R., Pfister, J.A. LATE SEASON TOXIC ALKALOID CONCENTRATIONS IN TALL LARKSPUR (DELPHINIUM SPP.). JOURNAL OF RANGE MANAGEMENT. 2000.
• PFISTER, J.A., PROVENZA, F., PANTER, K.E., STEGELMEIER, B.L., LAUNCHBAUGH, K.L. RISK MANAGEMENT TO REDUCE LIVESTOCK LOSSES FROM TOXIC PLANTS. JOURNAL OF RANGE MANAGEMENT. 2002.
• Ralphs, M.H., Gardner, D.R., Turner, D.L., Pfister, J.A., Thacker, E.T. 2002. Predicting toxicity of tall larkspur (delphinium barbeyi) and measurement of the variation in toxic alkaloid concentration between plants and among yearss. Journal of Chemical Ecology.
• Ralphs, M.H., Woolsey, L., Bowns, J.E. 2004. Individual plant control of tall larkspur with tebuthiuron. Weed Technology.
• LEE, S.T., SCHOCH, T.K., GARDNER, D.R., STEGELMEIER, B.L. DEVELOPMENT OF ENZYME-LINKED IMMUNOSORBENT ASSAYS FOR TOXIC LARKSPUR ALKALOIDS. JOURNAL OF AGRICULTURAL FOOD & CHEMICAL. 2000.
• Ralphs, M.H. 2002. Ecological relationships between poisonous plants and rangeland condition. Journal of Range Management.
• GARDNER, D.R., MANNERS, G.D., PANTER, K.E., LEE, S.T., PFISTER, J.A. THREE NEW TOXIC NORDITERPENOID ALKALOIDS FROM THE LOW LARKSPUR DELPHINIUM NUTTALLIANUM PRITZ. JOURNAL OF NATURAL PRODUCTS. 2000.
• Ralphs, M.H., Gardner, D.R. 2001. Influence of defoliation on toxic alkaloid concentration and alkaloid poolsin tall larkspur. Journal of Chemical Ecology.
• GARDNER, D.R., PANTER, K.E., PFISTER, J.A., KNIGHT, A.P. ANAYLSIS OF TOXIC NORDITERPENOID ALKALOIDS IN DELPHINIUM SPECIES BY ELECTROSPRAY, ATMOSPHERIC PRESSURE CHEMCIAL IONIZATION AND SEQUENTIAL TANDEM MASS SPECTROMETRY. JOURNAL OF NATURAL PRODUCTS. 1999.

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

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Larkspur (Delphinium spp.) poisoning causes serious economic loss to livestock producers grazing cattle on many western ranges. The objectives of the multidisciplinary research are: (1) isolate and identify individual alkaloids from Delphinium species; test these alkaloids for toxicity; confirm structure activity relationships for new alkaloids; (2) develop analytical and diagnostic methods for larkspur alkaloids, including development of immunogenic alkaloid protein conjugates and antibody based detection methods and potential vaccine development; (3) investigate mechanisms of intoxication, including toxin metabolism and clearance, and treatment of intoxicated animals; and (4) determine plant, animal, or environmental factors influencing larkspur consumption and continue development of management strategies to reduce losses for all classes of larkspur 2. How serious is the problem? Why does it matter? Larkspurs (Delphinium spp.) are the most serious toxic plant problem on foothill and mountain rangeland in the western U.S. Cattle death losses average 2 to 5%, but may exceed 15% in areas where larkspurs are abundant. Total costs to the livestock industry from all larkspurs may exceed $20 million annually. In addition to dead livestock, significant amounts of forage are wasted, and management costs are increased as producers defer or avoid grazing larkspur-infested areas. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? Sixty percent of this CRIS research currently falls under the Food Safety Action Plan (103), the Toxic Chemical component, and the Toxic Plant problem area, specifically Toxins in Range Plants (3.1.1). New immunogenic and spectroscopic methods will allow for sensitive detection of toxic alkaloids in animal and plant tissue. Positive identification of poisoned animals will be useful in defining the scope of the problem, and will aid in developing improved strategies to minimize larkspur poisoning in relation to infectious and toxic diseases. New analytical and diagnostic techniques will provide for rapid and accurate analysis of plant tissue to determine when plants are toxic. This research will be used to develop management programs to reduce losses from larkspur. Research into the toxicokinetics and metabolism of larkspur alkaloids will ensure that animal products and tissues are not contaminated by alkaloids and thus help to ensure a safe and quality food supply for consumers. Information about how larkspurs poison cattle will help in devising and testing research strategies to reduce losses. Forty percent