Progress 02/11/08 to 02/10/13
Outputs
Progress Report Objectives (from AD-416): Objective I: Pine Needles 1.1 Determine if isocupressic acid (ICA; the abortifacient compound in pine needles) concentration in pine needles is modulated by the environment. 1.2 Identify the matabolites of isocupressic acid in pine needles that cause abortions in cattle. Determine the biological mechanism, develop diagnostic techniques, and therapeutic procedures. 1.3 Determine factors that influence cattle consumption of pine needles and develop management practices to prevent abortion. Objective II: Broom Snakeweed 2.1 Identify the toxic and abortifacient compounds in broom snakeweed. 2.2 Describe the ecology of broom snakeweed, develop management and control guidelines to reduce incidence of poisoning and abortion in livestock. Objective III: Lupine 3.1 Isolate, identify, and evaluate toxicity and teratogenicity of lupine alkaloids which cause birth defects in calves born to cows that graze these plants. 3.2 Evaluate the role of genotype and environment on lupine alkaloids, and thus the relative toxicity of various species and populations of lupine. 3.3 Determine the physiological mechanism of lupine-induced birth defects and evaluate the maternal and fetal toxicokinetics of alkaloids. 3.4 Evaluate the influence of climate on population cycles of lupine. 3.5 Determine the importance of lupines as nutritional components for cattle during critical times of the year. 3.6 Identify conditions under which cattle graze various lupine species. Objective IV: Veratrum 4.1 Develop models to study the toxicokinetics, including clearance times, and toxicity of steroidal alkaloids in Veratrum californicum. Approach (from AD-416): 1.1 Data on environmental conditions will be collected at each site using local weather stations. ICA levels and environmental conditions will be correlated to determine if any patterns emerge. Soil samples will be collected at each site for future evaluation. 1.2 Samples of maternal and fetal tissues will be collected for histologic analysis and determination of ICA concentrations using existing ELISA�s and GC/MS methods. Proteomic analyses via LC/MS/MS techniques will be done. 1.3 Pen and field studies using cattle in high, medium and low body condition will be done to determine effects on needle consumption and grazing times. Nutrient supplements will be offered to determine if pine needle consumption will be altered. 2.1 The diterpene acid �fingerprint� of broom snakeweed from various populations in Arizona, New Mexico and Utah will be determined by chemical analysis. Subsequent in vitro and in vivo studies will be done to determine abortifacient activity. 2.2 A grazing study will be conducted to determine if various management practices can be implemented to force cattle to graze snakeweed as a biological control. A clipping study will be conducted to further describe the effects of defoliation on snakeweed and the surrounding plant community. 3.1 Alkaloids will be isolated by chemical methods and identified by chromatography, NMR, mass spectrometry, and elemental analysis. Toxicology will be evaluated using a mouse bioassay and cell lines that express nicotinic acetylcholine receptors. 3.2 A chemical fingerprint of Lupinus sulphureus collected from different locations will be generated using chemical methods. Fingerprints will be analyzed via cluster analysis and phylogenetic analysis will be performed using AFLPs (Amplified Fragment Length Polymorphisms) to determine the genetic relationship of the populations. 3.3 Pregnant goats in late gestation will be used to determine the rate of absorption, distribution and elimination of the teratogenic alkaloids. The pharmacokinetic profiles of the alkaloids will be compared between maternal and fetal systems. 3.4 Established transects will be monitored over the next 5 years to determine the influence of weather patterns on lupine density. Correlations of lupine age, class, density, and trends will be made with seasonal precipitation and temperature. 3.5 Consumption of lupines by cattle on rangelands dominated by low quality forages may be related to nutrient content. Twelve yearling heifers in a field study will be supplemented with different levels of protein to compare lupine ingestion. 3.6 Short-duration and high intensity grazing studies in early, mid, and late summer will be used to determine what role grazing pressure has on lupine intake during different seasons of the year. 4.1 A monogastric model (swine) will be used to determine the kinetics (clearance and metabolism) of a well known teratogenic alkaloid from Veratrum. This pilot project will be a model for testing the clearance of other plant toxins from animal tissues to evaluate food safety of animal products. Clearance rates between the monogastric model and small ruminant model will be compared. This is the final report for project 5428-31320-005-00D. In collaboration with the Forage and Range Research Laboratory, Logan, UT, scientists at the Poisonous Plant Research Laboratory continued evaluations of replicated plots of various grass species and forage Kochia in Eastern Washington for forage production. Results from the first year of grazing studies on these plots with cattle were evaluated and a second year of grazing studies was conducted. A three year research project was initiated to determine whether the abortifacient compounds in western juniper trees change throughout the year, or from year to year. To this end needles, bark, and berries were collected bimonthly from western juniper trees at three locations across the state of Oregon. Additional work was performed to determine if metabolites of isocupressic acid detected in stomach and thoracic fluids from aborted calves can be used a diagnostic tool for identification of pine needle abortion cases. The replacement project is 5428-32630-012-00D. Accomplishments 01 A rodent model to identify teratogenic compounds in plants. Mimosa tenuiflora is a perennial tree or shrub native to Brazil, and is also distributed throughout tropical portions of South and Central America and Mexico. The plant causes deformed offspring (teratogenesis) when eaten by pregnant livestock, however, the toxin(s) is not known. Collaborative work by ARS scientists at Logan, UT was undertaken with scientists from Brazil to identify the teratogenic compounds using a rat model. Various purified compounds and mixtures of purified compounds were evaluated for their ability to cause fetal deformation and/or to inhibit fetal movement. A mixture of two purified compounds did cause birth defects; however, clear-cut teratogenic relationships were not established. Consequently, the mechanism by which Mimosa tenuiflora produces birth defects remains unknown. Identification of the compound(s) responsible for causing birth defects will allow for the quantitation of risk for specific plant populations so that better management recommendations can be provided to Brazilian livestock producers to prevent livestock losses. 