Understanding and Mitigating the Adverse Effects of Poisonous Plants on Livestock Production Systems

Goals / Objectives
Objective 1: Develop and implement novel management protocols for establishing improved forage species on sites infested with known poisonous plants to reduce the risk of livestock mortality and morbidity, improve livestock performance, and improve rangeland resiliency and diversity. Specifically, develop science-based guidelines for grazing livestock on rangelands infested with Lupinus, Senecio, Delphinium and swainsonine and selenium-containing plants. Objective 2: Reduce the risks of livestock losses due to variations in quantitative and qualitative differences in toxin accumulation over time and plant species by quantifying the influence of endophytes, climate changes, and genotype on plant toxin accumulation (particularly swainsonine-containing plants and Delphinium and Lupinus species). Objective 3: Enhance feed and food safety by improving risk assessment and diagnosis of plant-induced poisoning to livestock by improving analytical methods for analyzing plant and animal tissues for toxins; measuring toxicokinetics, assessing carcinogenic and genotoxic potential, and identifying toxin metabolites and biomarkers of toxicoses. Objective 4: Develop improved procedures with guidelines for diagnostic and prognostic evaluation to reduce negative impacts of poisonous plants on livestock reproduction and embryo/fetal growth by improving early identification of poisoned animals, predicting poisoning outcomes, and management and treatment options through improved understanding of clinical, morphological and molecular alterations of plant-induced toxicosis. Objective 5: Develop guidelines to aid producers and land managers in making genetic-based herd management decisions to improve livestock performance and safety on grazed rangelands infested with poisonous plants through the use of identified animal genes, physiological pathways, and molecular mechanisms of action that underlie Conium, Cicuta, Delphinium, Lupinus, and Nicotiana, and other neurotoxic plant effects.

Project Methods
Livestock poisoning by plants results in over $503,000,000 lost to the livestock industry annually in the 17 western United States from death losses and abortions alone (Holechek, 2002). Plant poisonings extend worldwide to include 333 million poisonous plant-infested hectares in China and 60 million hectares in the central western region of Brazil, to name a few. There are over 6,000 species of pyrrolizidine alkaloid (PA)-containing plants, and over 350 individual PAs causing diseases in animals and humans have been identified. Economic losses are much larger as significant amounts of nutritious forage are wasted and management costs are increased due to the threat of toxic plant-related livestock losses. The Poisonous Plant Research Laboratory (PPRL) has provided worldwide leadership in poisonous plant research to the livestock industry and consumers including numerous solutions to toxic plant problems using an integrated, interdisciplinary approach (see Figure below). The research team investigates plant poisonings in a systematic matter by identifying the plant, describing the effects in animals, determining the toxin(s) and evaluating the mechanisms of action. The ultimate goal is to develop research-based solutions to reduce livestock losses from toxic plants. There are five coordinated objectives in this project plan providing guidelines for potential genetic-based management. This research will reduce livestock losses from plants and enhance the economic well-being of rural communities, improve rangeland health by combating invasive plant species, and help to provide safe animal products free from potential plant toxins for consumers.

Progress 02/11/13 to 01/30/18

Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop and implement novel management protocols for establishing improved forage species on sites infested with known poisonous plants to reduce the risk of livestock mortality and morbidity, improve livestock performance, and improve rangeland resiliency and diversity. Specifically, develop science-based guidelines for grazing livestock on rangelands infested with Lupinus, Senecio, Delphinium and swainsonine and selenium-containing plants. Objective 2: Reduce the risks of livestock losses due to variations in quantitative and qualitative differences in toxin accumulation over time and plant species by quantifying the influence of endophytes, climate changes, and genotype on plant toxin accumulation (particularly swainsonine-containing plants and Delphinium and Lupinus species). Objective 3: Enhance feed and food safety by improving risk assessment and diagnosis of plant-induced poisoning to livestock by improving analytical methods for analyzing plant and animal tissues for toxins; measuring toxicokinetics, assessing carcinogenic and genotoxic potential, and identifying toxin metabolites and biomarkers of toxicoses. Objective 4: Develop improved procedures with guidelines for diagnostic and prognostic evaluation to reduce negative impacts of poisonous plants on livestock reproduction and embryo/fetal growth by improving early identification of poisoned animals, predicting poisoning outcomes, and management and treatment options through improved understanding of clinical, morphological and molecular alterations of plant-induced toxicosis. Objective 5: Develop guidelines to aid producers and land managers in making genetic-based herd management decisions to improve livestock performance and safety on grazed rangelands infested with poisonous plants through the use of identified animal genes, physiological pathways, and molecular mechanisms of action that underlie Conium, Cicuta, Delphinium, Lupinus, and Nicotiana, and other neurotoxic plant effects. Approach (from AD-416): Livestock poisoning by plants results in over $503,000,000 lost to the livestock industry annually in the 17 western United States from death losses and abortions alone (Holechek, 2002). Plant poisonings extend worldwide to include 333 million poisonous plant-infested hectares in China and 60 million hectares in the central western region of Brazil, to name a few. There are over 6,000 species of pyrrolizidine alkaloid (PA)- containing plants, and over 350 individual PAs causing diseases in animals and humans have been identified. Economic losses are much larger as significant amounts of nutritious forage are wasted and management costs are increased due to the threat of toxic plant-related livestock losses. The Poisonous Plant Research Laboratory (PPRL) has provided worldwide leadership in poisonous plant research to the livestock industry and consumers including numerous solutions to toxic plant problems using an integrated, interdisciplinary approach (see Figure below). The research team investigates plant poisonings in a systematic matter by identifying the plant, describing the effects in animals, determining the toxin(s) and evaluating the mechanisms of action. The ultimate goal is to develop research-based solutions to reduce livestock losses from toxic plants. There are five coordinated objectives in this project plan providing guidelines for potential genetic-based management. This research will reduce livestock losses from plants and enhance the economic well-being of rural communities, improve rangeland health by combating invasive plant species, and help to provide safe animal products free from potential plant toxins for consumers. This is the final report for this project which was bridged by 2080-32630- 013-00D, �Understanding and Mitigating the Adverse Effects of Poisonous Plants on Livestock Production Systems�, in January 2018, while peer panel review of the next five-year project plan is undergoing assessment by the Office of Scientific Quality Review. For additional information, see the report for the bridging project. Progress was made on all five Objectives and their Sub-objectives, all of which fall under National Program 215, Component I, Improved Rangeland Management for Enhanced Livestock Production, Conservation and Ecological Services. Progress on this project focuses on Problem A, the need for developing economic livestock grazing systems for rangelands that meet global food security objectives while being adaptable to changing climate and varying environmental conditions and preserve the natural resources integrity. Results have been communicated through peer-reviewed publications and to stakeholders through various means. Objective 1: Grass and forb species were identified that improve range conditions by increasing the quantity and quality of other available forage, thus reducing the risk of livestock poisoning by plants. It was found that changes in livestock reproductive capacity and production efficiency were negatively affected because of chronic, low-dose selenium intake. Progress to determine the weather factors that influence the population dynamics and alkaloid composition of Delphinium andersonii was made. Objective 2: It was determined that the concentrations of abortifacient labdane acids in western juniper vary among different locations and over time. Fungicide application and clipping do not alter swainsonine concentrations or the transmission of the endophyte. Swainsonine concentrations in locoweeds are not influenced by elevated carbon dioxide (CO2) but other measures like biomass and crude protein change as a result of elevated CO2. The alkaloid composition of other Delphinium species, including D. stachydeum and D. ramosum was determined. Swainsonine was identified in several North and South American Astragalus species, previously not known to contain it, Oxytropis species from North America, and Swainsona species from Australia. The gene cluster responsible for swainsonine biosynthesis in endophytes and other fungi that are reported to contain swainsonine was identified. Objective 3: The comparative toxicity of the different forms of selenium in plants and their toxicokinetics and pathology in sheep and cattle were identified and characterized. It was demonstrated that the putative toxin tremetone is likely not responsible solely for the toxicity of rayless goldenrod and white snakeroot. A polymerase chain reaction (PCR)-based method was developed to detect poisonous plants like larkspur in mixed feeds and the gastrointestinal contents of poisoned animals. The toxicity and carcinogenic potential of pyrrolizidine alkaloids (PAs) like riddelliine and their metabolites were identified and characterized. Methods were developed for the detection of toxic metabolites (pyrroles) in liver samples from animals poisoned with pyrrolizidine alkaloids. Progress was made in the identification of the major diterpene acids in broom snakeweed and their rumen and sera metabolites. Methods were developed to detect metabolites of ponderosa pine identified from the sera of poisoned animals in fetal tissues from diagnostic cases, and methods were developed for the detection and quantitation of toxic principles including monofluoroacetate. Objective 4: Species susceptibility to locoweed poisoning and other swainsonine-containing plants (Ipomoea carnea) was described and compared. The effects of western juniper trees on cattle reproduction was characterized and management guidelines to reduce losses from abortions due to ponderosa pine and related species were further refined. The teratogenic potential of piperidine and quinolizidine alkaloids in cell culture, rodent, and goat models was evaluated to calculate the relative risk of each alkaloid for causing fetal deformities. The abortifacient effects of western juniper were characterized. It was found that pre- conditioned and na�ve cattle metabolize the abortifacient compounds from ponderosa pine differently and that the serum toxicokinetics of larkspur toxins differs among cattle, sheep and goats. Livestock exposed to multiple toxins from different toxic plants may not be at greater risk if the toxins affect different body systems. The effects of larkspur in cattle ingesting the plant over several days to simulate natural grazing conditions was characterized. It was found that the rumen microbiome changes with ingestion of ponderosa pine in cattle. Objective 5: It was found that cattle within and between breeds may differ greatly in resistance or susceptibility to Delphinium alkaloids, and that differences in response may be genetic and heritable. ARS researchers characterized the physiologic and toxicokinetic effects of varying dose, duration, and frequency of toxic plant alkaloids in livestock including larkspur where the clearance time (half-life) for the major toxic alkaloid in Delphinium was 20 hours. The biological mechanisms of action of several piperidine and quinolizidine alkaloids that may or not be teratogenic were also characterized. Accomplishments 01 Two Delphinium ramosum chemotypes, their biogeography and potential toxicity. Larkspurs (Delphinium, Ranunculaceae) are poisonous plants found on rangelands throughout western North America. Two main structural groups of norditerpene alkaloids, the N-(methylsuccinimido) anthranoyllycoctonine type (MSAL-type) and the non-MSAL type, are responsible for larkspur-induced poisoning. Information on the alkaloid composition is lacking for several Delphinium species, including D. ramosum, which grows throughout parts of Colorado and northern New Mexico. ARS scientists in Logan, Utah, investigated the alkaloid composition of D. ramosum throughout its geographical distribution using both field and herbarium specimens. They identified two alkaloid profiles and found that each had a unique geographical distribution. One profile contained significantly greater concentrations of the MSAL- type alkaloids than the other. This information has important implications in grazing management decisions on D. ramosum-infested rangelands and demonstrates that botanical classification alone is not a fully adequate indicator of relative risk of toxicity. 02 Selenium (Se) concentrations in plants and soil. Selenium concentrations in western aster (Symphyotrichum ascendens Lindl.) are dependent upon Se species and concentrations in Se-contaminated soil. Selenium accumulation in vegetation has resulted in poisoning of livestock that graze on historic phosphate mine soils in southeastern Idaho. ARS scientists in Logan, Utah, demonstrated that soluble and phosphate-extractable Se (selenite and selenate) were determined to be �bioavailable fractions� for western aster. Thus, simple water extractions can be used for quick assessment of Se bioavailability to western aster and provide a means to identify potentially hazardous areas. This information is helpful to regulatory agencies and mining officials when determining the potential risk of grazing livestock on reclaimed mining areas. 03 Cell culture investigations of teratogenic compounds. Are cell culture studies alone an accurate predictor of the ability of toxins to cause birth defects in livestock? ARS scientists from Logan, Utah, compared different piperidine alkaloid toxins from poisonous plants and their effects on neurological receptors. Specialized cell lines (TE-671 and SHSY-5Y cells) and pregnant goats were used to characterize the mechanism and increase understanding of why these plants cause birth defects and animal deaths. Results suggest that, while experiments with isolated cells provide valuable information and predictions of the actions of plant alkaloids, animal experiments are still required to determine the ability of an alkaloid to inhibit fetal movement in livestock species. Moreover, other pharmacological properties such as receptor differences between mammalian species and differences in the toxicological properties of the alkaloids are also likely to weaken teratologic predictions based solely on the cell culture data. 04 Fungicide treatment and clipping of locoweed (Oxytropis sericea) does not disrupt swainsonine concentrations. Swainsonine, an indolizidine alkaloid, is an a-mannosidase and mannosidase II inhibitor that causes significant livestock losses via lysosomal storage disease and altered glycoprotein processing. Swainsonine is found in several plant species worldwide and prolonged consumption of these plants by livestock causes a disease condition characterized by weight loss, depression, altered behavior, decreased libido, infertility, and death. ARS scientists in Logan, Utah, tested the hypothesis that fungicide application and clipping of plant vegetative tissues may alter swainsonine concentrations, thus rendering the plant less toxic. Plants were treated with four different fungicides and clipped to determine if swainsonine concentrations were altered. Treatment of Oxytropis sericea with any of four different fungicides did not alter swainsonine concentrations in plants at any of three harvest timings. These results demonstrate that clipping and fungicide treatment are not options that will render locoweeds less toxic and should not be pursued. 05 Analysis of monofluoroacetate in plants toxic to livestock and in rumen contents as a diagnostic tool. Monofluoroacetate is a potent toxin that occurs in several species in four plant families that may occur in Africa, Australia, and South America and is responsible for significant livestock deaths in these regions. ARS scientists in Logan, Utah, using different analytical methods detected monofluoroacetate in the South American plants Fridericia elegans, Niedenzuella multiglandulosa, Niendenzuella acutifolia, and Aenigmatanthera lasiandra. Monofluoroacetate was also detected in the rumen contents of sheep poisoned in Australia by Gastrolobium species. This is the first report of monofluoroacetate being detected in these plants, some of which have been reported to cause sudden death or toxicity in livestock. More importantly, this is the first time that monofluoroacetate has been detected in the rumen contents of a poisoned animal. Methods developed for the detection of monofluoroacetate may be used as tools to identify plants that pose a toxic risk to livestock as well as in diagnostic investigations of infected livestock. 06 Identification of pyrrolizidine alkaloids in various Senecio species from Uruguay. Senecio is a genus of the daisy family that includes ragworts and groundsels. Samples of several Senecio species were surveyed by an ARS scientist in Logan, Utah, in collaboration with scientists from Uruguay, for the potentially toxic compounds known as pyrrolizidine alkaloids (PAs). PAs are potent liver toxins that, under some conditions, can be carcinogenic. PAs were detected in these species and found to be unique to each type. The results provide tools for land managers in Uruguay to make better grazing management decisions due to the toxic risk of Senecio. 07 A survey of swainsonine content in Swainsona species. Swainsonine, an indolizidine alkaloid, is found in several plant species worldwide, and causes severe toxicosis when ingested by livestock. Some species of the Australian genus Swainsona have been reported to be toxic to livestock due to the toxin swainsonine but a systematic screen using modern chemical instrumentation is lacking. Swainsonine was detected by ARS scientists in Logan, Utah, and an Australian collaborator using both liquid chromatography-mass spectrometry (LCMS) and gas chromatography- mass spectrometry (GCMS). Nine of 41 species screened tested positive where 8 of them had been previously determined as negative for swainsonine. Herbarium specimens were collected to assist in identification. This data set provides a valuable tool for risk assessment and diagnostic purposes and highlights the utility of herbarium specimens in phytochemical studies.

Impacts
(N/A)