of the research currently falls under the Rangeland, Forages and Pastures Action Plan (205), the Integrated Management of Weeds and Pests, and the Poisonous Plant problem area. The presence of larkspurs on ranges and pastures reduces productivity of animals and decreases the utilization of rangelands for grazing livestock. Research into larkspur toxicity and consumption by grazing livestock is leading to development of management strategies to reduce losses to larkspur. This research provides information to improve utilization of rangelands, promote biodiversity, and enhance livestock production. Currently, efforts are under way to reallocate the Larkspur CRIS to Animal Health and to Rangeland, Forages and Pastures and to drop the Food Safety designation 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment 2003 Knowledge of the toxic alkaloid concentrations in larkspur plants, and when and why these concentrations change, is necessary to reduce the risk of poisoning. The pools of toxic alkaloids (biomass x concentration) were determined in larkspur plants and plant parts over the growing season. Results suggest that alkaloids are synthesized in the roots in early growth and translocated throughout the above-ground growth. Concentrations are diluted as the biomass increases during the growing season, and mature larkspur translocates alkaloids back into roots before senescence. B. Other Significant Accomplishment(s), if any Development continued on three competitive inhibition enzyme-linked immunosorbent assays (CI-ELISA) for toxic larkspur alkaloids. One assay is class specific toward the N-(methylsuccinimido)anthranoyllycoctonine (MSAL) alkaloids, while two assays are specific for individual alkaloids. With additional development these techniques are likely to be excellent tools to diagnose poisoned animals and identify highly toxic plants. Studies were done to determine if larkspur toxins conjugated to protein carriers would promote active immunity in mice. Mice were injected with several larkspur toxin-protein conjugates or adjuvant alone to determine if the resulting immunologic response altered animal susceptibility to methyllycaconitine, the major toxic larkspur alkaloid. Although vaccinations appeared to provide a mild protective effect against intravenous methyllycaconitine toxicity, overlapping confidence intervals did not provide evidence of differences between the vaccinated and control groups. In the lycoctonine conjugate (LYC )vaccinated group, mouse survival was related to serum titers for methyllycaconitine doses up to 4.5 mg/kg BW. When mice with low antibody titers were removed from the vaccinated groups in which titer was related to survival, the recalculated LD50s were 20% greater than the LD50s of the control group. These results suggest that vaccination altered methyllycaconitine toxicity in mice and that vaccination may be useful in decreasing the effects of larkspur toxins in animals. Research continued on the relationship between low larkspur population densities and environmental conditions (e.g., precipitation). This is a multi-state and multi-year project. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Developed the Fourier transform infrared spectroscopic method of analysis of toxic and total alkaloid levels in tall larkspur, thus allowing for rapid analysis of thousands of samples. Elucidated the comparative toxicity of 24 alkaloids from larkspurs and structure activity relationships. Understanding toxicity has important implications for developing rational treatments to reduce losses. Developed grazing strategies to reduce losses to tall larkspurs. Continued implementation of early- and late-season grazing strategies has resulted in reduced cattle losses for ranchers grazing on tall larkspur- infested pastures. Determined that cattle eat tall larkspur in a cyclic "toxification- detoxification" pattern after ingesting a daily threshold of 14-18 mg toxic alkaloid/kg b.w. This research helps to understand patterns of consumption and how much larkspur is necessary to poison cattle. Determined that when cattle eat larkspur, adverse postingestive consequences regulate consumption, not plant flavor (i.e., taste and smell). Increases in the toxic alkaloid concentration deter consumption by sheep, but