Publications

Santos-Barbosa, J.M., Lee, S.T., Cook, D., Gardner, D.R., Viana, L.H., Re, N. 2017. A gas chromatography-mass spectrometry (GC-MS) method for the detection and quantitation of monofluoroacetate in plants toxic to livestock. Journal of Agricultural and Food Chemistry. 65(7):1428�1433.
Panter, K.E., Taylor, J.B., Lee, S.T., Strong, N.K., Wierenga, T.L., Cook, D., Welsh, S.L. 2017. Lupine poisoning in sheep on the USDA-ARS U.S. Sheep Experiment Station (USSES), Dubois, Idaho. International Journal of Poisonous Plant Research. 4:79-87.
Colegate, S.M., Gardner, D.R., Resager, W.C., Bollar, N., Betz, J.M., Panter, K.E. 2017. Hydroxylic solvent-induced ring opening of the dehydropyrrolizidine alkaloids riddelliine and seneciphylline: Implications for toxicity and analytical studies. International Journal of Poisonous Plant Research. 4:1-15.
Butler, R., Tai, S., Allen, J.G., Cook, D., Lee, S.T. 2017. Gastrolobium spp. poisoning in sheep: A case report. International Journal of Poisonous Plant Research. 4:68-71.
Panter, K.E., Colegate, S.M., Davis, T.Z., Welsh, S.L., Gardner, D.R., Cuneo, P.S., Stegelmeier, B.L., Stonecipher, C.A. 2017. Fiddleneck (Amsinckia intermedia Lehmann Boraginaceae): Toxicity in cattle potentiated by burrow weed (Isocoma acradenia). International Journal of Poisonous Plant Research. 4(1):16-24.
Welsh, S.L., Panter, K.E., Colegate, S.M., Gardner, D.R., Cuneo, P.S., Davis, T.Z., Stegelmeier, B.L., Stonecipher, C.A. 2017. Amsinckia Lehmann (Boraginaceae): A summary taxonomic review. International Journal of Poisonous Plant Research. 4(1):25-35.
Lee, S.T., Gardner, D.R., Cook, D. 2017. Identification of indole diterpenes in Ipomoea asarifolia and Ipomoea muelleri, plants tremorgenic to livestock. Journal of Agricultural and Food Chemistry. 65:5266-5277.
Alhawatema, M.S., Gebril, S., Cook, D., Creamer, R. 2017. RNAi-mediated down-regulation of a melanin polyketide synthase (pks1) gene in the fungus Slafractonia leguminicola. World Journal of Microbiology and Biotechnology. 33(10):179.
de Oliveira Neto, T.S., Riet-Correa, F., Lee, S.T., Cook, D., Sousa Barbosa, F.M., da Silva Neto, J.F., Sim�es, S.V., Lucena, R.B. 2017. Poisoning in goats by the monofluoracetate-containing plant Palicourea aeneofusca (Rubiaceae). Toxicon. 135:12-16.
Pimentel, L.A., Maia, L.A., Campos, E.M., Dantas, A.F., Medeiros, R.M., Pfister, J.A., Cook, D., Riet-Correa, F. 2012. Conditioned food aversion to control outbreaks of intoxication by Ipomoea carnea and Turbina cordata in goats. Pesquisa Veterinaria Brasileira. 32(8):707-714.
Stegelmeier, B.L. 2016. Overview of sweet clover poisoning. In: Aiello, S. E., Moses, M.A, editors. Merck Veterinary Manual. 11th edition. Kenilworth, NJ: Merck and Company. Available:
Stegelmeier, B.L. 2016. Overview of bracken fern poisoning. In: Aiello S.E. , Moses, M.A, editors. Merck Veterinary Manual. 11th edition. Kenilworth, NJ: Merck and Company. Available:
Pfister, J.A., Villalba, J., Gardner, D.R. 2012. Effect of dietary protein level and quebracho tannin on consumption of pine needles (Pinus ponderosa) by beef cows. Professional Animal Scientist. 28(5):528-533.
Gotardo, A.T., Schumaher, B.H., Pfister, J.A., Traldi, A.S., Maiorka, P.C., Spinosa, H.S., Gorniak, S.L. 2012. The use of ultrasonography to study teratogenicity in ruminants: Evaluation of Ipomoea carnea in goats. Birth Defects Research Part B: Developmental and Reproductive Toxicology. 95(4) :289-295.
Estep, A.S., Becnel, J.J., Lee, S.T. 2016. Toxicity of compounds isolated from white snakeroot (Ageratina altissima) to adult and larval yellow fever mosquitoes (Aedes aegypti). Natural Product Communications. 11(11) :1675-1677.
Clemensen, A.K., Rottinghaus, G.E., Lee, S.T., Provenza, F.D., Villalba, J. J. 2017. How planting configuration influences plant secondary metabolites and total N in tall fescue (Festuca arundinacea Schreb.), alfalfa (Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatus L.). Grass and Forage Science. 1-7.
Favorito, J.E., Eick, M.J., Grossl, P.R., Davis, T.Z. 2017. Selenium geochemistry in reclaimed phosphate mine soils and its relationship with plant bioavailability. Plant and Soil. 418(1-2):541-555.
Panter, K.E., Welch, K.D., Gardner, D.R. 2014. Poisonous plants: Biomarkers for diagnosis. In: Gupta, R.C., editor. Biomarkers in Toxicology. Oxford, UK: Elsevier. p. 563-589.
Cook, D., Gardner, D.R., Roper, J.M., Ransom, C.V., Pfister, J.A., Panter, K.E. 2016. Fungicide treatment and clipping of Oxytropis sericea does not disrupt swainsonine concentrations. Toxins. 122:26-30.
Cook, D., Gardner, D.R., Welch, K.D., Allen, J.G. 2017. A survey of swainsonine content in Swainsona species. The Australian Rangeland Journal. 39:213-218.
Cook, D., Gardner, D.R., Pfister, J.A., Lee, S.T., Welch, K.D., Welsh, S.L. 2017. A screen for Swainsonine in select North American Astragalus species. Chemistry and Biodiversity. 14(4):e1600364.
Cook, D., Donzelli, B., Creamer, R., Baucom, D.L., Gardner, D.R., Pan, J., Moore, N., Krasnoff, S., Jaromczyk, J.W., Schardl, C.L. 2017. Swainsonine biosynthesis genes in diverse symbiotic and pathogenic fungi. G3, Genes/ Genomes/Genetics. 7(6):1791-1797.
Peixoto De Arruda, F., Bravim Caldeira, F.H., Ducatti, K., Bezerra, K.S., Marcolongo-Pereira, C., Lee, S.T., Cook, D., Riet-Correa, F., Colodel, E. 2017. Experimental poisoning by Niedenzuella stannea in cattle and corresponding detection of monofluoroacetate. Ciencia Rural. 47(03) :e20160761.
Stegelmeier, B.L., Brown, A.W., Welch, K.D. 2015. Safety concerns of herbal products and traditional Chinese herbal medicines: Dehydropyrrolizidine alkaloids and aristolochic acid. Journal of Applied Toxicology. 35(12):1433-1437.
Pfister, J.A., Lee, S.T., Arnett, D., Panter, K.E. 2017. Preference by horses for bedding pellets made from switchgrass (Panicum virgatum) straw. Professional Animal Scientist. 33(3):349�356.
Clemensen, A.K., Provenza, F.D., Lee, S.T., Gardner, D.R., Rottinghaus, G. E., Villalba, J.J. 2017. Plant secondary metabolites in alfalfa, birdsfoot trefoil, reed canarygrass, and tall fescue unaffected by two different nitrogen sources. Crop Science. 57(2):964�970.
Preliasco, M., Gardner, D.R., Moraes, J., Gonzalez, A.C., Uriarte, G., Rivero, R. 2017. Senecio grisebachii Baker: Pyrrolizidine alkaloids and experimental poisoning in calves. Toxicon. 133:68-73.
Riet-Correa, F., Medeiros, R.M., Pfister, J.A., Mendon�a, F.S. 2017. Toxic plants affecting the nervous system of ruminants and horses in Brazil. Pesquisa Veterinaria Brasileira. 37(12):1357-1368.
Cook, D., Gardner, D.R., Lee, S.T., Stonecipher, C.A., Pfister, J.A., Welch, K.D., Green, B.T. 2017. Two Delphinium ramosum chemotypes, their biogeographical distribution and potential toxicity. Biochemical Systematics and Ecology. 75:1-9.
Green, B.T., Lee, S.T., Keele, J.W., Welch, K.D., Cook, D., Pfister, J.A., Kem, W.R. 2018. Complete inhibition of fetal movement in the day 40 pregnant goat model by the piperidine alkaloid anabasine but not related alkaloids. Toxicon. 144:61-67.
Welch, K.D., Lee, S.T., Davis, T.Z. 2017. An evaluation of the toxicity of white snakeroot (Ageratina altissima) and rayless goldenrod (Isocoma pluriflora) in a lactating mouse model. International Journal of Poisonous Plant Research. 4(1):43-52. Available:
Lee, S.T., Davis, T.Z., Cook, D. 2017. Evaluation of the stability of benzofuran ketones in rayless goldenrod (Isocoma pluriflora) and white snakeroot (Ageratina altissima) under different storage conditions. International Journal of Poisonous Plant Research. 4(1):36-42.
Caldeira, F.H., Arruda, F.P., Shenkel, D.M., Marcolong-Pereira, C., Lee, S. T., Cook, D., Riet-Correa, F., Colodel, E.M. 2017. Intoxica��o experimental por Niedenzuella stannea (Malpighiaceae) em ovinos. Pesquisa Veterinaria Brasileira. 37(7):681-685.

Progress 10/01/16 to 09/30/17

Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop and implement novel management protocols for establishing improved forage species on sites infested with known poisonous plants to reduce the risk of livestock mortality and morbidity, improve livestock performance, and improve rangeland resiliency and diversity. Specifically, develop science-based guidelines for grazing livestock on rangelands infested with Lupinus, Senecio, Delphinium and swainsonine and selenium-containing plants. Objective 2: Reduce the risks of livestock losses due to variations in quantitative and qualitative differences in toxin accumulation over time and plant species by quantifying the influence of endophytes, climate changes, and genotype on plant toxin accumulation (particularly swainsonine-containing plants and Delphinium and Lupinus species). Objective 3: Enhance feed and food safety by improving risk assessment and diagnosis of plant-induced poisoning to livestock by improving analytical methods for analyzing plant and animal tissues for toxins; measuring toxicokinetics, assessing carcinogenic and genotoxic potential, and identifying toxin metabolites and biomarkers of toxicoses. Objective 4: Develop improved procedures with guidelines for diagnostic and prognostic evaluation to reduce negative impacts of poisonous plants on livestock reproduction and embryo/fetal growth by improving early identification of poisoned animals, predicting poisoning outcomes, and management and treatment options through improved understanding of clinical, morphological and molecular alterations of plant-induced toxicosis. Objective 5: Develop guidelines to aid producers and land managers in making genetic-based herd management decisions to improve livestock performance and safety on grazed rangelands infested with poisonous plants through the use of identified animal genes, physiological pathways, and molecular mechanisms of action that underlie Conium, Cicuta, Delphinium, Lupinus, and Nicotiana, and other neurotoxic plant effects. Approach (from AD-416): Livestock poisoning by plants results in over $503,000,000 lost to the livestock industry annually in the 17 western United States from death losses and abortions alone (Holechek, 2002). Plant poisonings extend worldwide to include 333 million poisonous plant-infested hectares in China and 60 million hectares in the central western region of Brazil, to name a few. There are over 6,000 species of pyrrolizidine alkaloid (PA)- containing plants, and over 350 individual PAs causing diseases in animals and humans have been identified. Economic losses are much larger as significant amounts of nutritious forage are wasted and management costs are increased due to the threat of toxic plant-related livestock losses. The Poisonous Plant Research Laboratory (PPRL) has provided worldwide leadership in poisonous plant research to the livestock industry and consumers including numerous solutions to toxic plant problems using an integrated, interdisciplinary approach (see Figure below). The research team investigates plant poisonings in a systematic matter by identifying the plant, describing the effects in animals, determining the toxin(s) and evaluating the mechanisms of action. The ultimate goal is to develop research-based solutions to reduce livestock losses from toxic plants. There are five coordinated objectives in this project plan providing guidelines for potential genetic-based management. This research will reduce livestock losses from plants and enhance the economic well-being of rural communities, improve rangeland health by combating invasive plant species, and help to provide safe animal products free from potential plant toxins for consumers. Progress was made on all five objectives and their sub-objectives, all of which fall under National Program 215, Component I, Rangeland Management Systems to Improve Economic Viability and Enhance the Environment. Progress was made on identifying sex differences in susceptibility to larkspur intoxication in Angus cattle. Beef producers in the western United States have reported that replacement heifers are most impacted by toxic larkspur (Delphinium) species. Initial findings have demonstrated that Angus heifer yearlings are over ten times more susceptible to toxic larkspur than Angus steers. These research results confirm field reports from ranchers of poisonings. This suggests that replacement heifers should not be grazed in pastures where toxic larkspur species grow. Beef producers in the western United States have reported that younger animals are most impacted by toxic larkspur species. Preliminary results showed Angus steers have a 20 percent greater tolerance to larkspur compared to yearlings by two years of age. This suggests that older animals have a greater tolerance to larkspur intoxication and with careful management can be better suited to graze in pastures that contain toxic larkspur species. Recent research efforts have demonstrated that great variation may exist in plant population density depending on factors such as climate and locale. Delphinium andersonii is a perennial forb on semi-arid rangelands, and population density is related to cattle deaths. A multi-year study was initiated in 2011 to measure toxicity, plant density and life history of D. andersonii populations in southern Idaho. In 4 out of 7 years, essentially no plants emerged and flowered because of drought conditions. During the 3 years when some plants emerged and flowered, plant densities were 2, 7, and 10 plants per square meter (m2); previous research has shown that plant densities greater than 5 plants/m2 pose a serious threat to grazing cattle. Approximately 8 percent of the marked plants remained dormant for 2-4 years, then emerged when precipitation was favorable. Understanding the life history and weather conditions that promote growth of dense Delphinium andersonii populations will enable livestock producers to better manage risk of cattle deaths. A survey of the toxic compounds in water hemlock has been performed in order to determine the variation in toxicity of different populations of water hemlock. Water hemlock is one of the most toxic plants known. Plant samples were collected from sites in Arizona, Utah, Nevada, Idaho, Wyoming, and Colorado. Initial analyses suggest that there is no difference in the toxicity of water hemlock at the various locations. Additional collections will be made. Further, samples of water hemlock will be collected from multiple sites over the growing season to determine if the toxicity of the plant changes as the plant grows and matures. This information will provide a better understanding of the toxic potential of water hemlock throughout the year as well as a better understanding of the variation in toxicity from locations throughout the western U.S. The plant Ipomoea asarifolia has been shown to cause a tremorgenic syndrome in goats. The tremorgenic compounds in I. asarifolia are reported to be indole diterpenes. The chemical profile of these indole diterpene compounds in I. asarifolia has been characterized. Initial efforts have been made to develop the methods to use a mouse model to study the tremorgenic nature of I. asarifolia. Once the mouse model has been developed and validated, the tremorgenic potential of individual purified indole diterpenes will be evaluated. Noxious annual grasses such as medusahead and ventenata are highly invasive and are displacing native vegetation throughout the western U.S. They result in stands of near monocultures reducing livestock and wildlife grazing capacity by 50 to 80%. Weedy and poisonous forbs, such as lupine, are often all that remain for livestock to graze resulting in increased consumption of poisonous plants. Herbicides with different modes of action are being tested to reduce seed production and to control ventenata and medusahead. Targeted grazing by livestock on herbicide treated ventenata and medusahead is being tested to determine if livestock can be used as a tool to remove the annual grasses and provide a seed bed for revegetation of desirable plant species. Providing alternative forages for livestock can help reduce the consumption of poisonous plants by livestock. Efforts are being conducted in collaboration with a foraging behavioral specialist at Utah State University (USU). Death camas is a bulbous perennial forb that occurs in a variety of habitats and is common in sagebrush foothills throughout the western United States. It starts growth early in the spring (typically one of the first plants to begin growth). The lack of alternative forage at this time can result in increased consumption by livestock and death losses, especially in sheep. Recent formulations of herbicides are being tested to determine their efficacy to control death camas while having minimal impacts on desirable forage. Death camas is also being tested to determine if herbicide application affects toxicity of the plant before it senesces. This information can be used to produce guidelines for managers to determine how to safely graze death camas infested rangelands treated with herbicides and what herbicide can most effectively control death camas. Efforts are being conducted in collaboration with a rangeland extension specialist and weed science specialist at USU. Long-term establishment of a competitive plant community on degraded rangelands within the arid Intermountain west is necessary to compete with invasive and/or poisonous plants. Broom snakeweed increases following disturbances such as overgrazing, drought, or wildfire and is toxic to livestock. Establishing a competitive plant community can help reduce the establishment and spread of broom snakeweed. Long-term monitoring is being conducted on research plots that were seeded in 2003 to determine if native and introduced grasses can persist in the arid environment of the Intermountain west and if the seeded species will provide competition to reduce poisonous plants. Efforts are being conducted in collaboration with a rangeland extension specialist at USU. Dehydropyrrolizidine alkaloids (PAs) are plant toxins that can poison livestock, wildlife and humans. Several have also been shown to be carcinogens. T demonstrated that riddelliine induced neoplasms at increased incidences regardless of dose or exposure duration. This work was important as it indicated that any PA exposure can increase the incidence of PA-related carcinogenesis. We have also shown the PAs, lasiocarpine, seneciophylline, senecionine and heliotridine were more toxic than riddelliine and thus potentially more carcinogenic. To further evaluate this hypothesis we have generated a colony of knockout mice and initiated studies to directly compare the carcinogenicity of these purified PAs. Additionally, we have further developed techniques to identify PA metabolites or adducts from livers and tissues of animals that were exposed to PAs by comparing adducts from tissues from cattle, horses, pigs and rodents. Initial results suggest the improved techniques will enhance detection of PA exposure and estimating initial dose and when used with histologic studies might be used to predict disease course and prognosis. Selenium concentration in forage may negatively impact reproduction in livestock. Bulls were fed alfalfa pellets containing selenium concentrations to mimic concentrations found in forages on some seleniferous soils. High levels of selenium in the diet had no effect on sperm motility or morphology. However, high selenium caused lameness and/ or congestive heart failure in some animals after 12 weeks of feeding the high selenium alfalfa pellets. These results suggest that cattle producers grazing in regions where forages are high in selenium should manage their herds to avoid exposure to high selenium forages for prolonged periods. Certain lupine (Lupinus) species in the western United States cause crooked calf syndrome (CCS). One alkaloid in particular, anagyrine, is very problematic in cattle and has been proposed to be metabolized to a teratogenic alkaloid that inhibits fetal movement, the putative mechanism behind CCS. In this cell culture research using two cell lines, an alternative hypothesis that anagyrine directly desensitizes fetal muscle was tested. Research results indicate that anagyrine is bioactive without metabolism and is directly a factor that causes malformation of an embryo (teratogenic) in cattle. Because un-metabolized anagyrine is bioactive, it can be used as a biomarker for the selection of cattle resistant to the teratogenic actions of quinolizidine alkaloids found in lupines. Soils high in selenium may promote the growth of toxic, high selenium forage. High selenium soil was treated with various amendments that could be used on reclaimed mine sites where poisonous selenium- accumulating forages grow. The addition of iron as an amendment to high selenium soil resulted in approximately a 90 percent decrease in the uptake of selenium by western aster, intermediate wheat grass, and alfalfa. The addition of the iron amendment also resulted in an increase in the biomass of intermediate wheat grass and alfalfa while decreasing the biomass of the highly toxic western aster. The information obtained in this study indicates iron can be used as an amendment to decrease selenium concentrations in forages growing on selenium-contaminated reclaimed mine sites. Accomplishments 01 Liver toxicity of Salvia compounds in cattle. ARS scientists in Logan, Utah, investigated a field case of cattle poisoning in Colorado where 170 cows in a herd of over 500 died after eating weed-infested alfalfa hay. Several hepatotoxic diterpenes were isolated from the contaminated alfalfa hay fed to the deceased animals. A bioassay guided chemical fractionation and extraction process to identify the toxins of the weedy hay was used. The identified diterpenoids known to be present in different Salvia species led to the identification of dried plant parts (stem and flower pods) of the toxic plant in bales of hay. A reexamination of the hay field location found a significant population of the toxic Salvia along the hay field edges and irrigation ditch banks. Historically, Salvia species have been suspected of causing toxicity in several areas worldwide, but this research is the first to link the diterpene compounds in Salvia with liver toxicity in livestock and will provide important information to veterinarians for diagnosis, and to livestock and hay producers to prevent poisonings in the future. 02 Effects of elevated carbon dioxide (CO2) on two Astragalus species. Understanding how locoweed responds to elevated CO2 is important for risk assessment as the climate changes. ARS researchers in Logan, Utah, in collaboration with other scientists, evaluated how two Astragalus species responded to elevated CO2 in a controlled environment. Swainsonine concentrations were not strongly affected by elevated CO2. Other traits such as biomass and water soluble carbohydrates responded positively while crude protein responded negatively to elevated CO2. Information on how toxic plants and their toxic principles respond to climate change is essential to risk assessment. 03 Rangeland restoration in central Washington to reduce losses from lupine-induced crooked calf syndrome (CCS). Lupine-induced crooked calf syndrome has caused devastating losses to cattle ranchers in central Washington, which may be exacerbated by declining forage conditions on rangelands due to invasive annual weeds. ARS Scientists at Logan, Utah, completed a three year analysis funded by a Western Sustainable Agriculture Research and Education (SARE) grant (OW13-005) to study the impacts of seeding rangelands infested with toxic lupine species. Results showed that degraded rangeland, heavily infested with undesirable plants such as cheatgrass and medusahead rye as well as lupine, could be substantially restored using targeted grazing followed by seeding with perennial grasses and forage Kochia. Improved rangeland conditions will reduce the need for cattle to graze lupine and other poisonous plants present on these ranges. More desirable forage production will improve cattle production in this region, including a reduction in CCS losses from lupines. 04 Detection of pyrrolizidine alkaloids in herbal products. ARS scientists in Logan, Utah, in cooperation with American Herbal Pharmacopoeia, analyzed about 70 samples, purported to be from the herbal plant �boneset� (Eupatorium perfoliatum), for the presence of dehydropyrrolizidine alkaloids. These alkaloids are potentially toxic, associated with liver and lung damage, congenital anomalies and various cancers. The results clearly show that �boneset� contains toxic dehydropyrrolizidine alkaloids. Further, there is misidentification of plants by some collectors, revealed by the difference in the alkaloids detected. Tinctures and water infusions of �boneset� also contain the alkaloids. Pyrrolizidine alkaloids are present in many herbal products, and potentially serious negative impacts on human heath, particularly liver toxicity. These findings will provide valuable information to the herbal products industry and consumer to reduce safety concerns and toxicity problems in humans. 05 Swainsonine biosynthesis genes in diverse symbiotic and pathogenic fungi. Understanding the genes responsible for swainsonine biosynthesis provides fundamental knowledge that may provide a means to render swainsonine-containing plants less toxic. ARS researchers in Logan, Utah, in collaboration with other scientists, identified the gene cluster responsible for swainsonine biosynthesis in two endophytes associated with two families of swainsonine-containing plants. This research extended the breadth of swainsonine-producing fungi to include human and other mammalian pathogens. Understanding swainsonine biosynthesis may provide critical knowledge to ultimately alter swainsonine production thus rendering plants less toxic. 06 Switchgrass pellets used as horse bedding material. Switchgrass (Panicum virgatum) is grown and harvested for biofuel, and switchgrass straw is used to make bedding pellets for livestock, but the material contains saponins. Saponins have been implicated in photosensitization in livestock, including horses. ARS scientists in Logan, Utah, examined the saponin concentration in switchgrass straw, and in bedding pellets made from switchgrass straw. It was determined the palatability of bedding pellets made from switchgrass straw to horses. Switchgrass straw and bedding pellets made from switchgrass straw contained low concentrations of saponins, however, horses still rejected eating switchgrass bedding pellets. These results indicate that the risk of intoxication from horses ingesting bedding pellets made with switchgrass straw is very low, or non-existent, and is important knowledge for commercial pellet makers and horse owners. 07 Consumption of larkspur by resistant and susceptible cattle. Larkspurs are a major cause of cattle losses on western ranges in North America, especially on foothill and mountain rangelands. ARS scientists at Logan, Utah, screened cattle to provide groups of resistant and susceptible animals. Cattle grazed for several seasons on a larkspur-infested rangeland in southeastern Idaho. Susceptible animals initially consumed more larkspur than did resistant animals, provoking serious and potentially fatal intoxication in susceptible animals. However, by the end of the grazing seasons, there were few differences in larkspur consumption between resistant and susceptible cattle. Resistant cattle may consume 2-3 times the amount of larkspur that susceptible animals consume with greatly reduced incidences of overt poisoning and death which is important to livestock producers who graze animals where larkspurs are an important component of the vegetation. 08 Identification of tremorgenic compounds in Ipomoea asarifolia and Ipomoea muelleri. I. asarifolia and I. muelleri have been associated with a tremorgenic syndrome in livestock in Brazil and Australia, respectively. ARS scientists at Logan, Utah, characterized the tremorgenic compounds in I. asarifolia and I. muelleri by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. This is the first detailed mass spectrometry analysis of the tremorgenic compounds in I. asarifolia and I. muelleri. These analytical methods will be important for determining the toxin concentration in plants and thus the toxic risks associated with individual plant populations. 09 Development of a gas chromatography-mass spectrometry (GC-MS) method for the analysis of monofluoroacetate in plants toxic to livestock. Monofluoroacetate is a potent toxin that occurs in over 50 plant species in Africa, Australia, and South America. It is responsible for significant livestock deaths in these regions. For example, monofluoroacetate containing plants are responsible for >50% of livestock losses in Brazil (>500,000 cattle annually). ARS scientists at Logan, Utah, developed a quantitative method for the analysis of monofluoroacetate in plants. Monofluoroacetate was detected in the following plants for the first time: Fridericia elegans, Niedenzuella multiglandulosa, Niedenzuella acutifolia, and Aenigmatanthera lasiandra. This method is another tool to mitigate livestock poisoning by monofluoroacetate-containing plants by increasing the ability of scientists to evaluate the poisoning risk of these plants. 10 A survey of North American Astragalus plant populations for swainsonine (the locoweed toxin). A systematic examination for swainsonine in these species provided a definitive reference in regard to species containing swainsonine and is a valuable reference for land managers. ARS researchers in Logan, Utah, conducted a systematic examination for swainsonine in multiple Astragalus species. Fifty-two Astragalus species were screened for swainsonine using chemical analytical techniques. Swainsonine was detected in twenty-nine species previously not reported to contain swainsonine. The expanded list of swainsonine- containing taxa will serve as an essential reference for risk assessment and avoidance of livestock poisoning on rangelands with Astragalus species. 11 The role of calystegines from Ipomoea carnea in poisoning goats. Swainsonine is a potent toxin found in numerous plant species around the world, including several types of locoweeds found in the United States. I. carnea contains swainsonine and calystegines (a similar toxin). ARS scientists in Logan, Utah, in cooperation with Brazilian scientists, evaluated the role of calystegines in goats poisoned by I. carnea. They determined that when locoweed (Astragalus) and I. carnea are fed to goats at equal swainsonine doses that there is minimal differences between the goats dosed with the two plants. These research results indicate that the Ipomoea produced calystegines contribute little to the development of Ipomoea-induced neurologic disease, rather swainsonine is the critical toxin in I. carnea. 12 The effect of ponderosa pine needles on the rumen microbiome of cattle. Consumption of ponderosa pine needles by cattle during the later stages of pregnancy can cause abortions to occur. Previous research by ARS scientists in Logan, Utah, demonstrated that there is a difference in metabolism of the compounds in the pine needles that cause abortions between cattle that are na�ve to ponderosa pine needles versus cattle that have been conditioned (i.e., exposed) to the pine needles. Recent results have now shown that rumen microbe populations change upon exposure to pine needles, which alters the metabolism of the compounds causing the abortions. However, the change is very transitory as the microbe populations revert back to baseline within one week after pine needle exposure stops. This data provides important knowledge regarding adaption of cattle to poisonous plants and the feasibility of potential preventative treatments. 13 Characterization of absorption and elimination of larkspur alkaloids in goats dosed orally versus intravenously. Larkspur (Delphinium) plants are acutely toxic to cattle, resulting in significant cattle losses every year. ARS scientists in Logan, Utah, compared the absorption and elimination of the toxic compounds in larkspur plants in goats that were dosed both orally and intravenously (IV). It was determined that the absorption of the toxins is the rate limiting step in the kinetic process. This knowledge will help provide a better understanding of how and when to treat cattle that have been poisoned by larkspur. 14 The effect of multiple doses of a less toxic larkspur plant to cattle. Larkspur (Delphinium) plants are acutely toxic to cattle, resulting in significant cattle losses every year. However, different species of larkspur, and even different populations of a given species of larkspur, have different toxic risks. In order to more fully characterize the toxic potential of larkspurs, ARS scientists in Logan, Utah, dosed cattle with a less toxic larkspur twice a day for four days. It was determined that the cattle can become poisoned when dosed with a less toxic larkspur multiple times in a short time period. This knowledge will help in defining the risk of poisoning for all larkspur plants, whether they are highly toxic or less toxic.

Impacts
(N/A)

Publications

Yang, M., Hall, J., Fan, Z., Regouski, M., Meng, Q., Rutigliano, H., Stott, R., Rood, K., Panter, K.E., Polejaeva, I. 2016. Oocytes from small and large follicles exhibit equal development competence following goat cloning despite their differences in meiotic and cytoplasmic maturation. Theriogenology. 86:2302-2311.
Pfister, J.A., Cook, D., Panter, K.E., Welch, K.D., James, L.F. 2016. USDA- ARS Poisonous Plant Research Laboratory: History and current research on western North American rangelands. Rangelands. 38(5):241-249.
Stegelmeier, B.L., Colegate, S.M., Brown, A.W. 2016. Dehydropyrrolizidine alkaloid toxicity, cytotoxicity, and carcinogenicity. Toxins. 8:356. doi:10.3390/toxins8120356.
Almeida, V.M., Rocha, B.P., Pfister, J.A., Medeiros, R.M., Riet-Correa, F., Chaves, H.A., Silva Filho, G.B., Mendonca, F.S. 2017. Spontaneous poisoning by Prosopis juliflora (Leguminosae) in sheep. Pesquisa Veterinaria Brasileira. 37(2):110-114.
Welch, K.D., Stonecipher, C.A., Green, B.T., Gardner, D.R., Cook, D., Pfister, J.A. 2017. Administering multiple doses of a non N- (methylsuccinimido) anthranoyllycoctonine (MSAL)-containing tall larkspur (Delphinium occidentale) to cattle. Toxicon. 128:46-49. doi: 10.1016/j. toxicon.2017.01.020.
Green, B.T., Lee, S.T., Welch, K.D., Cook, D. 2017. Anagyrine desensitization of peripheral nicotinic acetylcholine receptors. A potential biomarker of quinolizidine alkaloid teratogenesis in cattle.. Research in Veterinary Science. 115:195-200. doi: 10.1016/j.rvsc.2017.04. 019.
Welch, K.D., Gardner, D.R., Stonecipher, C.A., Green, B.T., Pfister, J.A. 2017. Serum toxicokinetics after intravenous and oral dosing of larkspur toxins in goats. Toxicon. 133:91-94. doi: 10.1016/j.toxicon.2017.05.008.
Ruiz-Santos, P., Pfister, J.A., Verdes, J.M. 2016. Conditioning and aversion to toxic Solanum bonariense (naranjillo) leaves in calves. Ciencia Rural. 46(4):669-673.
Welch, K.D., Stonecipher, C.A., Gardner, D.R., Cook, D., Pfister, J.A. 2017. Changes in the rumen bacterial microbiome of cattle exposed to ponderosa pine needles. Journal of Animal Science. 95:2314�2322. doi: 10. 2527/jas2016.1228.
Welch, K.D., Lee, S.T., Cook, D., Green, B.T., Panter, K.E. 2017. Natural toxins of plant origin (phytotoxins). In: Witczak, Al, Sikorshi, Z.E., editors. Toxins and Other Harmfuil Compounds in Foods (Chemical and Functional Properties of Food Components Series). 1st edition. Boca Raton, FL: CRC Press. p.7-52.
Panter, K.E., Welch, K.D., Gardner, D.R. 2017. Toxic plants: Effects on reproduction and fetal and embryonic development in livestock. In: Gupta, R.C., editor. Reproductive and Developmental Toxicology. 2nd edition. Cambridge, MA: Academic Prsss. p. 903-923.
Davis, T.Z., Tiwary, A.K., Stegelmeier, B.L., Pfister, J.A., Panter, K.E., Hall, J.O. 2017. Comparative oral dose toxicokinetics of sodium selenite and selenomethionine. Journal of Applied Toxicology. 37:231-238. doi: 10. 1002/jat.3350.

Progress 10/01/15 to 09/30/16

Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop and implement novel management protocols for establishing improved forage species on sites infested with known poisonous plants to reduce the risk of livestock mortality and morbidity, improve livestock performance, and improve rangeland resiliency and diversity. Specifically, develop science-based guidelines for grazing livestock on rangelands infested with Lupinus, Senecio, Delphinium and swainsonine and selenium-containing plants. Objective 2: Reduce the risks of livestock losses due to variations in quantitative and qualitative differences in toxin accumulation over time and plant species by quantifying the influence of endophytes, climate changes, and genotype on plant toxin accumulation (particularly swainsonine-containing plants and Delphinium and Lupinus species). Objective 3: Enhance feed and food safety by improving risk assessment and diagnosis of plant-induced poisoning to livestock by improving analytical methods for analyzing plant and animal tissues for toxins; measuring toxicokinetics, assessing carcinogenic and genotoxic potential, and identifying toxin metabolites and biomarkers of toxicoses. Objective 4: Develop improved procedures with guidelines for diagnostic and prognostic evaluation to reduce negative impacts of poisonous plants on livestock reproduction and embryo/fetal growth by improving early identification of poisoned animals, predicting poisoning outcomes, and management and treatment options through improved understanding of clinical, morphological and molecular alterations of plant-induced toxicosis. Objective 5: Develop guidelines to aid producers and land managers in making genetic-based herd management decisions to improve livestock performance and safety on grazed rangelands infested with poisonous plants through the use of identified animal genes, physiological pathways, and molecular mechanisms of action that underlie Conium, Cicuta, Delphinium, Lupinus, and Nicotiana, and other neurotoxic plant effects. Approach (from AD-416): Livestock poisoning by plants results in over $503,000,000 lost to the livestock industry annually in the 17 western United States from death losses and abortions alone (Holechek, 2002). Plant poisonings extend worldwide to include 333 million poisonous plant-infested hectares in China and 60 million hectares in the central western region of Brazil, to name a few. There are over 6,000 species of pyrrolizidine alkaloid (PA)- containing plants, and over 350 individual PAs causing diseases in animals and humans have been identified. Economic losses are much larger as significant amounts of nutritious forage are wasted and management costs are increased due to the threat of toxic plant-related livestock losses. The Poisonous Plant Research Laboratory (PPRL) has provided worldwide leadership in poisonous plant research to the livestock industry and consumers including numerous solutions to toxic plant problems using an integrated, interdisciplinary approach (see Figure below). The research team investigates plant poisonings in a systematic matter by identifying the plant, describing the effects in animals, determining the toxin(s) and evaluating the mechanisms of action. The ultimate goal is to develop research-based solutions to reduce livestock losses from toxic plants. There are five coordinated objectives in this project plan providing guidelines for potential genetic-based management. This research will reduce livestock losses from plants and enhance the economic well-being of rural communities, improve rangeland health by combating invasive plant species, and help to provide safe animal products free from potential plant toxins for consumers. Progress was made on all 5 objectives and their sub-objectives, all of which fall under National Program 215, Component I, Rangeland Management Systems to Improve Economic Viability and Enhance the Environment. ARS scientists and support staff at the Poisonous Plant Research Lab (PPRL) in Logan, Utah, made significant progress on all five basic objectives as outlined in the project plan: 1) identify plants that cause poisoning and describe the etiology of poisoning; 2) characterize the secondary compounds (toxins) through chemical analyses; 3) define how the toxins cause poisoning through toxicology and pharmacology studies; 4) improve diagnosis and prognosis of poisoning; and 5) develop strategies and management recommendations for livestock producers and land managers to reduce losses. Studies have been completed and published regarding the co-exposure of livestock to multiple poisonous plants. Death camas and low larkspur were the first plants to be evaluated as they often grow in the same habitats and cattle and sheep graze both as part of their diet. Initial results suggest that ingestion of multiple poisonous plants may exacerbate toxicity as one might expect, however results have also suggested that certain toxins affecting the same organ system may compete with each other thus reducing the effect of one toxin over another. Evaluation of improved perennial grass species and forage Kochia varieties to reduce and compete with invasive species is continuing. After 3 years of research on the Channeled Scablands of eastern Washington, it appears that successful establishment of perennial grass and kochia species will prevent reinvasion of annual grasses in both replicated plots and demonstration plots. The most successful grass species were Vavilov II (an improved Siberian wheat grass), Hycrest (an improved crested wheat grass), Sherman Big Blue (a native grass) and two forage Kochia varieties (Immigrant and Sahro)Evaluation of plots in the 4th year after establishment demonstrated that these species would competitively prevent reinvasion of cheat grass, medusahead rye and poisonous plants. Furthermore, after these plots were grazed by cattle, data suggested that forage quality and biomass was significantly improved. Last year two ranchers initiated larger scale plantings on their respective ranches and one rancher has implemented a five year plan to improve his entire ranch using this information and technology. Cattle and sheep losses have continued to occur on mine reclamation sites where soils contain high levels of selenium. Recent feeding trials in sheep determined that reproductive performance is significantly reduced when sheep ingest forage containing levels of selenium above the nutritional requirements and ranchers will need to remove animals from these grazing sites well in advance of the breeding season. Greenhouse studies were recently completed and data suggests that certain soil amendments may successfully reduce the level of selenium taken up by forage plants. Analytical methods developed by ARS scientists at Logan, Utah, were used to detect pyrrolizidine alkaloids (PAs) in contaminated food and feed supplies. Further progress has been made on the National Institute of Health (NIH) funded PA research including large scale isolation and purification of epimeric lycopsamine and intermedine and synthesis and purification of their amine oxides (N-oxides). Analytical methods to screen herbal products for PAs have been completed. Echimidine was isolated from Echium vulgare and purified and the N-oxide synthesized. Three separate commercial samples of butterbur root powder (Petasites spp.), an herbal remedy, were screened for PAs and one contained two dehydropyrrolizidine alkaloids (DHPAs). Locoweed poisoning was studied in numerous animal models and ARS scientists found that large differences in sensitivity, disease progression, lesions and lesion distribution exist between animal species. Recent research indicates this is largely due to swainsonine�s affinity to a species' mannosidase enzymes. Additional comparisons of mannosidase expression in tissues and correlation of that expression with lesion development continue. Dose response studies of swainsonine poisoning have been completed in goats and initial results suggest goat susceptibility is similar to that of horses and greater than sheep and cattle. Studies comparing the lesions in the brain and eyes of multiple livestock species is continuing and publications are being prepared. Additional progress and new information to aid livestock producers and land managers in making genetic-based grazing decisions to improve livestock performance and safety on grazed rangelands infested with poisonous plants is continuing. Results from genome sequencing experiments have calculated that cattle susceptibility to larkspur is 90% heritable and one-third of the heritability can be attributed to a single candidate gene. Animal genes, physiological pathways, and molecular mechanisms of action that underlie the effects of neurotoxic plants such as lupine, larkspurs, and poison hemlock have been identified. Different cattle herds are being tested for resistance or sensitivity and individual animals are being selected using the gene based criteria. The current focus is to identify male and female individuals that express the resistant gene and cross breed them to determine the heritability of this trait. This research is a collaborative project with Clay Center, Nebraska. Accomplishments 01 The effect of co-administration of death camas (Zigadenus spp.) and low larkspur (Delphinium spp.) in cattle. In most rangeland settings livestock are potentially exposed to multiple poisonous plants containing multiple toxins. ARS scientists at Logan, Utah, studied the effects of co-administration of death camas and low larkspur in several livestock species. It was determined that combinations of toxic plants and their toxins acted synergistically or competitively depending on the toxin and the organ systems they affect. The results from these studies provide an increased knowledge and understanding regarding the acute toxicity of death camas and low larkspur in sheep and cattle. This information will be useful in further developing livestock management recommendations for ranchers. 02 Comparison of the serum toxicokinetics of larkspur toxins in cattle, sheep and goats. Larkspur plants (Delphinium spp.) are acutely toxic to cattle, and as such they cause a significant number of cattle death losses every year. ARS Scientists in Logan, Utah, compared the serum toxicokinetic profiles of toxic larkspur alkaloids from D. barbeyi in cattle, goats, and sheep. Cattle and sheep are the two livestock species that are most often exposed to larkspur-infested rangelands, with cattle being more susceptible and sheep more resistant to larkspur toxicosis. Goats were also included, as goats are often used as a small ruminant model to study poisonous plants. It was determined that the increased resistance of goats and sheep to poisoning by larkspur could be due to differences in the toxicokinetics of the toxic larkspur alkaloids. This information will be used to evaluate risk when cattle graze larkspur. 03 Relative toxicity of the swainsonine-containing plants Ipomoea carnea and Astragalus lentiginosus in goats. The Brazilian plant Ipomoea carnea contains both the indolizidine alkaloid swainsonine and the nortropane alkaloids calystegines, whereas the U.S. locoweed A. lentiginosus contains only swainsonine. A dosing study was conducted by ARS scientists at Logan, Utah, in collaboration with Brazilian colleagues using goats to determine the relative contribution of swainsonine and calystegines to intoxication and resulting pathology in goats. The results of this study demonstrated that swainsonine contributed to the majority of the overt toxicity and histopathological lesions. Calystegines do not appear to be major toxins in livestock poisoning from Ipomoea carnea. This information is important to livestock producers in the U.S. and Brazil who graze animals on rangelands where Ipomoea carnea or locoweeds grow. 04 Consumption of larkspur by resistant and susceptible cattle. ARS Scientists in Logan, Utah, screened cattle to provide groups of resistant and susceptible cattle to tall larkspur poisoning. Cattle were grazed on a larkspur-infested rangeland in southeastern Idaho during the summer for 2 years. Susceptible animals initially consumed more larkspur than did resistant animals both years, provoking clinical signs of intoxication. However, results at the end of the first summer trials showed there were few differences in larkspur consumption between resistant and susceptible cattle; in the latter portion of the second grazing trial, resistant steers consumed more larkspur than did susceptible steers, but with no fatalities. This information is important to livestock producers in the U.S. who graze animals on rangelands where larkspurs are an important component of the vegetation. 05 Neurological effects of water hemlock on animals. ARS scientists at Logan, Utah, completed a toxicology study of water hemlock (Cicuta spp.) in a cultured cell line. The pharmacological mechanism of action was identified and characterized. Interestingly, the toxic effects were reversed with two drugs used in human and veterinary medicine that prevent seizures (midazolam and benzodiazepine). This information is important in diagnosing and preventing water hemlock poisoning in animals. 06 Biological mechanism of piperidine alkaloids from poisonous plants. ARS scientists in Logan, Utah, compared the toxic effects of different piperidine alkaloid toxins from poisonous plants on neurological selected receptors. Specialized cell lines (TE-671 and SHSY-5Y cells) were used to understand and characterize the mechanism of action. These cells have fetal characteristics and were used to model crooked calf syndrome. This information will increase understanding why these plants cause birth defects and animal deaths. 07 Breed specific resistant to teratogenic lupine species. ARS scientists at Logan, Utah, demonstrated that breed differences exist in how cattle respond to plant toxins that cause birth defects. Pregnant Holstein heifers appear to eliminate the toxins faster and have lower serum concentrations of the lupine teratogen anagyrine than Angus heifers when dosed one time orally with ground lupines. Under these conditions, fetal movement in Angus heifers was inhibited more and longer than Holstein heifers. This information is important to livestock producers and scientists to select individual animals or breeds that may be resistant to plant poisoning. 08 The use of biomarkers to identify cattle resistant to poisoning by plants. ARS scientists from Logan, Utah, and Clay Center, Nebraska, identified resistant Angus bulls and Angus steers to the larkspur toxins. This was accomplished using a large DNA genotyping assay to identify specific genes for resistance. Angus bulls expressing the resistant genes were selected for progeny testing. This information will benefit ranchers in regions where larkspur and other neurotoxic plants infest the rangelands and will allow producers to make genetic- based grazing decisions to select replacement heifers and bulls for resistance and reduce cattle losses. 09 A survey of North American Astragalus and Oxytropis taxa for swainsonine (the locoweed toxin). A systematic examination for swainsonine in these species provided a definitive reference in regard to species containing swainsonine and is a valuable reference for land managers. ARS researchers in Logan, Utah, conducted a systematic examination of swainsonine in multiple species. Twenty-two Astragalus species representing ninety-three taxa and four Oxytropis species representing eighteen taxa were screened for swainsonine using chemical analytical techniques. Swainsonine was detected in forty-eight Astragalus taxa representing thirteen species and five Oxytropis taxa representing four species. The list of swainsonine-containing taxa reported here will serve as an essential reference for risk assessment and avoidance of livestock poisoning. 10 Seasonal variation of the toxins in low larkspur. The toxic alkaloids in Delphinium (larkspur) species are divided into two classes based on the chemical structures, i.e., highly toxic or moderately toxic. ARS researchers in Logan, Utah, studied alkaloid concentrations of low larkspur (D. nuttallianum) and found that alkaloid concentrations differed between vegetative and reproductive tissues. The vegetative tissues had significantly lower alkaloid concentrations than reproductive tissues. Understanding how alkaloid concentrations change at different stages of plant growth and different plant parts in a given larkspur species is important to developing management strategies to reduce livestock losses. 11 Effects of selenium on reproduction in sheep. ARS scientists in Logan, Utah, fed alfalfa pellets containing selenium concentrations to mimic that found in forages on certain soil sites. High levels of selenium in the diet resulted in decreased reproductive rates in sheep by up to forty percent. Lower conception rates and early embryonic loss during the first thirty days of gestation resulted. The information obtained from this study suggests that producers grazing in regions where forages are high in selenium should manage their flocks to avoid grazing these forages prior to or during the breeding season. 12 Monofluoroacetate in Palicourea and Arrabidaea species in South America. Several plant species in Brazil cause sudden death in cattle due to the toxic principle monofluoroacetate. ARS researchers in Logan, Utah, in collaboration with scientists in Brazil, reported for the first time that two species of Palicourea cause sudden death in cattle and contain monofluoroacetate. In addition, they corrected literature that a species of Arrabidaea previously reported to cause sudden death does not contain monofluoroacetate. A knowledge of taxa that contain monofluoroacetate will serve as reference for risk assessment and diagnostic purposes for livestock producers, fellow scientists and veterinarians. 13 A massive death loss in cattle fed weed infested alfalfa hay. ARS scientists in Logan, Utah, investigated a case of cattle poisoning in Colorado where 170 cows of a herd of over 500 died after feeding weed infested alfalfa hay. The cause of death was determined to be acute liver failure incident to unknown toxicosis. Subsequent liver biopsies of some of the survivors indicated significant liver damage had occurred in all cows ingesting the hay. ARS scientists used a bioassay guided chemical extraction process to identify the toxin and the poisonous plant in the hay. This research will provide important information to veterinarians for diagnosis and to livestock and hay producers to prevent poisonings in the future. 14 Improvement of rangelands in eastern Washington State infested with medusahead, cheatgrass and lupine. Vegetation on the Channeled Scablands of eastern Washington has been altered to a community dominated by medusahead, cheatgrass, lupine and other invasive weeds. Lupine is poisonous and causes crooked calf syndrome in cattle, and this problem is exacerbated by the invasion of annual grasses. ARS scientists in Logan, Utah, demonstrated that improved perennial grasses will establish in this region and will compete with and replace medusahead and cheatgrass, thus improve range conditions. These cool- season grasses will improve forage quality, increase forage production, reduce consumption of lupine and enhance the economic viability of the rural livestock producers in this region. 15 Protein supplementation enhances forage utilization by cattle on annual grass-dominated rangelands. The annual invasive grass, medusahead, has replaced much of the native vegetation on the Channeled Scablands of eastern Washington resulting in poor range conditions, reduced forage availability for cattle, increased consumption of poisonous weeds and a decline in productivity. ARS scientists in Logan, Utah, supplemented cattle with protein during the summer grazing months. The protein supplementation increased consumption of medusahead by cattle. Therefore, protein supplements can be used by livestock producers to increase utilization of medusahead and potentially reduce the consumption of poisonous plants. 16 Detection of pyrrolizidine alkaloids in herbal products. ARS scientists in Logan, Utah, completed the development of analytical methods to screen herbal products for the liver toxins, dehydropyrrolizidine alkaloids (DHPA�s). Large scale isolation of three major DHPA�s and their amine oxides (N-oxides) was completed. DHPA�s were detected in one of three commercially available herbal products (butterbur root powder (petasites spp.)). This is in addition to the detection of the multiple DHPA�s in many comfrey products commonly found in herbal remedies. 17 Poisoning in cattle from fiddleneck (Amsinckia spp.) in Arizona. Fiddleneck (Amsinckia spp.) contain the liver toxins, pyrrolizidine alkaloids (PA�s). A case of poisoning in cattle where over fifty cows died was reported to ARS scientists in Logan, Utah. They investigated the poisoning, collected and identified the suspect plants as Amsinckia, and performed chemical analyses on the plant material and tissues from the dead cows. Multiple PA�s were detected in the plant material and this was compared with fiddleneck collected in Washington State. This information was provided to the livestock producers and veterinarians with recommendations to prevent further losses. 18 Toxicity of pyrrolizidine alkaloids (PAs). Pyrrolizidine alkaloids (PAs) often contaminate feed, food, and medicinal or herbal products poisoning livestock, wildlife and humans. ARS scientists in Logan, Utah, previously demonstrated that riddelliine induced tumors at increased incidences regardless of dose or exposure duration and the type of tumor differed by exposure. ARS scientists also identified PA metabolites or adducts from livers of animals that were exposed to PAs providing information useful in identifying clinically poisoned animals. This research provides important information to animal and human health care providers.