have little impact on short-term consumption by cattle. This research provides fundamental information about when and why cattle eat larkspurs. Determined that mineral supplementation does not affect the amount of larkspur consumed by grazing cattle, indicating that producers can save money by providing mineral supplements as necessary for nutritional needs, rather than using minerals to alter larkspur consumption. Developed aversive conditioning as a potentially viable tool for ranchers. This practice could potentially greatly reduce the risk of cattle losses for those livestock producers that implement aversive conditioning. Developed herbicidal control recommendations for tall larkspurs. Ranchers with dense populations of larkspur can greatly reduce losses and enhance economic returns through the use of herbicides. Verified that sheep grazing before cattle can reduce larkspur availability and acceptability to cattle grazing afterward. Collaborative work with Colorado State University investigated the mechanism of action of MLA, nudicauline, 12-deacetylnudicauline, barbanine, and deltaline. It was determined that the alkaloids block nicotinic acetylcholine (nAch) receptors postsynaptically. The potency of deltaline is much lower, indicating that deltaline does not bind as tightly as do the other alkaloids at nAch receptors. 6. What do you expect to accomplish, year by year, over the next 3 years? FY 2003 Complete study on defoliation effects on toxic alkaloid concentrations Continue support of alkaloid analyses for rangeland or toxicity studies Continue immunization and toxicokinetic studies with mice and sheep Complete low larkspur toxic alkaloid survey Determine toxicokinetics (absorption and elimination profiles) of larkspur alkaloids in a sheep model Continue to study relationship between precipitation and abundance of low larkspur populations Complete comparative toxicity study of MLA and Deltaline in mice, rats, sheep and calves. FY 2004 Continue ELISA and immunization studies Continue development of grazing management strategies for low/plains larkspurs Continue investigation of toxicokinetics (absorption and elimination profiles) of larkspur alkaloids in a sheep model, and analyze blood, urine and feces for alkaloids. Continue to study relationship between precipitation and abundance of low larkspur populations Evaluate the synergistic and/or antagonistic effects of various MDL larkspur alkaloids on the more toxic MSAL alkaloids in a mouse bioassay. FY 2005 Continue ELISA and immunization studies Continue to study relationship between precipitation and abundance of low larkspur populations Complete toxicokinetic study in sheep. 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? Toxic larkspur alkaloid analyses done on a number of plant samples for ranchers and extension personnel. This is a useful tool for ranchers to help determine if they can begin grazing with low risk, or continue to graze, a particular pasture or allotment. The management concepts referred to as "primary toxic window" and "early season grazing" have been developed and transmitted to numerous ranchers and USFS personnel. These concepts are based on plant chemistry and cattle diet selection patterns, and are being used to reduce losses on summer-grazed Forest Service allotments where tall larkspur is problematic. 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). Presentation to Idaho Weed Association on "Management of toxic plants" Presentation to Western Wyoming Weed Association on "Management of Toxic plants" Presentation to Animal Science and Veterinary Science faculty and students, University of Sao Paulo, Brazil "Poisonous Plant Research at PPRL" and "Behavioral effects of toxic plant ingestion in livestock" Welch, S.L. and M.H. Ralphs. 2002. Some tall larkspurs (Delphinium- Ranunculaceae), a taxonomic review. Biochemical Systematics Ecology 30:103-112.

Impacts
(N/A)

Publications

• GARDNER, D.R., RALPHS, M.H., TURNER, D., WELSH, S. TAXONOMIC IMPLICATIONS OF DITERPENE ALKALOIDS IN THREE TOXIC TALL LARKSPUR SPECIES (DELPHINIUM SPP.). BIOCHEMICAL SYSTEMATICS AND ECOLOGY. 2002.
• LI, X., GARDNER, D.R., RALPHS, M.H., WANG, R. DEVELOPMENT OF STS AND CAPS MARKERS FOR IDENTIFICATION THREE TALL LARKSPURS (DELPHINIUM SPP.). GENOME. 2002.