Impacts
(N/A)

Publications

Adrien, M.L., Gardner, D.R., Pfister, J.A., Marcolongo-Pereira, C., Riet- Correa, F., Schild, A.L. 2014. Conditioning food aversions to Ipomoea carnea var. Fistulosa in sheep. Ci�ncia Rural. 44(2):362-367.
Beck, J.J., Porter, N., Cook, D., Griffith, C., Gee, W.S., Rands, A.D., Truong, T.V., Smith, L., San Roman, I. 2015. In-field volatile collection and analysis method utilizing a portable GC-MS: in situ headspace analysis of intact and damaged yellow starthistle flower heads. Phytochemical Analysis. 26:395-403.
Becker, M., Carneiro, F.M., Oliveira, L.P., Silva, M.I., Riet-Correa, F., Lee, S.T., Pescador, C.A., Nakazato, L., Colodel, E.M. 2016. Induction and transfer of resistance to poisoning by Amorimia pubiflora in sheep with non-toxic doses of the plant and ruminal content. Ciencia Rural. 46(4):674- 680.
Brito, L.B., Albuquerque, R.F., Rocha, B.P., Albuquerque, S.S., Lee, S.T., Medeiros, R.M., Riet-Correa, F., Mendonca, F.D. 2016. Spontaneous and experimental poisoning of cattle by Palicourea aeneofusca in the region of Pernambuco and introduction of conditioned food aversion. Ciencia Rural. 46(1):138-143.
Brown, A.W., Stegelmeier, B.L., Colegate, S.M., Gardner, D.R., Panter, K.E. , Knoppel, E.L., Hall, J.O. 2016. The comparative toxicity of a reduced, crude comfrey (Symphytum officinale) alkaloid extract and the pure, comfrey-derived pyrrolizidine alkaloids, lycopsamine and intermedine in chicks (Gallus gallus domesticus). Journal of Applied Toxicology. 36:716- 725.
Brown, A.W., Stegelmeier, B.L., Colegate, S.M., Panter, K.E., Knoppel, E.L. , Hall, J.O. 2015. Heterozygous P53 knockout mouse model for dehydropyrrolizidine alkaloid-induced carcinogenesis. Journal of Applied Toxicology. 35(12):1557-1563.
Carvalho, F.K., Cook, D., Lee, S.T., Taylor, C.M., Oliveira, J.B., Riet- Correa, F. 2016. Determination of toxicity in rabbits and corresponding detection of monofluoroacetate in four Palicourea (Rubiaceae) species from the Amazonas state, Brazil. Toxicon. 109:42-44.
Colegate, S.M., Boppre, M., Monzon, J., Betz, J.M., Panter, K.E. 2015. Pro- toxic dehydropyrrolizidine alkaloids in the traditional Andean herbal medicine "asmachilca". Journal of Ethnopharmacology. 172:179-194.
Colegate, S.M., Gardner, D.R., Betz, J.M., Fischer, O.W., Liede-Schumann, S., Boppre, M. 2016. Pro-toxic 1,2-Dehydropyrrolizidine alkaloid esters, including unprecedented 10-membered macrocyclic diesters, in the medicinally-used Alafia cf. caudata and Amphineurion marginatum (Apocynaceae: Apocynoideae: Nerieae and Apocyneae). Phytochemical Analysis. 27(5):257-276.
Collett, M.G., Stegelmeier, B.L., Tapper, B.A. 2014. Could nitrile derivatives of turnip (Brassica rapa) glucosinolates be hepato- or cholangiotoxic in cattle? Journal of Agricultural and Food Chemistry. 62(30):7370-7375.
Cook, D., Gardner, D.R., Lee, S.T., Pfister, J.A., Stonecipher, C.A., Welsh, S.L. 2016. A swainsonine survey of North American Astragalus and Oxytropis taxa implicated as locoweeds. Toxicon. 118:104-111.
Cook, D., Gardner, D.R., Pfister, J.A., Grum, D.S. 2014. Biosynthesis of natural products in plants by fungal endophytes with an emphasis on swainsonine. Recent Advances in Phytochemistry. 44:23-42.
Cook, D., Slominski, A., Gardner, D.R., Pfister, J.A., Irwin, R.E. 2016. Seasonal variation in the secondary chemistry of foliar and reproductive tissue of Delphinium nuttallianum. Biochemical Systematics and Ecology. 65:93-99.
Davis, T.Z., Stegelmeier, B.L., Lee, S.T., Green, B.T., Evans, T.J., Grum, D.S., Buck, S., Meyerholtz, K.A. 2016. White snakeroot poisoning in goats: Variations in toxicity with different plant chemotypes. Research in Veterinary Science. 106:29-36.
De Lima, F.G., Lee, S.T., Pfister, J.A., Miyagi, E.S., Costa, G.L., De Silva, R.D., Fioravanti, M.S. 2015. The effect of ensiling and haymaking on the concentrations of steroidal saponin in two Brachiaria grass species. Ciencia Rural. 45(5):858-863.
Duarte, A.L., Medeiros, R.M., Carvalho, F.K., Lee, S.T., Cook, D., Pfister, J.A., Costa, V.M., Riet-Correa, F. 2014. Induction and transfer of resistance to poisoning by Amorimia (Mascagnia) septentrionalis in goats. Journal of Applied Toxicology. 34(2):220-223.
Galbraith, M.L., Vorachek, W.R., Estill, C.T., Whanger, P.D., Bobe, G., Davis, T.Z., Hall, J.A. 2016. Rumen microorganisms decrease bioavailability of inorganic selenium supplements. Biological Trace Element Research. 171(2):338-343.
Gardner, D.R., Cook, D. 2016. Analysis of swainsonine and swainsonine N- oxide as trimethylsilyl derivatives by Liquid Chromatography-Mass Spectrometry and their relative occurrence in plants toxic to livestock. Journal of Agricultural and Food Chemistry. 64(31):6156-6162.
Golo, P.S., Gardner, D.R., Grilley, M.M., Takemoto, J.Y., Krasnoff, S., Pires, M.S., Fernandes, E.K., Bittencourt, V.R., Roberts, D.W. 2014. Production of destruxins from metarhizium spp. fungi in artificial medium and in endophytically colonized cowpea plants. PLoS One. 9(8):e104946.
Gotardo, A.T., Pfister, J.A., Raspantini, P.C., Gorniak, S.L. 2016. Maternal ingestion of Ipomoea carnea: Effects on goat-kid bonding and behavior. Toxins. 8(3):74.
Green, B.T., Brown, D.R. 2016. Interactions between bacteria and the gut mucosa: Do enteric neurotransmitters acting on the mucosal epithelium influence intestinal colonization or infection? Advances in Experimental Medicine and Biology. 874:121-141.
Green, B.T., Goulart, C., Welch, K.D., Pfister, J.A., Mccollum, I.J., Gardner, D.R. 2015. The non-competitive blockade of GABAA receptors by an aqueous extract of water hemlock (Cicuta douglassi) tubers. Toxicon. 108:11-14.
Green, B.T., Lee, S.T., Welch, K.D., Cook, D., Kem, W.R. 2016. Activation and desensitization of peripheral muscle and neuronal nicotinic acetylcholine receptors by selected, naturally-occurring pyridine alkaloids. Toxins. doi: 10.1016/j.toxicon.2015.09.015.
Green, B.T., Lee, S.T., Welch, K.D., Gardner, D.R., Stegelmeier, B.L., Davis, T.Z. 2015. The serum concentrations of lupine alkaloids in orally- dosed Holstein cattle. Research in Veterinary Science. 100:239-244.
Green, B.T., Panter, K.E., Lee, S.T., Welch, K.D., Pfister, J.A., Gardner, D.R., Stegelmeier, B.L., Davis, T.Z. 2015. Differences between Angus and Holstein cattle in the Lupinus leucophyllus induced inhibition of fetal activity. Toxicon. 106:1-6.
Lima, E.F., Medeiros, R.M., Cook, D., Lee, S.T., Kaehler, M., Santos- Barbosa, J.M., Riet-Correa, F. 2016. Studies in regard to the classification and putative toxicity of Fridericia japurensis (Arrabidaea japurensis) in Brazil. Toxicon. 115:22-27.
Lopes, J.R., Riet-Correa, F., Cook, D., Pfister, J.A., Medeiros, R.M. 2014. Elimination of the tremorgenic toxin of Ipomoea asarifolia by milk. Pesquisa Veterinaria Brasileira. 34(11):1085-1088.
Maia, L.A., Macedo Pessoa, C.R., Rodrigues, A.F., Colegate, S.M., Dantas, A.M., Medeiros, R.T., Riet-Correa, F. 2014. Duration of an induced resistance of sheep to acute poisoning by Crotalaria retusa seeds. Ci�ncia Rural. 44(6):1054-1059.
Mott, I.W., Cook, D., Lee, S.T., Stonecipher, C.A., Panter, K.E. 2016. Phylogenetic examination of two chemotypes of Lupinus leucophyllus. Biochemical Systematics and Ecology. 65:57-65.
Nascimento, E.M., Medeiros, R.M., Lee, S.T., Riet-Correa, F. 2014. Poisoning by Poiretia punctata in cattle and sheep. Pesquisa Veterinaria Brasileira. 34(10):963-966.
Oliveira, C.A., Riet-Correa, G., Lima, E., Leite, D.M., Pfister, J.A., Cook, D., Riet-Correa, F. 2015. Feeding preferences of experienced and na�ve goats and sheep for the toxic plant Ipomoea carnea subsp. fistulosa. Ciencia Rural. 45(9):1634-1640.
Pfister, J.A., Panter, K.E., Lee, S.T., Moterram, E. 2014. Crude protein supplementation to reduce lupine consumption by pregnant cattle in the scablands of eastern Washington. International Journal of Poisonous Plant Research. 3(1):26-32.
Rincon, D.F., Diaz, G.J., Gardner, D.R. 2016. Detection of Ptaquilosides in different phenologic stages of Bracken fern (Pteridium aquilinum) and analysis of milk samples in farms with hematuria in Tolima, Colombia. Revista de Medicina Veterinaria. 11(1):72-77.
Rocha, B.P., Reis, M.O., Driemeier, D., Cook, D., Camargo, L.M., Riet- Correa, F., Evencio-Neto, J., Mendonca, F.S. 2016. Liver biopsy as diagnostic method for poison�ing by swainsonine-containing plants. Pesquisa Veterinaria Brasileira. 36(5):373-377.
Silva Negreiros Neto, T.D., Gardner, D.R., Hallwass, F., Jessica Matias Leite, A., Guimaraes De Ameida, C., Nunes Silva, L., Araujo Roque, A.D., Gobbi De Bietncourt, F., Guimaraes Barbosa, E., Tasca, T., Jose Macedo, A., Veira De Almeida, M., Brandt Giordani, R. 2016. Activity of pyrrolizidine alkaloids against biofilm formation and Trichomonas vaginalis. Biomedicine and Pharmacotherapy. 83:323-329.
Stewart, W.C., Whitney, R.R., Scholljegerdes, E.J., Naumann, H.D., Cherry, N.M., Muir, J.P., Lambert, B.T., Walker, J.W., Adams, R.P., Welch, K.D., Gardner, D.R., Estell, R.E. 2015. Effects of juniperus species and stage of maturity on nutritional, in vitro digestibility, and plant secondary compound characteristics. Journal of Animal Science. 93(8):4034-4047.
Stonecipher, C.A., Panter, K.E., Villalba, J.J. 2016. Effect of protein supplementation on forage utilization by cattle in annual grass-dominated rangelands in the Channel Scablands of Eastern Washington. Journal of Animal Science. 94(6):2572-2582.
Welch, K.D., Cook, D., Green, B.T., Gardner, D.R., Pfister, J.A., Mcdaneld, T.G., Panter, K.E. 2015. Adverse effects of larkspur (Delphinium spp.) on cattle. Agriculture. 5:456-474.
Welch, K.D., Gardner, D.R., Green, B.T., Stonecipher, C.A., Cook, D., Pfister, J.A. 2016. Comparison of the serum toxicokinetics of larkspur toxins in cattle, sheep and goats. Toxicon. 119:270-273.
Welch, K.D., Green, B.T., Gardner, D.R., Cook, D., Pfister, J.A. 2015. The effect of administering multiple doses of tall larkspur (Delphinium barbeyi) to cattle. Journal of Animal Science. 93(8):4181-4188.
Welch, K.D., Green, B.T., Gardner, D.R., Stonecipher, C.A., Pfister, J.A., Cook, D. 2016. The effect of co-administration of death camas (Zigadenus spp.) and low larkspur (Delphinium spp.) in cattle. Toxins. doi: 10.3390/ toxins8010021.
Welch, K.D., Lee, S.T., Panter, K.E., Gardner, D.R. 2014. A study on embryonic death in goats due to Nicotiana glauca ingestion. Toxicon. 90:64- 69.
Welch, K.D., Parsons, C., Gardner, D.R., Deboodt, T., Shreder, P., Cook, D. , Pfister, J.A., Panter, K.E. 2015. Evaluation of the seasonal and annual abortifacient risk of western juniper trees on Oregon rangelands: Abortion risk of western juniper trees. Rangelands. 37(4):139-143.
Oliveira, C.A., Riet-Correa, G., Tavares, C., Souza, E., Cerqueira, V.D., Pfister, J.A., Cook, D., Riet-Correa, F. 2014. Conditioned food aversion to control poisoning by Ipomoea carnea subsp. fistulosa in goats. Ciencia Rural. 44(7):1240-1245.
Panter, K.E., Welch, K.D., Gardner, D.R., Green, B.T. 2013. Poisonous plants: Effects on embryo and fetal development. Birth Defects Research Part C: Embryo Today: Reviews. 99:223-234.
Pfister, J.A., Green, B.T., Welch, K.D., Provenza, F.D., Cook, D. 2016. Impacts of toxic plants on the welfare of grazing livestock. In:Villalba, J.J., editor. Animal Welfare in Extensive Production Systems. Sheffield, U. K.:5m Publishing. p. 78-102.

Progress 10/01/14 to 09/30/15

Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop and implement novel management protocols for establishing improved forage species on sites infested with known poisonous plants to reduce the risk of livestock mortality and morbidity, improve livestock performance, and improve rangeland resiliency and diversity. Specifically, develop science-based guidelines for grazing livestock on rangelands infested with Lupinus, Senecio, Delphinium and swainsonine and selenium-containing plants. Objective 2: Reduce the risks of livestock losses due to variations in quantitative and qualitative differences in toxin accumulation over time and plant species by quantifying the influence of endophytes, climate changes, and genotype on plant toxin accumulation (particularly swainsonine-containing plants and Delphinium and Lupinus species). Objective 3: Enhance feed and food safety by improving risk assessment and diagnosis of plant-induced poisoning to livestock by improving analytical methods for analyzing plant and animal tissues for toxins; measuring toxicokinetics, assessing carcinogenic and genotoxic potential, and identifying toxin metabolites and biomarkers of toxicoses. Objective 4: Develop improved procedures with guidelines for diagnostic and prognostic evaluation to reduce negative impacts of poisonous plants on livestock reproduction and embryo/fetal growth by improving early identification of poisoned animals, predicting poisoning outcomes, and management and treatment options through improved understanding of clinical, morphological and molecular alterations of plant-induced toxicosis. Objective 5: Develop guidelines to aid producers and land managers in making genetic-based herd management decisions to improve livestock performance and safety on grazed rangelands infested with poisonous plants through the use of identified animal genes, physiological pathways, and molecular mechanisms of action that underlie Conium, Cicuta, Delphinium, Lupinus, and Nicotiana, and other neurotoxic plant effects. Approach (from AD-416): Livestock poisoning by plants results in over $503,000,000 lost to the livestock industry annually in the 17 western United States from death losses and abortions alone (Holechek, 2002). Plant poisonings extend worldwide to include 333 million poisonous plant-infested hectares in China and 60 million hectares in the central western region of Brazil, to name a few. There are over 6,000 species of pyrrolizidine alkaloid (PA)- containing plants, and over 350 individual PAs causing diseases in animals and humans have been identified. Economic losses are much larger as significant amounts of nutritious forage are wasted and management costs are increased due to the threat of toxic plant-related livestock losses. The Poisonous Plant Research Laboratory (PPRL) has provided worldwide leadership in poisonous plant research to the livestock industry and consumers including numerous solutions to toxic plant problems using an integrated, interdisciplinary approach (see Figure below). The research team investigates plant poisonings in a systematic matter by identifying the plant, describing the effects in animals, determining the toxin(s) and evaluating the mechanisms of action. The ultimate goal is to develop research-based solutions to reduce livestock losses from toxic plants. There are five coordinated objectives in this project plan providing guidelines for potential genetic-based management. This research will reduce livestock losses from plants and enhance the economic well-being of rural communities, improve rangeland health by combating invasive plant species, and help to provide safe animal products free from potential plant toxins for consumers. Poisonous plants continue to cause significant economic loss to the livestock industry and negatively impact the nearby rural communities and regions. Losses include direct death losses, birth defects in offspring, reduced reproductive performance, lost production capacity, increased management costs, lost forage value and lost opportunities. Toxins from poisonous plants may contaminate food supplies resulting in health risks to the human population. ARS researchers at the Poisonous Plant Research Lab (PPRL) in Logan, Utah, continue to perform interdisciplinary research through a concerted team approach to provide new information and tools for livestock producers and land managers to mitigate poisonous plant losses. Five objectives have been defined in the project plan beginning with plant identification, progressing through chemical analysis and characterization of secondary compounds, to toxicology and pharmacology of toxins, description of the etiology of plant poisoning, diagnosis and prognosis of poisoning and finally to develop strategies and management approaches for livestock producers to reduce losses. Evaluation of improved rangeland grass species and forage Kochia varieties seeded and established into research, demonstration and ranch scale plots on the channeled scablands of east central Washington State continues. The most successful species include Vavilov II (an improved Siberian wheat grass), Hycrest (an improved crested wheat grass), Sherman Big Blue (a native grass) and two forage Kochia varieties (Immigrant and Sahro). After 4 years these species show the most promise to establish and persist, providing the potential to improve forage quality while successfully competing with cheat grass, medusahead rye and poisonous plants. Plots were recently grazed by cattle and data suggest these selected species and varieties have great potential to improve the rangelands in this harsh environment. Last year two ranchers initiated larger scale plantings on their respective ranches using the technology provided by ARS researchers. One rancher seeded a quarter section of rangeland and has put into place a 5 year plan to reseed his entire ranch using this information and technology. Locoweed seedlings were treated with different fungicides to evaluate the effects on swainsonine production by the endophyte and to determine if swainsonine levels could be reduced to lower the risk of locoweed poisoning from these otherwise nutritious plants. Data analysis was completed on the effects of elevated CO2 levels on locoweed growth and swainsonine concentrations. Additional progress has been made on elucidating the biosynthetic pathway of swainsonine. This research is in collaboration with scientists at the University of Kentucky and New Mexico State University. Genome sequencing of two swainsonine producing fungal endophytes continues and ARS scientists in collaboration with researchers at Indiana University have identified several Convolvulaceae taxa species that contain swainsonine. Recent cattle and sheep losses have been reported after grazing forages growing on seleniferous soils. Pen studies were conducted to determine the effects of high selenium in the diet on reproductive performance. Feeding trials to compare the effects of high selenium in the diet on feed preferences in sheep, cattle and elk and to determine the rate of absorption and elimination of different chemical forms of selenium were completed and published. Tremetone was reported to be the toxin in rayless goldenrod in the Southwest and white snakeroot in the Midwest since the 1900's. Chemical studies have isolated and characterized numerous benzofuran ketones (BFK) including tremetone from these plants. Subsequent animal studies suggest that tremetone is not the primary toxin. It is likely that other benzofuran ketones act together with tremetone to cause toxicity. ARS scientists at PPRL are using different animal models and chemical extraction techniques to determine the active principle in these plants. Numerous plant populations from different geographical locations with variable BFK concentrations are being evaluated to determine why poisoning in animals occurs in some locations while the same or similar plant species in another location may not cause poisoning. Larkspur specific primers have been developed and validated for diagnostic purposes to assist veterinarians in identifying the cause of plant associated death losses in cattle. Initial research using PCR-based methods to detect larkspur in rumen cultures and in the rumens of live cannulated cows shows promise. Analytical methods developed by ARS scientists at Logan, Utah, were used to detect pyrrolizidine alkaloids (PAs) in contaminated food and feed supplies. We determined that a traditional Peruvian herbal tea that is available internationally contains toxic levels of the dehydropyrrolizidine alkaloids (DHPAs) and is a risk to cause liver disease in people. An aquatic plant (Senegal tea) that produces toxic DHPAs suggests a potential risk of water contamination for humans and animals. We continue to monitor subsistence grain supplies from the Tigray region of Ethiopia, honey and pollen samples from the islands of Hawaii and contaminated chicken feed from Colombia. The analytical methods for detection of DHPA metabolites in liver and blood from diagnostic samples were significantly improved and are being used to investigate the relative toxicity of individual alkaloids in cell culture and small animal models. Further progress has been made on the National Institute of Health (NIH) funded DHPA research including large scale isolation and purification of epimeric lycopsamine and intermedine and synthesis and purification of their N-oxides. Analytical methods to screen herbal products for PAs have been completed. Echimidine was isolated from Echium vulgare and purified and the N-oxide synthesized. Three separate commercial samples of butterbur root powder (Petasites spp.), an herbal remedy, were screened for PAs and one contained two DHPAs. Collaborative research with a scientist funded through the Department of Defense was completed and research results published, including histologic evaluation of DHPA carcinogenicity studies in P53 heterozygous mice. Both acute exposures and chronic low dose exposure to riddelliine caused increased incidence of PA-induced neoplasms. The neoplasm type differed by exposure, i.e. chronic low dose exposures produced hepatic vascular neoplasms while acute high dose exposures produced neoplasms outside the hepatic vasculature. This suggests that any PA exposure can increase the incidence of PA-related carcinogenesis. Research using cell cultures and chick models to better document PA toxicity and correlate that toxicity to their carcinogenicity continues, and specific PAs have been ranked according to their toxicity to cells. Four PAs�lasiocarpine, seneciphylline, senecionine and heliotrine�are more toxic than riddelliine. Twenty purified DHPAs from different plant sources have been evaluated and ranked relative to their toxicity using cell culture models and highly sensitive chick models. Studies to evaluate the carcinogenic potential of most toxic alkaloids in a mouse P-53 model have begun. Analyses to detect DHPA metabolites or adducts in tissues have been refined. We found that riddelliine consistently produces nearly 10 times more adducts than the other alkaloids in our chick model. How these adducts are involved in riddelliine-induced carcinogenesis remains to be determined. Locoweed poisoning was studied in numerous animal models and ARS scientists found that huge differences in sensitivity, disease progression, lesions and lesion distribution exist between animal species. Recent research indicates this is largely due to swainsonine's affinity to a species' mannosidase enzymes. Additional comparisons of mannosidase expression in tissues and correlation of that expression with lesion development continue. Dose response studies of swainsonine poisoning have been completed in goats and initial results suggest goat susceptibility is similar to that of cows and sheep. Comparative studies of species specific locoweed-induced ophthalmic lesions have been completed and are being prepared for publication. We found that locoweed related changes in vision are due to neurological lesions in the brain and not lesions in the eye. Additional progress and new information to aid livestock producers and land managers in making genetic-based grazing decisions to improve livestock performance and safety on grazed rangelands infested with poisonous plants has been completed. Results from genome sequencing experiments have calculated that cattle susceptibility to larkspur is 90% heritable and one-third of the heritability can be attributed to a single candidate gene. Animal genes, physiological pathways, and molecular mechanisms of action that underlie the effects of neurotoxic plants such as lupine, larkspurs, and poison hemlock have been identified. Five cattle breeds have been tested and resistant and sensitive animals identified in all 5 breeds. The current focus is to identify individuals in the Angus breed from multiple ranches, test these animals for sensitivity to the neurotoxins and associate responses with the gene marker(s). Studies were completed and published regarding the co-exposure of cattle to death camas and low larkspur. Evaluation of the chemistry of juniper bark and needles from young and mature trees determined that there are no differences in the concentration of the abortifacient compounds between young and mature western juniper trees. Samples of bark and needles have been collected bi-monthly for two years to evaluate seasonal variations in the abortifacient compounds in western juniper trees. Recent studies determined that consumption of western juniper bark does not affect the estrous cycle in beef cattle. Accomplishments 01 A biomarker to identify cattle resistant to poisoning by neurotoxic plants. ARS scientists from Logan, Utah, and Clay Center, Nebraska, used a large DNA assay to identify genes for resistance to larkspur poisoning from five breeds of cattle. The ARS scientists identified a gene region that is associated with larkspur resistance. The research team then tested DNA from 32 Angus cattle to identify further genetic variation associated with larkspur resistance. Results from these experiments suggest that cattle resistance to larkspur is highly heritable and can be attributed to one or two gene regions. These experiments will allow us to work towards development of commercially available DNA markers for larkspur tolerance in cattle. This information will benefit ranchers in regions where larkspur and other neurotoxic plants infest the rangelands and will allow producers to make genetic-based grazing decisions to select replacement heifers and bulls for resistance and reduce cattle losses. 02 Rangeland improvements on the channeled scablands. ARS scientists at Logan, Utah, seeded research plots, demonstration plots and ranch scale plots on the channeled scablands of east central Washington to determine if improved perennial grasses and selected forbs could be established on this harsh landscape and if these improvements would out- compete annual grasses and ultimately reduce the utilization of lupine to mitigate the impact of lupine-induced "crooked calf syndrome" in this region. Over the last 4 years, ARS scientists demonstrated that improved perennial grasses and forage Kochia could be established and they have persisted in the research plots and demonstration plots. These improved species have also prevented the reinvasion of annual grasses in these plots. The improved grasses include Hycrest II, Vavilov II, Bozoisky II, and Sherman Big bluegrass (a native) and immigrant forage Kochia. This technology has now been expanded to ranch scale plots and two ranchers have put into place long range plans to improve their entire ranches using this information. This research has the potential to transform the severely degraded rangelands in east central Washington to a sustainable and productive ecosystem for livestock and wildlife. 03 Relative risk of Ipomoea carnea to grazing livestock on an annual basis. Ipomoea carnea is known to cause a neurologic disease in grazing livestock in Brazil and other parts of the world. To better understand the relative toxicity and nutritional content of I. carnea, ARS researchers in Logan, Utah, and their Brazilian colleagues investigated the toxins swainsonine and calystegine and crude protein amounts in leaves of I. carnea on a monthly basis for a year in northern and northeastern Brazil. Swainsonine concentrations were detected at amounts that could potentially poison an animal throughout the year although there was some variation between months. Swainsonine concentrations were generally the highest during the rainy season or the months immediately following the rainy season at one location. Total calystegine amounts were similar to those reported previously while crude protein amounts are similar to those found in other Ipomoea species and are such that they may explain why I. carnea becomes desirable to grazing livestock as forage becomes limited during the dry season. Understanding swainsonine, calystegine, and protein concentrations in Ipomoea carnea on an annual basis may help make better management decisions to mitigate livestock losses. 04 Determination of the relative toxicity of different rayless goldenrod (Isocoma) species. Rayless goldenrod is toxic to livestock; similar species are also suspected to be toxic but their toxicity has never been scientifically demonstrated. ARS researchers in Logan, Utah, tested the relative toxicity of four Isocoma species in a recently developed goat model and determined that Isocoma pluriflora and Isocoma tenuisecta were toxic. Isocoma acradenia and Isocoma rusbyii were not toxic even though they contained significantly more of the benzofuran ketone compounds than Isocoma pluriflora. These results demonstrate that there is an additional compound(s) that contributes to, or is responsible for, the toxicity of the Isocoma spp. 05 Pyrrolizidine alkaloid (PA) carcinogenesis. Pyrrolizidine alkaloids (PAs) often contaminate feed, food, and medicinal or herbal products and can poison livestock, wildlife and humans. ARS scientists at Logan Utah, used cell culture and a sensitive chick model to determine the toxicity of four PAs. Research was published showing that riddelliine- induced tumors occurred most often in high-dose, short exposures and chronic, low-dose exposures. The type of tumor differed by exposure. This work indicates that any PA exposure may increase the incidence of PA-related liver cancer. 06 Evaluation of dehydropyrrolizidine alkaloids (DHPAs) in an herbal tea. ARS scientists at Logan, Utah, chemically evaluated "asmachilca", a Peruvian traditional herbal tea mixture. This herbal tea is commercially available throughout the world. Two previously undescribed major alkaloids in this herbal preparation were isolated and their structures identified. This research has significant health implications for people using herbal products. 07 Late term abortions in cattle ingesting juniper. Pinus and Juniperus spp. have been shown to cause late term abortions in cattle. ARS scientists at Logan, Utah, recently characterized the abortifacient effects of western juniper trees in cattle, demonstrating that bark and needles will cause late term abortions. Additionally, the effects of western juniper consumption on the estrous cycle in beef cattle were studied, with no adverse impact. ARS scientists also evaluated the variation in abortifacient compounds in western juniper trees across the state of Oregon as well as seasonal differences over multiple years. These results indicate that western juniper trees throughout their geographical distribution are a risk to cause late term abortions in cattle, however, there does not seem to be any adverse effect on the estrous cycle. This information is important to cattle producers who graze their cattle on rangelands where western juniper trees grow. 08 Transfer of rayless goldenrod (RGR) toxins into milk. Rayless goldenrod (Isocoma pluriflora) poisons livestock in the southwestern United States. The toxin has historically been thought to be tremetone. ARS scientists at Logan, Utah, have shown that tremetone is only one of several toxins in rayless goldenrod. These toxins are transferred through the milk of lactating goats and poison the nursing baby goats. This research is significant to cattle, horse, and goat breeders in the southwest where RGR grows and has implications for animal and human health. 09 Certain lupines reduce fetal movement in pregnant cattle. ARS scientists at Logan, Utah, determined that lupines contain toxins that will reduce fetal movement in pregnant heifers and there appears to be a breed difference in sensitivity. Pregnant Angus and Holstein heifers were fed the same amount of dried ground lupine. There were significant differences between the two breeds when fetal movement was monitored with ultrasound and there were differences in toxin levels in the blood that paralleled the lack of fetal activity. This information is helpful to cow calf producers, suggesting that there is the potential to make breed-based grazing decisions to select animals that are more resistant to lupines for grazing on pastures infested with toxic lupine species. 10 Detection of toxic plants in poisoned animals. Toxic plants are a significant cause of livestock losses worldwide. Correctly determining the causative agent responsible for the death of an animal whether by disease, poisonous plant, or other means is critical in developing strategies to prevent future losses. ARS researchers at Logan, Utah, are developing an alternative diagnostic tool to detect the genetic material from a specific plant within a complex matrix such as rumen (stomach) contents. A pair of primers specific to larkspur was developed; using these primers, a Polymerase Chain Reaction (PCR) product was detected in samples from in vivo, in vitro, and in vivo/in vitro coupled digestions of larkspur. Lastly, larkspur was detected in a matrix of ruminal material where the amount of larkspur was far less than what one would expect to find in the rumen contents of a poisoned animal. The PCR-based technique holds promise to diagnose larkspur and perhaps other toxic plant-caused losses. 11 Presence of toxic dehydropyrrolizidine alkaloids (DHPAs) in Senegal tea and comfrey. ARS scientists at Logan, Utah, determined that the aquatic plant Senegal tea produces potentially toxic DHPAs. This information on Senegal tea suggests there is a potential environmental threat and that it could be a health risk to humans and livestock from contaminated water sources. Four additional DHPAs and their N-oxides were identified in comfrey. This research provides valuable information and resources for other researchers and has significant implications for the division of dietary supplements at NIH and the herbal industry. 12 Consumption of larkspur pellets by cattle that are resistant or sensitive to toxicity. Consumption of larkspur alkaloids causes muscle weakness and collapse in cattle and may result in death. ARS scientists at Logan, Utah, conducted a study to quantify consumption of larkspur alkaloids and relate plant intake and clinical effects to blood alkaloid levels and beef cattle genetics. A 25% larkspur pellet was offered to all the cattle and while they readily ate the pellets the first day, they refused to eat any more pellets on subsequent days. The genetically sensitive cattle showed clinical signs while the genetically resistant cattle did not. Blood levels of the major larkspur alkaloid, methyllycaconitine (MLA), were measured and in the genetically susceptible animals was approximately 250 ng/mL, lower than the previously determined toxic threshold. This is the first in a series of studies using larkspur resistant and sensitive cattle that will provide important information for livestock producers and veterinarians regarding larkspur toxicosis and provide further information for genetic-based grazing decisions. 13 Evaluation of the N-oxide form of the locoweed toxin swainsonine. The N-oxide form of the locoweed toxin swainsonine has been reported to be present in locoweeds by previous qualitative analyses. ARS scientists in Logan, Utah, developed a new method for quantitative analysis of swainsonine N-oxide. This method uses a simple one step extraction process followed by a derivatization step and then analysis by liquid chromatography-mass spectrometry. This allows rapid screening of plants containing the locoweed toxin swainsonine in plant species found worldwide. This research is important for other scientists quantifying the toxins in plants containing N-oxides of swainsonine and will improve the screening of plants for poisoning potential. 14 Effect of larkspur ingestion in cattle over multiple days. Larkspurs are a major cause of cattle losses on western ranges in the U.S., especially on foothill and mountain rangelands. To simulate natural grazing conditions on the range, ARS scientists at Logan, Utah, performed multiple dose experiments over a period of days in order to determine the effect of repeated doses of larkspur in a controlled pen setting. The no adverse effect level was determined, which provides an approximation of how much plant can be safely consumed without adverse effects. Additionally, several dosing scenarios were modeled using computer software. These data provide a more detailed understanding of the amount of larkspur cattle can safely consume without becoming severely poisoned. This will be helpful for livestock owners to effectively manage their cattle in larkspur infested rangelands. 15 Relative toxicity of different larkspur species. Larkspurs (Delphinium spp.) are poisonous plants on rangelands throughout the western United States and Canada. ARS scientists at Logan, Utah, determined the alkaloid (toxin) composition of spiked larkspur and compared its toxicity in cattle to two tall larkspur species. The results indicate that predictions of larkspur toxicity can't be accurately made based on the ratio of one alkaloid type to another. Understanding the relative risk of each larkspur species is important to developing management strategies to mitigate livestock losses. 16 Carbon application to the soil reduces competition from medusahead. Medusahead is an invasive annual grass that decreases biological diversity, reduces livestock forage production, degrades ecological function of native plant communities, increases the frequency of wildfires on rangelands, and predisposes animals to ingest poisonous plants. ARS scientists at Logan, Utah established plots on two ranches located in Adams County in eastern Washington. Additional carbon (C) or nitrogen (N) was added to the soil to determine the effects of carbon nitrogen balance on suppression of medusahead populations to improve establishment and persistence of perennial grasses and forbs. Vegetation and soil will be monitored over two growing seasons. This technology will add another tool for the rancher to fight the invasion of annual grasses and improve rangeland conditions to enhance the economic viability of rural communities. 17 Comparative toxicity of water hemlock in animals. Water hemlock is considered one of the most toxic plants in North America. ARS scientists at Logan, Utah, demonstrated that tubers and green seed of water hemlock are very toxic whereas stalks, stems, leaves, flowers, and mature seeds are of low risk for poisoning. Chemical analysis demonstrated a direct correlation between the water hemlock toxins in the various plant parts and their toxicity. Furthermore, recent cell culture studies were performed to characterize the mechanism of action of the water hemlock toxins as well as to identify potential treatments, or antidotes. This information is useful for livestock producers and veterinarians as well as medical personnel who treat humans.