• Pfister, J.A., Ralphs, M.H. Gardner, D.R., Stegelmeier, B.L., Manners, G.D. , Panter, K.E. and Lee, S.T. 2002. Management of three toxic Delphinium species based on alkaloid concentrations. Biochemical Systematics Ecology 30:129-138
• PANTER, K.E., MANNERS, G.D., JAMES, L.F., GARDNER, D.R., RALPHS, M.H., PFISTER, J.A., STEGELMEIER, B.L., LEE, S.T. LARKSPUR POISONING: TOXICOLOGY AND ALKALOID STRUCTURE ACTIVITY RELATIONSHIPS. BIOCHEMICAL SYSTEMATICS AND ECOLOGY. 2002.
• PANTER, K.E., JAMES, L.F., GARDNER, D.R., RALPHS, M.H., PFISTER, J.A., STEGELMEIER, B.L., LEE, S.T. REPRODUCTIVE LOSSES TO POISONOUS PLANTS: INFLUENCE OF MANAGEMENT STRATEGIES. JOURNAL OF RANGE MANAGEMENT. 2002.
• Pfister, J.A., Gardner, D.R., Stegelmeier, B.L., Knight, A.P., Waggoner, Jr., J.W. and Hall, J.O. 2002. Plains larkspur (Delphinium geyerii) grazing by cattle in Wyoming. Journal of Range Management 55:350-359.
• Li, X., Ralphs, M.H., Gardner, D.R., Wang, R.R.-C. 2002. Genetic variation within and among 22 accessions of three tall larkspur species (Delphinium spp.) based RAPD markers. Biochemical Systematics Ecology, 30:91-102.

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

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Larkspur (Delphinium spp.) poisoning causes serious economic loss to livestock producers grazing cattle on many western ranges. The objectives of the multidisciplinary research are: (1) isolate and identify individual alkaloids from Delphinium species; test these alkaloids for toxicity; confirm structure activity relationships for new alkaloids; (2) develop analytical and diagnostic methods for larkspur alkaloids, including development of immunogenic alkaloid protein conjugates and antibody based detection methods and potential vaccine development; (3) investigate mechanisms of intoxication, including toxin metabolism and clearance, and treatment of intoxicated animals; and (4) determine plant, animal, or environmental factors influencing larkspur consumption and continue development of management strategies to reduce losses for all classes of larkspur. 2. How serious is the problem? Why does it matter? Larkspurs (Delphinium spp.) are the most serious toxic plant problem on foothill and mountain rangeland in the western U.S. Cattle death losses average 2 to 5%, but may exceed 15% in areas where larkspurs are abundant. Total costs to the livestock industry from all larkspurs may exceed $20 million annually. In addition to dead livestock, significant amounts of forage are wasted, and management costs are increased as producers defer or avoid grazing larkspur-infested areas. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? Sixty percent of this CRIS research currently falls under the Food Safety Action Plan (103), the Toxic Chemical component, and the Toxic Plant problem area, specifically Toxins in Range Plants (3.1.1). New immunogenic and spectroscopic methods will allow for sensitive detection of toxic alkaloids in animal and plant tissue. Positive identification of poisoned animals will be useful in defining the scope of the problem, and will aid in developing improved strategies to minimize larkspur poisoning in relation to infectious and toxic diseases. New analytical and diagnostic techniques will provide for rapid and accurate analysis of plant tissue to determine when plants are toxic. This research will be used to develop management programs to reduce losses from larkspur. Research into the toxicokinetics and metabolism of larkspur alkaloids will ensure that animal products and tissues are not contaminated by alkaloids and thus help to ensure a safe and quality food supply for consumers. Information about how larkspurs poison cattle will help in devising and testing research strategies to reduce losses. Forty percent of the research currently falls under the Rangeland, Forages and Pastures Action Plan (205), the Integrated Management of Weeds and Pests, and the Poisonous Plant problem area. The presence of larkspurs on ranges and pastures reduces productivity of animals and decreases the utilization of rangelands for grazing livestock. Research into larkspur toxicity and consumption by grazing livestock is leading to development of management strategies to reduce losses to larkspur. This research provides information to improve utilization of rangelands, promote biodiversity, and enhance livestock production. Currently, efforts are under way to reallocate the Larkspur CRIS to Animal Health and to Rangeland, Forages and Pastures and to drop the Food Safety designation. 