Impacts
(N/A)

Publications

Colegate, S.M., Gardner, D.R., Betz, J.M., Panter, K.E. 2014. Semi- automated separation of the epimeric dehydropyrrolizidine alkaloids lycopsamine and intermedine: Preparation of their N-oxides and NMR comparison with diastereoisomeric rinderine and echinatine. Phytochemical Analysis. 25(5):429-38.
Goodman, L.E., Cibils, A.F., Lopez, S.C., Steiner, R.L., Graham, J.D., Mcdaniel, K.C., Abbott, L.B., Stegelmeier, B.L., Hallford, D.M. 2014. Targeted grazing of white locoweed: Short-term effects of herbivory regime on vegetation and sheep. Rangeland Ecology and Management. 67(6):680-92.
Field, R.A., Stegelmeier, B.L., Colegate, S.M., Brown, A.W., Green, B.T. 2015. An in vitro comparison of the cytotoxic potential of selected dehydropyrrolizidine alkaloids and some N-oxides. Toxicon. 97:36-45.
Cook, D., Pfister, J.A., Constantino, J., Roper, J.M., Gardner, D.R., Welch, K.D., Hammond, Z.J., Green, B.T. 2015. Development of a PCR-based method for detection of Delphinium species in poisoned cattle. Journal of Agricultural and Food Chemistry. 63:1120-1125.
Cook, D., Welch, K.D., Green, B.T., Gardner, D.R., Pfister, J.A., Constantino, J., Stonecipher, C.A. 2015. The relative toxicity of Delphinium stachydeum in mice and cattle. Toxicon. 99:36-43.
Welch, K.D., Panter, K.E., Lee, S.T., Gardner, D.R. 2015. The effect of intermittent dosing of Nicotiana glauca on teratogenesis in goats. Toxicon. 93:119-124.
Edgar, J.A., Molyneux, R.J., Colegate, S.M. 2014. Pyrrolizidine alkaloids: Potential role in the etiology of cancers, pulmonary hypertension, congenital anomalies, and liver disease. Chemical Research in Toxicology. 28(1):4-20.
Welch, K.D., Stonecipher, C.A., Gardner, D.R., Panter, K.E., Parsons, C., Deboodt, T., Johnson, B. 2015. The effect of western juniper on the estrous cycle in beef cattle. Research in Veterinary Science. 98:16-18.
Boppre, M., Colegate, S.M. 2015. Recognition of pyrrolizidine alkaloid esters in the invasive aquatic plant Gymnocoronis spilanthoides (Asteraceae). Phytochemical Analysis. 26(3):215-225.
Cook, D., Oliveira, C.A., Gardner, D.R., Pfister, J.A., Riet-Correa, G.A., Riet-Correa, F. 2015. Changes in swainsonine, calystegine, and nitrogen concentrations on an annual basis in Ipomoea carnea. Toxicon. 95:62-66.
Snider, D.B., Gardner, D.R., Janke, B.H., Ensley, S.M. 2014. Pine needle abortion biomarker detected in bovine fetal fluids. Journal of Veterinary Diagnostic Investigation. 27(1):74-79.
Pfister, J.A., Davis, T.Z., Hall, J.O., Stegelmeier, B.L., Panter, K.E. 2015. Elk (Cervus elaphus canadensis) preference for feeds varying in selenium concentration. Journal of Animal Science. doi: 10.2527/jas2015- 9008.
Stegelmeier, B.L., Brown, A., Welch, K.D. 2015. Safety concerns of herbal products and traditional Chinese herbal medicines: Dehydopyrrolizidine alkaloids and aristolochic acid. Journal of Applied Toxicology. doi: 10. 1002/jat.3192.
Lee, S.T., Cook, D., Davis, T.Z., Gardner, D.R., Johnson, R.L., Stonecipher, C.A. 2015. A survey of tremetone, dehydrotremetone and structurally related compounds in Isocoma spp. (Goldenbush) in the southwestern United States. Journal of Agricultural and Food Chemistry. 63(3):872-879.
Davis, T.Z., Lee, S.T., Collett, M.G., Stegelmeier, B.L., Green, B.T., Buck, S.R., Pfister, J.A. 2015. Toxicity of White Snakeroot (Ageratina altissima) and chemical extracts of White Snakeroot in goats. Journal of Agricultural and Food Chemistry. 63(7):2092-2097.

Progress 10/01/13 to 09/30/14

Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop and implement novel management protocols for establishing improved forage species on sites infested with known poisonous plants to reduce the risk of livestock mortality and morbidity, improve livestock performance, and improve rangeland resiliency and diversity. Specifically, develop science-based guidelines for grazing livestock on rangelands infested with Lupinus, Senecio, Delphinium and swainsonine and selenium-containing plants. Objective 2: Reduce the risks of livestock losses due to variations in quantitative and qualitative differences in toxin accumulation over time and plant species by quantifying the influence of endophytes, climate changes, and genotype on plant toxin accumulation (particularly swainsonine-containing plants and Delphinium and Lupinus species). Objective 3: Enhance feed and food safety by improving risk assessment and diagnosis of plant-induced poisoning to livestock by improving analytical methods for analyzing plant and animal tissues for toxins; measuring toxicokinetics, assessing carcinogenic and genotoxic potential, and identifying toxin metabolites and biomarkers of toxicoses. Objective 4: Develop improved procedures with guidelines for diagnostic and prognostic evaluation to reduce negative impacts of poisonous plants on livestock reproduction and embryo/fetal growth by improving early identification of poisoned animals, predicting poisoning outcomes, and management and treatment options through improved understanding of clinical, morphological and molecular alterations of plant-induced toxicosis. Objective 5: Develop guidelines to aid producers and land managers in making genetic-based herd management decisions to improve livestock performance and safety on grazed rangelands infested with poisonous plants through the use of identified animal genes, physiological pathways, and molecular mechanisms of action that underlie Conium, Cicuta, Delphinium, Lupinus, and Nicotiana, and other neurotoxic plant effects. Approach (from AD-416): Livestock poisoning by plants results in over $503,000,000 lost to the livestock industry annually in the 17 western United States from death losses and abortions alone (Holechek, 2002). Plant poisonings extend worldwide to include 333 million poisonous plant-infested hectares in China and 60 million hectares in the central western region of Brazil, to name a few. There are over 6,000 species of pyrrolizidine alkaloid (PA)- containing plants, and over 350 individual PAs causing diseases in animals and humans have been identified. Economic losses are much larger as significant amounts of nutritious forage are wasted and management costs are increased due to the threat of toxic plant-related livestock losses. The Poisonous Plant Research Laboratory (PPRL) has provided worldwide leadership in poisonous plant research to the livestock industry and consumers including numerous solutions to toxic plant problems using an integrated, interdisciplinary approach (see Figure below). The research team investigates plant poisonings in a systematic matter by identifying the plant, describing the effects in animals, determining the toxin(s) and evaluating the mechanisms of action. The ultimate goal is to develop research-based solutions to reduce livestock losses from toxic plants. There are five coordinated objectives in this project plan providing guidelines for potential genetic-based management. This research will reduce livestock losses from plants and enhance the economic well-being of rural communities, improve rangeland health by combating invasive plant species, and help to provide safe animal products free from potential plant toxins for consumers. Poisonous plants continue to cause large economic losses to the livestock industry from death losses, birth defects, reduced reproductive performance and lost forage value. Natural toxins may also contaminate human food supplies. ARS researchers at the Poisonous Plant Research Lab (PPRL) in Logan, UT continue to mitigate poisonous plant losses through a concerted interdisciplinary team approach. Five objectives have been defined in the project plan to develop research-based solutions to reduce livestock losses from poisonous plants. Research progress over the last year includes the continued evaluation of improved rangeland grass species and forage Kochia varieties seeded into research, demonstration and ranch scale plots on the channeled scablands of central Washington State. Two improved and one native grass species and one forage Kochia variety successfully established. Current data suggest that annual grass and poisonous plant reinvasion is suppressed where these improved varieties have established. Vavilov II, Hycrest wheat grasses, Sherman Big Blue grass and Immigrant forage Kochia were the most successful species and show promise to establish and provide improved forage quality while successfully competing with cheat grass, medusa head rye and poisonous plants in this harsh environment of the Washington scablands. Analytical methods developed by ARS scientists at PPRL were used to detect pyrrolizidine alkaloids (PAs) in contaminated food and feed supplies. Examples include the continued monitoring of subsistence grain supplies from the Tigray region of Ethiopia, honey and pollen samples from the islands of Hawaii and contaminated chicken feed from Colombia. In addition, the analytical method for detection of pyrrolizidine alkaloid metabolites in liver and blood diagnostic samples was significantly improved and used in the investigation of the relative toxicity of the various alkaloids as tested in a small animal assay model. Significant progress has been made on the NIH funded PA research over the last five years including large scale isolation and purification of epimeric lycopsamine and intermedine and synthesis and purification of their N-oxides. Analytical methods to screen herbal products including comfrey for PAs have been completed. Echimidine was isolated from Echium vulgare and purified and the N-oxide synthesized. Three separate commercial samples of butterbur root powder (Petasites spp.), an herbal remedy, were screened for PAs and one contained two dehydropyrrolizidine alkaloids. Histologic evaluation of PA carcinogenicity studies in P53 heterozygous mice were concluded. Both acute exposures and chronic low dose exposure to riddelliine caused increased incidence of PA-induced neoplasms. The neoplasm type differed by exposure, i.e. chronic low dose exposures produced hepatic vascular neoplasms while acute high dose exposures produced neoplasms outside the hepatic vasculature. This work suggests that any PA exposure can increase the incidence of PA-related carcinogenesis. Studies of the carcinogenic potential of other PAs are currently being evaluated. Additional work using in vitro cell cultures and in vivo models to better document PA toxicity and correlate that toxicity to their carcinogenicity has been extended, and specific PAs have been ranked according to their toxicity to cells. Four PAs�lasiocarpine, seneciophylline, senecionine and heliotridine�are more toxic than riddelliine and will be evaluated for their potential carcinogenicity. A sensitive small animal model for in vivo PA toxicity has been developed. This model has been used to directly compare the toxicity of 20 purified PAs from different plant sources. Initial findings support the cellular toxicity results, indicating that lasiocarpine, seneciophylline, senecionine, heliotridine and monocrotaline are more toxic than riddelliine. This model is important because with relatively small amounts of pure alkaloid and/or their metabolites, comparative toxicity can be evaluated. These methodologies are important to aid in the diagnosis of PA exposure in animals and to determine how metabolite concentrations correlate with disease progression and PA exposure. Research to aid producers and land managers in making genetic-based herd management decisions to improve livestock performance and safety on grazed rangelands infested with poisonous plants has progressed. Animal genes, physiological pathways, and molecular mechanisms of action that underlie the effects of neurotoxic plants such as lupine, larkspurs, and poison hemlock have been identified. Of the cattle breeds tested to date, differences have been identified and Brahman cattle are most sensitive and dairy breeds most resistant. Cell-based assays have been completed to determine the molecular mechanism of the plant neuro toxins and the neuro toxin lamprolobine from lupine was evaluated. A plant extract-based assay to study the action of Cicuta (water hemlock) toxins at the GABAA receptors in the brain was developed. Since the 1900�s tremetone was reported to be the toxin in rayless goldenrod in the Southwest and white snakeroot in the Midwest. Recent chemical studies have isolated and identified many benzofuran ketones including tremetone from these plants. Subsequent animal studies suggest that tremetone is not the primary toxin. Recent studies indicate that other benzofuran ketones act together with tremetone to cause toxicity. Different plant populations with variable benzofuan ketone concentrations are being evaluated to determine why poisoning in animals occurs in some locations while the same or similar plant species in another location may not cause poisoning. Seleniferous forages are toxic to livestock and are suspected to cause decreased reproductive rates in some livestock species. Studies were conducted to determine the effects of high selenium in the diet on reproductive performance. Additional studies were completed to determine the effects of high selenium in the diet on feed preferences of sheep, cattle and elk and to study the rate and processes of elimination of the selenium compounds. Larkspur specific primers have been developed and validated for all the larkspur species in the Intermountain West that have resulted in cattle losses. They have been used to detect larkspur that has been incubated in rumen cultures and in cannulated cows as a potential PCR-based diagnostic tool. A new group of locoweed seedlings has been started to evaluate fungicide applications as a treatment to reduce swainsonine (produced by the endophyte in locoweed plants) and render the plants less toxic. Data analysis has been completed on the effects of elevated CO2 levels on locoweed growth and swainsonine concentrations. In collaboration with researchers at the University of Kentucky and New Mexico State University, research to elucidate the swainsonine biosynthetic pathway in swainsonine-producing endophyte is progressing. Genome sequencing of two swainsonine producing fungal endophytes is underway. In collaboration with researchers at Indiana University, we have screened several Convolvulaceae taxa for swainsonine using herbarium specimens and identified several species that contain swainsonine. Locoweed poisoning was studied in various animal models and we found that huge differences in susceptibility, disease progression, lesions, and lesion distribution exist between animal species. Initial work indicated this is largely due to swainsonine affinity to a species� mannosidase enzymes. Additional comparisons of mannosidase expression in tissues and correlation of that expression with lesion development are ongoing. Goats and horses are highly susceptible to locoweed poisoning, developing severe neurologic disease. Early reproductive studies in goats determined that the goat fetus is very sensitive to the effects of locoweed, resulting in fetal loss. Comparative studies of species specific locoweed-induced ophthalmic lesions have been completed and we found that locoweed blindness was due to neurological lesions and not lesions in the eye. Studies were conducted to determine if co-exposure of cattle to death camas will exacerbate low larkspur toxicity. Samples of bark and needles were collected from young and mature juniper trees to determine if there is a difference in the concentration of the abortifacient compounds between and young and mature trees. Additionally, samples of bark and needles have been collected bi-monthly for two years to evaluate seasonal variations in the abortifacient compounds in western juniper trees. Accomplishments 01 Reduction in annual grass and poisonous plant invasion. Annual grasses have extensively invaded the rangelands of the channeled scablands of central Washington State which are already infested with lupines. When the grasses dry out in early summer, lupines become the preferred forage, exacerbating lupine-induced �crooked calf syndrome�. ARS scientists from Logan, Utah established small plots on the channeled scablands of central Washington State to determine which grass species is best adapted for this harsh environment and if forage Kochia would grow in this area. Data suggest that Vavilov II, Hycrest and Sherman Big Blue are the grasses most likely to germinate and establish, and demonstration and range scale plots were planted. Immigrant Kochia germinated and established over a three year period on the demonstration and range scale plots. Preliminary information suggests that these improved perennial grasses and forage Kochia will germinate, persist in the short term (three years to date) and reduce the re- invasion of the annual grasses and poisonous plants to provide an improved forage base for livestock and wildlife. 02 Pyrrolizidine alkaloids (PAs) found in honey and eggs. Pyrrolizidine alkaloid (PAs) exposure in humans is most often via contamination of a primary food source such as in grains. The potential for long term, low level exposure in foods, including pollen and honey as well as eggs, milk and perhaps meat, is unknown. ARS scientists in Logan, Utah provided analytical support for two studies. Significant concentrations of PAs were detected in experimental hives near fields infested with fireweed. No PAs were detected in honey and pollen from hives physically isolated from fireweed-contaminated fields. Secondly, ARS scientists in cooperation with the National University of Colombia investigated the effect of dietary supplementation of chicken feed with Crotalaria seeds during a 35 day feeding of commercial laying hens. Eggs collected during the 35 days of the treatment feedings were found to contain significant concentrations of PAs with decreased concentrations within seven days after removal of the contaminated feed. Information from this research is being used to direct the agriculture industries to avoid PA contamination of food supplies. 03 Discovery of pyrrolizidine alkaloids (PAs) in herbal products (NIH funded project). ARS scientists at Logan, Utah completed large scale isolation of two important PAs (lycopsamine and intermedine) found in animal feeds and herbal products. Echimidine, a PA found in Symphytum uplandicum (comfrey), was isolated from Echium vulgare (Paterson's curse). Samples of three commercial herbal products of butterbur root powder (Petasites spp.) have been analyzed for dehydropyrrolizidine alkaloids and one showed the presence of two PAs, senecionine and integerrimine. The screening and analytical methods developed will be standardized and provided for reference labs around the world to monitor safety of herbal products and food sources. 04 Pyrrolizidine alkaloid (PA) may cause an increased risk of cancer. PAs often contaminate feed, food, and medicinal or herbal products, poisoning livestock, wildlife and humans. Several PAs have been linked with cancer but only one, riddelliine, has been officially classified as a potential human carcinogen. ARS scientists at Logan, UT showed that both high-dose, short exposure and chronic, low-dose exposure increased the incidence of PA-induced tumors. The type of tumor differed by exposure. Acute high dose exposures produced many tumors in the liver and chronic low dose exposures produce tumors in the blood vessels of the liver. This work is important as it indicates that any PA exposure can increase the incidence of PA-related cancers. 05 Toxicity of pyrrolizidine alkaloids (PAs) in liver cell cultures. ARS scientists at Logan, Utah developed cell cultures and animal models to better document PA toxicity and correlate toxicity with cancer formation. Specific PAs were ranked according to their toxicity to cells. Four PAs�lasiocarpine, seneciophylline, senecionine and heliotridine�are more toxic than riddelliine. As the mechanisms of toxicity and cancer induction are probably similar, these four PAs are likely to cause liver cancer, similar to riddelliine, and they should be further evaluated to determine their potential to cause cancer. This information is critical for making science based decisions about the safety of herbal products and certain food sources. 06 Effects of plant toxins on the nervous system and muscle function. Plants containing toxins that affect the nervous system are commonly found on open rangelands where livestock can be poisoned when they consume too much of these plants. A significant clinical sign observed in livestock poisoned by these plants is the disruption of coordinated muscle function. ARS scientists at Logan, Utah characterized the muscle function and coordination deficiencies that occur upon exposure to a non-lethal dose of several plant toxins. Experiments using rodent models provided valuable information regarding the acute toxicity of these plants and identifying their toxins. ARS scientists found that animals which survive poisoning episodes from neurotoxic plant alkaloids will fully recover without lasting muscular dysfunction or coordination deficits. The results obtained from these studies provide a basic understanding of acute toxicity and adverse effects, including motor function and coordination, of specific toxins. This information is used to better design experiments using livestock species to help in providing management recommendations to livestock producers, extension agents, and government regulatory agencies. 07 Blood profiles of several plant toxins. The metabolism, rate of absorption, and elimination (toxicokinetics) of plant toxins in livestock is often unknown. ARS scientists at Logan, Utah characterized the blood profiles of numerous toxins from several plants that cause significant livestock losses. The blood profiles were shown to correlate directly with physiologic changes in livestock. The blood profile of the larkspur alkaloids was compared between susceptible and resistant breeds of cattle, which may be a factor in the differences in susceptibility to larkspur poisoning. This research provides important data regarding feed safety for animals as well as food safety for humans. Knowledge of the absorption and elimination of plant toxins in animals provides the necessary information for ARS scientists to develop safe grazing practices and pasture rotations to ensure that animal losses are minimized as they better understand how long these toxins remain in the animals. This information helps to ensure that livestock exposed to poisonous plants are safe for human consumption. It also provides livestock owners, extension agents, and veterinarians with valuable information regarding management and treatment of intoxicated animals (for example, how soon they can safely move an intoxicated animal to safe pastures). 08 The genetic association of resistance to plant toxins in cattle. The selection of genetically superior cattle for grazing on rangelands is important because losses from toxic plants such as larkspur and lupine cost ranchers millions of dollars each year. ARS scientists at Logan, Utah have identified both breed-associated and individual differences in resistance to intoxication. ARS researchers are currently identifying resistant Angus and susceptible Hereford steers with the Illumina BovineHD Genotyping platform to identify genetic markers for resistance to plant toxins. This research will ultimately result in genetic markers that can be used to make genetic-based herd management decisions. 09 Identification of cattle gene markers for resistance to lupine-induced birth defects. There are differences in the rates of lupine toxin elimination between individuals and breeds of cattle. ARS scientists at Logan, Utah have shown that selected dairy breeds eliminate larkspur toxins faster than selected beef breeds and there is individual animal to animal variation within each breed as well as breed differences. This research determined the elimination rates of four lupine toxins from velvet lupine orally dosed to four Holstein steers as an initial step to identify why certain animals are resistant to lupine poisoning. ARS scientists determined that dairy breeds are inherently more resistant to plant toxins that affect the nervous system than beef breeds. This research will be important in selection of cattle for replacements and bulls for herd sires. 10 Individual animal variation in the susceptibility to larkspur toxins. Research by ARS scientists at Logan, Utah demonstrated that there is variation in the susceptibility to larkspur toxins between different strains of mice. Using mice as a model system for cattle, several potential susceptibility factors were identified that could explain the differences in susceptibility of the different strains of mice to larkspur poisoning. Studies determined that genetically engineered mice lacking a specific gene receptor are not more resistant to larkspur poisoning, indicating that this receptor (alpha 7) does not play an integral role in the acute toxic effect of larkspur. However, several other potential genetic markers have been identified which will provide the basis for future experiments to identify genetic factors that correlate with sensitivity to larkspur poisoning in cattle. Once this research is validated in cattle, this knowledge will provide livestock producers with specific information that will be useful in breeding, culling, and grazing management programs to reduce or prevent larkspur poisoning on rangelands. 11 Characterization of pine needle-induced late term abortions in cattle. ARS scientists at Logan, Utah discovered that cattle pre-conditioned to ponderosa pine needles metabolize the abortifacient toxins in the needles more quickly than na�ve cattle. Additionally, they demonstrated that western juniper trees can also cause late term abortions in cattle. Research into the effects of western juniper exposure on reproduction demonstrated that western juniper does not affect the reproductive cycle of cattle nor their ability to become pregnant. The results from this work provide an increased knowledge and understanding regarding the ability of pine and juniper trees to cause late-term abortions as well as differences between na�ve and conditioned cattle in their susceptibility to the adverse outcomes, e.g., abortions. This information is useful in further developing livestock management recommendations for ranchers. Government action agencies, extension agents, veterinarians, and livestock owners can use this information in formulating management recommendations and procedures regarding the grazing of cattle on rangelands with pine and juniper trees to limit reproductive losses. 12 Evaluation of co-exposure of multiple poisonous plants in animals. In most cases where livestock are poisoned by plants on rangelands, they are grazing on multiple species of poisonous plants. Two poisonous plants often found growing simultaneously in the same area are death camas and low larkspur. ARS scientists at Logan, Utah demonstrated that the co-administration of toxins from death camas and low larkspur have an additive effect (they both contribute to the toxicity), resulting in enhanced toxicity in a rodent model. However, similar experiments performed in sheep support previous findings that sheep are resistant to larkspur poisoning. Thus co-exposure of low larkspur does not enhance toxicity of death camas in sheep. These results provide an increased knowledge and understanding regarding the acute toxicity of death camas and risk of co-exposure of multiple plant toxins. This information is useful in developing livestock management recommendations for ranchers. Government action agencies, extension agents, veterinarians, and livestock owners can use this information in formulating management recommendations and procedures regarding the grazing of cattle in larkspur and death camas-infested rangelands. 13 Survey of Palicourea plants in South America for the toxin monofluoroacetate (MFA). The genus Palicourea represents one of the most important poisonous plants in Brazil. Two species of Palicourea cause sudden death and contain MFA. ARS scientists at Logan, Utah developed a rapid method to analyze MFA and used it to screen herbarium specimens of 46 Palicourea species from South America. Ten species of Palicourea were identified that contained MFA, two previously reported and eight newly reported. This research impacts livestock production systems in South America as it provides a representative list of Palicourea species that contain MFA and thus pose a risk to grazing livestock. 14 Chemistry of plant toxins with unique structures. ARS scientists at Logan, Utah developed cell-based assays for the evaluation of toxins that occur in plants as right and left handed pairs. There is a difference in activity depending on which of the pairs is dominant in the plant. The results from this research suggest that it is the concentration of each toxin present in the plant that determines toxicity, not just total toxin concentration. Prior to this work, livestock management decisions were based on total toxin concentrations and ignored the presence and impact of these pairs of toxins which vary from plant population to plant population. This information will enhance current risk assessment by scientists and land managers to reduce livestock losses from these plants. If land managers do not consider the ratio of each toxin when developing management plans, then the toxicity of a plant population may be underestimated. 15 Determination of the toxins in rayless goldenrod and white snakeroot. ARS scientists at Logan, Utah demonstrated that the toxic component in rayless goldenrod and white snakeroot is a mixture of toxins and not a single toxin as historically thought. These plants continue to poison animals and pose a risk of contaminating milk in the Southwest and Midwest. This information is important when assessing the risk of plant populations and diagnosing cases of livestock poisoning. 16 Excess selenium decreases reproductive rates in sheep. ARS scientists at Logan, Utah completed pen studies that determined a 20 to 40% decrease in pregnancy rates in ewes fed high selenium forages for three weeks before being exposed to rams. This information is beneficial to manage sheep grazing on selenium contaminated ranges so that producers can maximize the reproductive efficiency of their flocks. 17 Locoweed poisoning in goats and other species. Locoweed poisoning costs the livestock industry millions of dollars each year. ARS scientists at Logan, Utah determined there are substantial animal differences in locoweed-induced disease, lesions and subsequent costs. An early indicator of poisoning in pregnant animals is fetal toxicity and fetal loss. This research demonstrates that all livestock species are at risk; however, horses and goats are extremely sensitive and should not be allowed to graze in pastures where any level of locoweed is found. 18 Locoweed-induced eye pathology. Locoweed poisoning in cattle and other livestock has historically been associated with dull, glassy appearing eyes, and there are some reports of blindness. ARS scientists at Logan, Utah found that in horses, cows, sheep and goats; locoweed poisoning does not cause true blindness. Eye examinations of these animals are normal including normal reflexes. However the tear ducts showed abnormal changes in all four species and the tear production in poisoned horses was about 50% below normal. Visible mucous strands were often observed. This indicates that the dull appearing eyes of poisoned animals is due to changes in tear duct secretion and the tear film. Poisoned animals are not truly blind but the reported �blindness� behavior is most likely due to the effects of locoweed poisoning on the brain. 19 Wild parsnip causes sunburn and inflammation to the skin. Wild parsnip (Pastinaca sativa) is a biennial introduced weed that is often associated with sunburn of livestock and humans. ARS scientists at Logan, Utah found that parsnip toxins are quickly excreted in the urine by cattle, sheep and goats. Generally, sunburn is due to repeated exposures to the photo-active toxins on the skin such as what happens in walking through or grazing in patches of wild parsnip. Reports of skin inflammation in horses fed parsnip contaminated hay suggests its metabolism in horses may be different than in cattle. This research is only preliminary and more work is needed to better define these differences.

Impacts
(N/A)

Publications

Adrien, M.L., Riet-Correa, G., Oliveira, C.A., Pfister, J.A., Cook, D., Souza, E.G., Riet-Correa, F., Schild, A.L. 2013. Conditioned food aversion to Ipomoea carnea var. fistulosa induced by Baccharis coridifolia in goats. Pesquisa Veterinaria Brasileira. 33(8):999-1003.
Albuquerque, S.S., Rocha, B.P., Almeida, V.M., Oliveira, J.S., Riet-Correa, F., Lee, S.T., Neto, J.E., Mendoca, F.S. 2014. Cardiac fibrosis associated to the poisoning of Amorimia septentrionalis in cattle. Pesquisa Veterinaria Brasileira. 34(5):433-437.
Almeida, M.B., Schild, A.L., Pfister, J.A., Assis-Brasil, N.D., Pimental, M., Forster, K.M., Riet-Correa, F. 2013. Methods of inducing conditioned food aversion to Baccharis coridifolia (mio-mio) in cattle. Electronic Publication. 43(10):1866-1871.
Beck, J.J., Mahoney, N.E., Cook, D., Gee, W.S., Baig, N. 2014. Comparison of the volatile emission profiles of ground almond and pistachio mummies: part 1 � addressing a gap in knowledge of current attractants of navel orangeworm. Phytochemistry Letters. 9:102-106. DOI.10.1016/j.phytol.2014. 04.010.
Becker, M., Caldeira, F.H., Carneiro, F.M., Oliveira, L.P., Tokarnia, C.H., Riet-Correa, F., Lee, S.T., Colodel, E.M. 2013. Epidemiological aspects of field intoxication by Amorimia pubiflora (Malpighiaceae) in cattle in Mato Grosso and experimental reproduction of intoxication in cattle and sheep. Pesquisa Veterinaria Brasileira. 33(9):1049-1056.
Colegate, S.M., Welsh, S.L., Gardner, D.R., Betz, J.M., Panter, K.E. 2014. Profiling of dehydropyrrolizidine alkaloids and their N-oxides in herbarium-preserved specimens of Amsinckia species using HPLC-esi(+)MS. Journal of Agricultural and Food Chemistry. 62(30):7382-7392.
Cook, D., Gardner, D.R., Pfister, J.A. 2014. Swainsonine-containing plants and their relationship to endophytic fungi. Journal of Agricultural and Food Chemistry. 62(30):7326-7334.
Cook, D., Lee, S.T., Taylor, C.M., Bassuner, B., Riet-Correa, F., Pfister, J.A., Gardner, D.R. 2014. Detection of toxic monofluoroacetate in Palicourea species. Toxicon. 80:9-16.
Dale, L.M., Thewis, A., Boudry, C., Rotar, I., Pacurar, F.S., Abbas, O., Dardenne, P., Baeten, V., Pfister, J.A., Fernandez Pierna, J.A. 2013. Discrimination of grassland species and their classification in botanical families by laboratory scale hyperspectral imaging NIR: preliminary results. Talanta. 116:149-154.
Davis, T.Z., Green, B.T., Stegelmeier, B.L., Lee, S.T., Welch, K.D., Pfister, J.A. 2013. Physiological and serum biochemical changes associated with rayless goldenrod (Isocoma pluriflora) poisoning in goats. Toxicon. 76:247-254.
Davis, T.Z., Stegelmeier, B.L., Green, B.T., Welch, K.D., Hall, J.O. 2013. Evaluation of the respiratory elimination kinetics of selenate and Se- methylselenocysteine after oral administration in lambs. Research in Veterinary Science. 95(3):1163-1168.
Davis, T.Z., Stegelmeier, B.L., Hall, J.O. 2014. Analysis in horse hair as a means of evaluating selenium toxicoses and long-term exposures. Journal of Agricultural and Food Chemistry. 62(30):7393-7397.
Davis, T.Z., Stegelmeier, B.L., Lee, S.T., Green, B.T., Hall, J.O. 2013. Experimental rayless goldenrod (Isocoma pluriflora) toxicosis in horses. Toxicon. 73:88-95.
Davis, T.Z., Stegelmeier, B.L., Welch, K.D., Pfister, J.A., Panter, K.E., Hall, J.O. 2013. Comparative oral dose toxicokinetics of selenium compounds commonly found in selenium accumulator plants. Journal of Animal Science. 91(9):4501-4509.
Duarte, A.L., Medeiros, R.M., Carvalho, F.K., Lee, S.T., Cook, D., Pfister, J.A., Costa, V.M., Riet-Correa, F. 2014. Induction and transfer of resistance to poisoning by Amorimia (Macagnia) septentrionalis in goats. Journal of Applied Toxicology. 34(2):220-223.
Gardner, D.R., Riet-Correa, F., Lemos, D., Welch, K.D., Pfister, J.A., Panter, K.E. 2014. Teratogenic effects of Mimosa tenuiflora in a rat model and possible role of N-methyl and N,N-dimethyltryptamine. Journal of Agricultural and Food Chemistry. 62(30):7398-7401.
Green, B.T., Lee, S.T., Welch, K.D., Panter, K.E. 2013. Plant alkaloids that cause developmental defects through the disruption of cholinergic neurotransmission. Birth Defects Research Part C: Embryo Today: Reviews. 99:235-246.
Green, B.T., Welch, K.D., Gardner, D.R., Stegelmeier, B.L., Lee, S.T. 2013. A toxicokinetic comparison of two species of low larkspur (Delphinium spp. ) in cattle. Research in Veterinary Science. 95(2):612-615.
Green, B.T., Welch, K.D., Panter, K.E., Lee, S.T. 2013. Plant toxins that affect nicotinic acetylcholine receptors: A review. Chemical Research in Toxicology. 26(8):1129-1138.
Green, B.T., Welch, K.D., Pfister, J.A., Chitko-Mckown, C.G., Gardner, D.R. , Panter, K.E. 2014. Mitigation of larkspur poisoning on rangelands through the selection of cattle. Rangelands. 36(1):10-15.
Grum, D.S., Cook, D., Baucom, D., Mott, I.W., Gardner, D.R., Creamer, R., Allen, J.G. 2013. Production of the alkaloid swainsonine by a fungal endophyte in the host Swainsona canescens. Journal of Natural Products. 76(10):1984-1988.
Irwin, R.E., Cook, D., Richardson, L.L., Manson, J.S., Gardner, D.R. 2014. Secondary compounds in floral rewards of toxic rangeland plants: Impacts on pollinators. Journal of Agricultural and Food Chemistry. 62(30):7335- 7344.
Porto, M.R., Saturnino, K.C., Lima, E.M., Lee, S.T., Lemos, R.A., Marcolongo-Pereira, C., Riet-Correa, F., Castro, M.B. 2013. Evaluation of solar exposure on the experimental intoxication by Brachiaria decumbens in sheep. Pesquisa Veterinaria Brasileira. 33(8):1009-1015.
Latorre, A.O., Caniceiro, B.D., Fukumasu, H., Gardner, D.R., Lopes, F.M., Wyospchi Jr, H.L., Da Silva, T.C., Haraguchi, M., Bressan, F.F., Gorniak, S.L. 2013. Ptaquiloside reduces NK cell activities by enhancing metallothionein expression, which is prevented by selenium. Toxicology. 304:100-108.
Lee, S.T., Cook, D., Molyneux, R.J. 2014. Identification of the quinolizidine alkaloids in Sophora leachiana. Biochemical Systematics and Ecology. 54:1-4.
Lima, F.G., Haraguchi, M., Pfister, J.A., Guimaraes, V.Y., Andrade, D.D., Ribeiro, C.S., Costa, G.L., Araujo, A.L., Fioravanti, M.C. 2013. Weather and plant age affect the levels of steroidal saponin and Pithomyces chartarum spores in Brachiaria grass. International Journal of Poisonous Plant Research. 2:45-53.
Maia, L.A., De Lucena, R.B., Da T Nobre, V.M., Dantas, A.F., Colegate, S.M. , Riet-Correa, F. 2013. Natural and experimental poisoning of goats with the pyrrolizidine alkaloid-producing plant Crotalaria retusa L. Journal of Veterinary Diagnostic Investigation. 25(5):592-595.
Manson, J.S., Cook, D., Gardner, D.R., Irwin, R.E. 2013. Direct and pollinator-mediated effects of nectar alkaloids on Delphinium barbeyi. Journal of Ecology. 1001:1604-1612.
Panaccione, D.G., Beaulieu, W.T., Cook, D. 2014. Bioactive alkaloids in vertically transmitted fungal endophytes. Functional Ecology. 28(2):299- 314.
Penno, M.A., Colegate, S.M., Michalski, W.P., Hoffmann, P. 2012. Detection and measurement of carbohydrate deficient transferrin in serum using immuno-capture mass spectrometry: Diagnostic applications for annual regrass toxicity and corynetoxin exposure. Research in Veterinary Science. 93(2):611-617.
Pessoa, C.R., Pessoa, A.F., Maia, L.A., Medeiros, R.M., Colegate, S.M., Barros, S.S., Soares, M.P., Borges, A.S., Riet-Correa, F. 2013. Pulmonary and hepatic lesions caused by the dehydropyrrolizidine alkaloid-producing plants Crotalaria juncea and Crotalaria retusa in donkeys. Toxicon. 71:113- 120.
Pfister, J.A., Cook, D., Gardner, D.R., Baker, S.D. 2013. Early season grazing by cattle of waxy larkspur (Delphinium glaucescens) in Central Idaho. Rangelands. 35(4):2-5.
Lee, S.T., Cook, D., Pfister, J.A., Allen, J.G., Colegate, S.M., Riet- Correa, F., Taylor, C.M. 2014. Monofluoroacetate-containing plants that are potentially toxic to livestock. Journal of Agricultural and Food Chemistry. 62(30):7345-7354.
Lee, S.T., Welch, K.D., Panter, K.E., Gardner, D.R., Garrossian, M., Chang, C.T. 2014. Cyclopamine: From cyclops lambs to cancer treatment. Journal of Agricultural and Food Chemistry. 62(30):7355-7362.
Pfister, J.A., Davis, T.Z., Hall, J.O. 2013. Effect of selenium concentration on feed preferences by cattle and sheep. Journal of Animal Science. 91(12):5970-5980.
Pimentel, L., Maia, L.A., Carvalho, F.K., Campos, E.M., Pfister, J.A., Cook, D., Medeiros, R.M., Riet-Correa, F. 2013. Conditioned food aversion for control of poisoning by Ipomoea carnea subsp. fistulosa. Pesquisa Veterinaria Brasileira. 33(6):719-723.
Welch, K.D., Green, B.T., Gardner, D.R., Stonecipher, C.A., Panter, K.E., Pfister, J.A., Cook, D. 2013. The effect of low larkspur (Delphinium spp.) co-administration on the acute toxicity of death camas (Zigadenus spp.) in sheep. Toxicon. 76:50-58.
Welch, K.D., Green, B.T., Panter, K.E., Gardner, D.R., Pfister, J.A., Cook, D. 2014. If one plant toxin is harmful to livestock, what about two? Journal of Agricultural and Food Chemistry. 62(30):7363-7369.
Welch, K.D., Lee, S.T., Panter, K.E., Gardner, D.R., Knoppel, E.L., Green, B.T., Hammond, C.K., Hammond, Z.J., Pfister, J.A. 2014. Studies on the teratogenicity of anabasine in a rat model. Toxicon. 87:32-37.