4. What was your most significant accomplishment this past year? Knowledge of the toxic alkaloid concentrations in larkspur plants, and when and why these concentrations change, is necessary to reduce the risk of poisoning. The pools of toxic alkaloids (biomass x concentration) were determined in larkspur plants and plant parts over the growing season. Results suggest that alkaloids are synthesized in the roots in early growth and translocated throughout the above-ground growth. Concentrations are diluted as the biomass increases during the growing season, and mature larkspur translocates alkaloids back into roots before senescence. 4B. Other Accomplishments: Development continued on three competitive inhibition enzyme-linked immunosorbent assays (CI-ELISA) for toxic larkspur alkaloids. One assay is class specific toward the N-(methylsuccinimido)anthranoyllycoctonine (MSAL) alkaloids, while two assays are specific for individual alkaloids. With additional development these techniques are likely to be excellent tools to diagnose poisoned animals and identify highly toxic plants. Studies were done to determine if larkspur toxins conjugated to protein carriers would promote active immunity in mice. Mice were injected with several larkspur toxin-protein conjugates or adjuvant alone to determine if the resulting immunologic response altered animal susceptibility to methyllycaconitine, the major toxic larkspur alkaloid. Although vaccinations appeared to provide a mild protective effect against intravenous methyllycaconitine toxicity, overlapping confidence intervals did not provide evidence of differences between the vaccinated and control groups. In the lycoctonine conjugate (LYC )vaccinated group, mouse survival was related to serum titers for methyllycaconitine doses up to 4.5 mg/kg BW. When mice with low antibody titers were removed from the vaccinated groups in which titer was related to survival, the recalculated LD50s were 20% greater than the LD50s of the control group. These results suggest that vaccination altered methyllycaconitine toxicity in mice and that vaccination may be useful in decreasing the effects of larkspur toxins in animals. Research continued on the relationship between low larkspur population densities and environmental conditions (e.g., precipitation). This is a multi-state and multi-year project. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? Developed the Fourier transform infrared spectroscopic method of analysis of toxic and total alkaloid levels in tall larkspur, thus allowing for rapid analysis of thousands of samples. Elucidated the comparative toxicity of 24 alkaloids from larkspurs and structure activity relationships. Understanding toxicity has important implications for developing rational treatments to reduce losses. Developed grazing strategies to reduce losses to tall larkspurs. Continued implementation of early- and late-season grazing strategies has resulted in reduced cattle losses for ranchers grazing on tall larkspur- infested pastures. Determined that cattle eat tall larkspur in a cyclic "toxification- detoxification" pattern after ingesting a daily threshold of 14-18 mg toxic alkaloid/kg b.w. This research helps to understand patterns of consumption and how much larkspur is necessary to poison cattle. Determined that when cattle eat larkspur, adverse postingestive consequences regulate consumption, not plant flavor (i.e., taste and smell). Increases in the toxic alkaloid concentration deter consumption by sheep, but have little impact on short-term consumption by cattle. This research provides fundamental information about when and why cattle eat larkspurs. Determined that mineral supplementation does not affect the amount of larkspur consumed by grazing cattle, indicating that producers can save money by providing mineral supplements as necessary for nutritional needs, rather than using minerals to alter larkspur consumption. Developed aversive conditioning as a potentially viable tool for ranchers. This practice could potentially greatly reduce the risk of cattle losses for those livestock producers that implement aversive conditioning. Developed herbicidal control recommendations for tall larkspurs. Ranchers with dense populations of larkspur can greatly reduce losses and enhance economic returns through the use of herbicides. Verified that sheep grazing before cattle can reduce larkspur availability and acceptability to cattle grazing afterward. Collaborative work with Colorado State University investigated the mechanism of action of MLA, nudicauline, 12-deacetylnudicauline, barbanine, and deltaline. It was determined that the alkaloids block nicotinic acetylcholine (nAch) receptors postsynaptically. The potency of deltaline is much lower, indicating that deltaline does not bind as tightly as do the other alkaloids at nAch receptors. 6. What do you expect to accomplish, year by year, over the next 3 years? FY 2003: Complete study on defoliation effects on toxic alkaloid concentrations Continue support of alkaloid analyses for rangeland or toxicity studies Continue immunization and toxicokinetic studies with mice and sheep Complete low larkspur toxic alkaloid survey Determine toxicokinetics (absorption and elimination profiles) of larkspur alkaloids in a sheep model Continue to study relationship between precipitation and abundance of low larkspur populations Complete comparative toxicity study of MLA and Deltaline in mice, rats, sheep and calves. FY 2004: Continue ELISA and immunization studies Continue development of grazing management strategies for low/plains larkspurs Continue investigation of toxicokinetics (absorption and elimination profiles) of larkspur alkaloids in a sheep model, and analyze blood, urine and feces for alkaloids. Continue to study relationship between precipitation and abundance of low larkspur populations Evaluate the synergistic and/or antagonistic effects of various MDL larkspur alkaloids on the more toxic MSAL alkaloids in a mouse bioassay. FY 2005: Continue ELISA and immunization studies Continue to study relationship between precipitation and abundance of low larkspur populations Complete toxicokinetic study in sheep. 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? Toxic larkspur alkaloid analyses done on a number of plant samples for ranchers and extension personnel. This is a useful tool for ranchers to help determine if they can begin grazing with low risk, or continue to graze, a particular pasture or allotment. The management concepts referred to as "primary toxic window" and "early season grazing" have been developed and transmitted to numerous ranchers and USFS personnel. These concepts are based on plant chemistry and cattle diet selection patterns, and are being used to reduce losses on summer-grazed Forest Service allotments where tall larkspur is problematic. 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) Presentation to Idaho Weed Association on "Management of toxic plants" Presentation to Western Wyoming Weed Association on "Management of Toxic plants" Presentation to Animal Science and Veterinary Science faculty and students, University of Sao Paulo, Brazil "Poisonous Plant Research at PPRL" and "Behavioral effects of toxic plant ingestion in livestock"

Impacts
(N/A)

Publications

• Gardner, D. R., Ralphs, M.H., Turner, D. L., Welsh, S. L. Taxonomic implications of diterpene alkaloids in three toxic tall larkspur species (Delphinium spp.). Biochemical Systematics Ecology, 30:77-90 (2002).
• Li, X., Ralphs, M. H., Gardner, D. R., Wang R. R.-C. Genetic variation within and among 22 accessions of three tall larkspur species (Delphinium spp.) based RAPD markers. Biochemical Systematics Ecology, 30: 91-102 (2002).
• Li, X.M., Gardner, D.R., Ralphs, M.H,, et al. Development of STS and CAPS markers for identification of three tall larkspurs (Delphinium spp.). Genome, 45(2): 229-235 (2002).
• Pfister, J.A., M.H. Ralphs, D.R. Gardner, B.L. Stegelmeier, G.D. Manners, K.E. Panter, and S.T. Lee. 2002. Management of three toxic Delphinium species based on alkaloid concentrations. Biochemical Systematics Ecology 30:129-138.
• Panter, K.E., G.D. Manners, B.L. Stegelmeier, S.T. Lee, D.R. Gardner, M.H. Ralphs, J.A. Pfister, and L.F. James. 2002. Larkspur poisoning: toxicology and alkaloid structure-activity relationships. Biochemical Systematics Ecology 30:113-128.
• Pfister, J.A., F.D. Provenza, K.E. Panter, B.L. Stegelmeier, and K.L. Launchbaugh. 2002. Risk management to reduce livestock losses from toxic plants. Journal Range Management 55:291-300.
• Panter, K.E., L.F. James, D.R. Gardner, M.H. Ralphs, J.A. Pfister, B.L. Stegelmeier, and S.T. Lee. 2002. Influence of management strategies on reproductive losses to poisonous plants. Journal Range Management 55:301- 308.
• Pfister, J.A., D.R. Gardner, B.L. Stegelmeier, A.P. Knight, J.W. Waggoner, Jr., and J.O. Hall. 2002. Plains larkspur (Delphinium geyerii) grazing by cattle in Wyoming. Journal Range Management 55:350-359.
• Welch, S.L. and M.H. Ralphs. 2002. Some tall larkspurs (Delphinium- Ranunculaceae), a taxonomic review. Biochemical Systematics Ecology 30:103-112.