Progress 10/01/12 to 09/30/13

Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop science-based guidelines for grazing livestock on rangelands infested with toxic plants (particularly Lupinus, Senecio, Delphinium and swainsonine and selenium-containing plants) and evaluate the potential for establishing improved forage species on infested sites to improve livestock gains, reduce the risk of livestock loss, and improve other rangeland ecosystem services. Objective 2: Reduce the risks of livestock losses due to variations in quantitative and qualitative differences in toxin accumulation over time and plant species by quantifying the influence of endophytes, climate changes, and genotype on plant toxin accumulation (particularly swainsonine-containing plants and Delphinium and Lupinus species). Objective 3: Enhance feed and food safety by improving risk assessment and diagnosis of plant-induced poisoning to livestock by improving analytical methods for analyzing plant and animal tissues for toxins; measuring toxicokinetics, assessing carcinogenic and genotoxic potential, and identifying toxin metabolites and biomarkers of toxicoses. Objective 4: Develop improved procedures with guidelines for diagnostic and prognostic evaluation to reduce negative impacts of poisonous plants on livestock reproduction and embryo/fetal growth by improving early identification of poisoned animals, predicting poisoning outcomes, and management and treatment options through improved understanding of clinical, morphological and molecular alterations of plant-induced toxicosis. Objective 5: Develop guidelines to aid producers and land managers in making genetic-based herd management decisions to improve livestock performance and safety on grazed rangelands infested with poisonous plants through the use of identified animal genes, physiological pathways, and molecular mechanisms of action that underlie Conium, Cicuta, Delphinium, Lupinus, and Nicotiana, and other neurotoxic plant effects. Approach (from AD-416): Livestock poisoning by plants results in over $503,000,000 lost to the livestock industry annually in the 17 western United States from death losses and abortions alone (Holechek, 2002). Plant poisonings extend worldwide to include 333 million poisonous plant-infested hectares in China and 60 million hectares in the central western region of Brazil, to name a few. There are over 6,000 species of pyrrolizidine alkaloid (PA)- containing plants, and over 350 individual PAs causing diseases in animals and humans have been identified. Economic losses are much larger as significant amounts of nutritious forage are wasted and management costs are increased due to the threat of toxic plant-related livestock losses. The Poisonous Plant Research Laboratory (PPRL) has provided worldwide leadership in poisonous plant research to the livestock industry and consumers including numerous solutions to toxic plant problems using an integrated, interdisciplinary approach (see Figure below). The research team investigates plant poisonings in a systematic matter by identifying the plant, describing the effects in animals, determining the toxin(s) and evaluating the mechanisms of action. The ultimate goal is to develop research-based solutions to reduce livestock losses from toxic plants. There are five coordinated objectives in this project plan providing guidelines for potential genetic-based management. This research will reduce livestock losses from plants and enhance the economic well-being of rural communities, improve rangeland health by combating invasive plant species, and help to provide safe animal products free from potential plant toxins for consumers. Losses to livestock producers continue to plague the industry reducing the economic viability of rural ranching communities. ARS researchers at the Poisonous Plant Research Lab in Logan, UT continue to mitigate losses through team research using an integrated, interdisciplinary approach. Five coordinated objectives have been defined to develop research based solutions to reduce livestock losses from poisonous plants. Research progress over the last year include establishment of seeding plots using introduced grass and forb species to improve rangeland conditions and provide alternative high quality forages for livestock to reduce grazing of poisonous plants. An endophyte (Undifillum) was shown to produce the locoweed toxin swainsonine. In the last year similar endophytes that produce swainsonine have been discovered in other plant species including Ipomoea in Brazil, Swainsona in Australia and Sida and Turbina in Africa and South America. Breed and individual animal to animal variation in response to larkspur poisoning suggests a genetic basis for these differences and 5 breeds of cattle have now been screened. Juniper species have been shown to cause abortion in cattle and chemical analysis of juniper collections in Oregon demonstrated that the toxin (isocupressic acid) was the same as that found in Ponderosa pine needles. Pyrrolizidine alkaloids poison animals and people throughout the world. Research screening individual PA�s through cell culture methods and using a cancer P53 transgenic mouse model has provided new information on mechanism and structure activity relationship of selected PA�s. Over 15 benzofuran ketones have been identified in white snakeroot and rayless goldenrod and the toxicity and role of each ketone is being evaluated in a Spanish goat small ruminant model. Plants that accumulate selenium have been evaluated for the chemical form, concentration of selenium in plant parts and the effect of different chemical forms on toxicity in sheep, cattle and horses. Significant Activities that Support Special Target Populations: A stake holder meeting was held in Ritzville, Washington in May 2013 with more than 50 ranchers attending. Seeding plots and demonstration plots were evaluated and discussed with those in attendance and the potential of improving rangeland conditions using improved grass and forb germplasm to reduce the impact of lupine was presented and discussed. This information, in addition to new methods (intermittent grazing), were presented to livestock producers to reduce the impact from lupine-induced crooked calf syndrome. An International Symposium on Poisonous Plants was held in HohHot, Inner Mongolia, China in July 2013 with 106 participants. (8) ARS Scientists from the Poisonous Plant Laboratory chaired plenary and poster sessions, gave presentations, and were responsible for co-chairing the event. This forum is held every (3-4) years to discuss common poisonous plant problems throughout the world and the research that is currently being done to understand and mitigate these issues. Countries represented include: the United States, New Zealand, Australia, France, Brazil, and multiple provinces from China including Inner Mongolia, Tibet, and Shanxi. Accomplishments 01 Rangeland restoration to reduce losses to livestock from poisonous plants. ARS researchers at Logan, UT established seeding and demonstration plots using improved rangeland grasses and forbs to demonstrate that restoration of rangelands to a healthy ecosystem will reduce or prevent losses from poisonous plants. Replicated seeding plots and larger demonstration plots were established on the Channeled Scablands in eastern Washington State where lupine-induced �crooked calf syndrome� is endemic and occasional catastrophic losses occur. Evaluation of replicated plots and demonstration plots indicated that varieties of wheat grasses including Hycrest and Siberian, mixed with palatable forbs such as forage Kochia, will establish and persist in this harsh environment. Seeding plots have been evaluated over two years including biomass and nutrient value. This research is important to ranchers over the 2000 square mile rangeland called the Channeled Scablands and will improve forage quality, biomass and reduce losses from lupine-induced crooked calf syndrome. 02 Analysis of pyrrolizidine alkaloid (PA) in contaminated feeds. Pyrrolizidine alkaloid (PA)-containing plants have global distribution, and they often poison animals and humans by contaminating pastures, feeds, food, herbal products, and medicinal products. Toxicity is characterized by liver failure; several PAs are also carcinogenic. ARS scientists in Logan, UT developed in vitro and in vivo models of poisoning including a transgenic mouse model of carcinogenesis and confirmed that several PA are carcinogenic. This research explains a disease called �Unidentified Liver Disease� in poor populations in the northwest region of Tigray, Ethiopia and it is suspected that the disease is related to natural toxins in food. In cooperation with the Centers for Disease Control and Prevention, samples were collected and screened from the Tigray region of Ethiopia, including millet, teff, sorghum, maize, sesame, honey and an alcoholic drink called tella. Samples were analyzed by ARS scientists in Logan, UT for the presence of toxic PA�s which will cause liver disease. Because PA-containing plants are worldwide in distribution and impact, this research is important to national and international animal and human health organizations. 03 Poisoning in livestock by plants that accumulate selenium. Selenium is an essential micronutrient in the diet of animals and people. ARS researchers in Logan, UT, determined the blood profiles of various seleno-compounds in sheep and cattle. Pathology in poisoned animals was described and characterized based on the different types of selenium. The elimination of selenium following exposure to high Se levels in plants is slow requiring a relatively long period for selenium to clear from the animal�s system. Additionally, high levels of selenium in the diet of sheep suppressed reproductive performance. This research is important to assess risk on ranges and reclamation sites where soils are high in selenium. 04 Diagnosis of pine needle and juniper induced abortions. Sera samples from aborted fetuses and their damns were analyzed for metabolites of isocupressic acid (ICA), the abortifacient toxin in pine needles. In a number of cases diagnostic metabolites were detected and established cause of premature parturition or abortion. In particular ARS scientists in Logan, UT established that common juniper was the cause of plant induced abortion in the Rioja mountain region of Spain. This research is important to cattle producers in the western U.S. and Spain and identified the cause of cattle abortions from juniper. 05 Identification of swainsonine producing endophytes in Swainsona canescens. Legumes belonging to the Astragalus, Oxytropis and Swainsona genera cause livestock poisoning in the Americas (locoism), Asia (locoism) and Australia (peastruck). The toxin in S. canescens is swainsonine, and was shown to be produced by a fungus related to the Undifilum found in Astragalus and Oxytropis. The fungus cultured from S. canescens was shown to produce swainsonine in vitro and represents a new species of Undifilum and has significant importance to solving the problem of livestock poisoning in the U.S., Asia and Australia. 06 Genetic predisposition to larkspur poisoning in cattle. ARS Scientists at Logan, UT have conducted research on breed differences for susceptibility to larkspur poisoning. Five breeds have been tested thus far and while there are significant breed differences there are also differences between individuals within each breed. The number of larkspur sensitive animals may vary in each individual herd/breed due to the genetic background of that herd/breed. These differences are likely due to each individual animal�s genetic predisposition for larkspur poisoning which could potentially be predicted from its DNA sequence. We intend to exploit this variation between breeds and individuals within breeds to eventually provide a gene based method to select animals with resistance to larkspur poisoning and other neurological plant toxins to ultimately reduce or prevent cattle losses to larkspur. 07 Common Juniper trees contain the pine needle abortion toxin ICA. Surveys of western juniper trees across the state of Oregon demonstrated that western juniper trees will cause late-term abortions in cattle, similar to ponderosa pine trees. The risk is not well characterized and it is unclear how much variation there is in the abortifacient compounds in western juniper trees. Thus, ARS researchers in Logan, UT collected samples of bark, needles, and berries from western juniper trees from 35 locations across the state of Oregon in order to determine the variation in abortifacient compounds in western juniper trees. Results indicated that western juniper trees throughout the state of Oregon should be considered a risk to induce late-term abortions in cattle. This knowledge will allow livestock owners and others to better understand the potential abortion risk from western juniper trees. 08 Selenium (Se)-containing plants are reputed to be unpalatable to livestock. A study was conducted by ARS Scientists to determine if sheep and cattle could discriminate between forages and feeds with different concentrations of Se. Initially, freshly-harvested forages (intermediate wheatgrass, Thinopyrum intermedium; alfalfa, Medicago sativa; western aster, Symphyotrichum ascendens) with different Se concentrations were offered to cattle and sheep in preference trials. The Se concentrations ranged from 0.8 to 50 parts per million (ppm) in grass, 1.4 to 275 ppm in alfalfa, and 4 to 4,455 ppm in aster. The Se concentration had no influence on the initial or subsequent preferences of sheep or cattle for grass or alfalfa. Cattle developed an aversion to aster after consuming 95% of the offered plant material during the first brief exposure, and subsequently refused to eat any aster plants. Sheep consumption of aster was variable, but their preference was not driven by Se concentration. After initial exposure, cattle may have generalized an aversion based on shared flavors in the low- and high- Se asters. In the second trial, cattle and sheep were offered pellets containing various concentrations of Se. A trial with pellets was conducted using concentrations of 0, 5, 25, 45, 110 ppm Se. When given Se pellets, initial responses were variable, but animals adjusted their intake over time to allow for detoxification when over ingesting Se. A more thorough understanding of diet selection as a function of selenium concentration will provide information to land managers and stakeholders about the risk associated with selenium containing forage. 09 Understanding the metabolism and toxicity of pyrrolizidine alkaloids (PA�s) in fireweed. Fireweed (Senecio madagascariensis) is an invasive weed on the island of Hawaii that causes liver disease in cattle and results in large losses because of liver condemnation at the slaughter plants. ARS scientists from Logan, UT in collaboration with scientists from the U. of Hawaii isolated and characterized a mixture of PA�s from fireweed and determined how much ingested plant would result in toxicoses. Synthesis of PA adducts and their pyrrole metabolites were completed for future research. Detection and identification of PA metabolites in the liver of diseased animals is an important diagnostic tool for all diagnostic labs in the U.S. This research impacts the international scientific community also and resulted in collaborations between PPRL and the Institute for Risk Assessment in Germany to validate and screen for PA�s in other food products consumed by animals and people. 10 Identification of swainsonine-producing endophytes in other plant species. Some plant species within the Convolvulaceae (morning glory family) from South America, Africa, and Australia cause a neurologic disease in grazing livestock similar to locoweed poisoning in the western U.S. The morning glory plant, Ipomoea carnea, contains the toxins swainsonine and calystegines, inhibitors of key enzymes in protein metabolism. Swainsonine, the locoweed toxin, has been shown to be produced by a fungus that grows within the Astragalus and Oxytropis genera, and causes a neurologic disease in grazing livestock called locoism. Research by ARS scientists in Logan, Utah determined that the swainsonine producing fungus was detected in I. carnea by cell culture methods and PCR. It was also determined that plants derived from fungicide-treated seeds did not produce swainsonine. This research impacts livestock production systems in the western U.S. as well as large agricultural areas in South America, Africa and Australia. 11 Determining seed germination rates in seleniferous soils. Historical reclaimed mine sites contain high concentrations of selenium making it difficult to establish and maintain forages safe for grazing when they are reclaimed. Seeds of eleven different plant species were germinated in seleniferous soils containing 16, 35 and 70 ppm Se. ARS researchers in Logan, UT determined that there were no differences in germination rates of the different plant species in soils containing 16, 35 ppm Se. However, in soils containing 70 ppm Se there were differences as the mean rate of seedling emergence were highest in the intermediate wheat grass > Russian Wildrye > big bluegrass > Siberian wheat grass > prairie junegrass > mountain brome > western wheat grass> slender wheat grass > meadow brome > western aster > tufted hairgrass. This information will be helpful in determining forages that will establish in seleniferous soils commonly found on old phosphate mine sites and provide safe forage for livestock and wildlife. 12 White snakeroot and rayless goldenrod poisoning in livestock. White snakeroot (Ageratina altissima var. altissima) and rayless goldenrod (Isocoma pluriflora) are common North American plants that contain potent toxins that poison livestock and people. ARS scientists in Logan, UT identified and characterized these toxins as benzofuran ketones. Methods to detect low concentrations of these toxins in plants, feed, and animal tissues were developed. Research trials were completed to determine which chemical fractions contained the most bioactive compounds. This information will be used by livestock producers, veterinarians, diagnosticians, and human health scientists across the U.S. to monitor feeds and food for contamination, diagnose poisoning, and better predict the prognosis of poisoned animals. This research is essential to evaluate risk for livestock producers and veterinarians to identify and avoid exposure to toxic populations of white snakeroot and rayless goldenrod.

Impacts
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Publications

Lee, S.T., Green, B.T., Welch, K.D., Jordan, G.T., Zhang, Q., Panter, K.E., Hughes, D., Cheng-Wei, T.C., Pfister, J.A., Gardner, D.R. 2013. Stereoselective potencies and relative toxicities of y-Coniceine and N- Methylconiine enantiomers. Chemical Research in Toxicology. 23:616-21.
Lima, D.D., Albuquerque, R.F., Rocha, B.P., Barros, M.E., Gardner, D.R., Medeiros, R.M., Correa-Riet, F., Mendonca, F.S. 2013. Doen�a de dep�sito lisossomal induzida pelo consumo de Ipomoea verbascoidea (Convolvulaceae) em caprinos no semi�rido de Pernambuco. Pesquisa Veterinaria Brasileira. 33(7): 867-72.
Welch, K.D., Pfister, J.A., Gardner, D.R., Green, B.T., Panter, K.E. 2013. The role of the a7 subunit of the nicotinic acetylcholine receptor on motor coordination in mice treated with methyllcaconitine and anabasine. Journal of Applied Toxicology. 33(9):1017-26.
Cook, D., Beaulieu, W.T., Mott, I.W., Grum, D.S., Riet-Correa, F., Gardner, D.R., Pfister, J.A., Clay, J., Marcolongo-Pereira, C. 2013. Production of the alkaloid swainsonine by a fungal endosymbiont of the ascomycete order Chaetothyriales in the host Ipomoea carnea. Journal of Agricultural and Food Chemistry. 61: 3797-803.