02 The actions of piperidine and pyridine alkaloids on fetal movement in pregnant goats. Plants containing alkaloids that affect normal fetal development in livestock are found on US rangelands. The developing fetus can be poisoned when the pregnant female consumes plants which cause fetal deformities. The Nicotiana and Lupinus genera contain a variety of toxic piperidine and pyridine alkaloids. However, there is little information available on the actions of these alkaloids on fetal development. ARS scientists at Logan, Utah determined that the piperidine alkaloids anabasine and lobeline are more effective than the pyridine alkaloid myosmine at inhibiting fetal movement in a goat model. Unexpectedly, the pyridine alkaloid myosmine was more effective at inhibiting fetal movement than lobeline. These results suggest that the alkaloids tested are agonists at fetal muscle-type nicotinic receptors and that they have the potential to reduce or inhibit fetal movement in goats to cause fetal deformities. A more thorough understanding of the mechanism of action of the plant toxins will aid in the prevention and treatment of birth defects in livestock. 03 Characterization of the alkaloid profiles of seven Sophora and Dermatophyllum species. Species of Sophora are known to contain quinolizidine alkaloids that are toxic and potentially teratogenic. Recently several Sophora species have been reclassified as Dermatophyllum species. ARS scientists in Logan, Utah characterized the quinolizidine alkaloid composition in seven Sophora and Dermatophyllum species throughout their geographical distribution using field collections and herbarium specimens. Several species were found to contain quinolizidine alkaloids, including the teratogen anagyrine. None of the species contained the neurotoxin swainsonine as had been suggested. The results from these studies provide valuable information regarding the potential risk of Sophora and Dermatophyllum species to cause birth defects in livestock species. Characterization of the alkaloid profiles in these species provides valuable information to evaluate potential risks associated with these plants. This knowledge will result in management recommendations to help livestock producers to prevent livestock losses associated with these plants. Additionally, the chemotaxonomic data from these studies support the recent reclassification of the Dermatophyllum species. 04 A grazing strategy to reduce impact of lupine induced crooked calf syndrome. ARS scientists at Logan, Utah determined that intermittent grazing of lupine could potentially mitigate or substantially reduce the impact of lupine to cause crooked calf syndrome. Pregnant cows were fed lupine in a ten day on 5 day off regiment for 30 days and compared with cows fed lupine daily for 30 days. Those cows fed lupine for 30 days uninterrupted gave birth to severely deformed calves all of which were humanely euthanized. Those cows fed lupine on the intermittent regiment produced calves that were marginally affected i.e. slight contractures which resolved spontaneously or were normal. This method confirms that removing cattle from lupine exposure, even for a short period, will allow calves to regain normal fetal movement and will prevent the most severe contracture birth defects. This method has been implemented by some cattle ranchers in Washington State with great success allowing producers to utilize these productive high risk pastures and reduce the impact from lupine.
Impacts
(N/A)
Publications
- Green, B.T., Lee, S.T., Welch, K.D., Pfister, J.A., Panter, K.E. 2013. Fetal-muscle type nicotinic acetylcholine receptor activation in TE-671 cells, and inhibition of fetal movement in a day 40 pregnant goat model by optical isomers of the piperidine alkaloid coniine. Journal of Pharmacology and Experimental Therapeutics. 344:1-13.
- Green, B.T., Lee, S.T., Welch, K.D., Pfister, J.A., Panter, K.E. 2013. Piperidine, pyridine alkaloid inhibition of fetal movement in a day 40 pregnant goat model. Food and Chemical Toxicology. 58:8-13.
- Lee, S.T., Cook, D., Molyneux, R.J., Davis, T.Z., Gardner, D.R. 2013. Alkaloid profiles of Dermatophyllum arizonicum, Dermatophllum gypsophilum, Dermatophyllum secundiflorum, Styphnolobium affine, and Styphnolobium japonicum previously classified as Sophora species. Biochemical Systematics and Ecology. 49:87-93.
- Panter, K.E., Gay, C.C., Clinesmith, R., Platt, T.E. 2013. Management practices to reduce lupine-induced Crooked Calf Syndrome in the Northwest. Rangelands. 35(2): 12-6.
- Welch, K.D., Cook, D., Gardner, D.R., Parsons, C., Pfister, J.A., Panter, K.E. 2013. A comparison of the abortifacient risk of western juniper trees in Oregon. Rangelands. 35(1):40-4.
- Welch, K.D., Gardner, D.R., Pfister, J.A., Panter, K.E., Zieglar, J., Hall, J. 2012. A comparison of the metabolism of the abortifacient compounds from Ponderosa Pine needles in conditioned versus naive cattle. Journal of Animal Science. 90(12): 4611-7.
- Welch, K.D., Panter, K.E., Stegelmeier, B.L., Lee, S.T., Gardner, D.R., Cook, D. 2012. Veratrum-induced placental dysplasia in sheep. International Journal of Poisonous Plant Research. 2:54-62.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): Objective I: Pine Needles 1.1 Determine if isocupressic acid (ICA; the abortifacient compound in pine needles) concentration in pine needles is modulated by the environment. 1.2 Identify the matabolites of isocupressic acid in pine needles that cause abortions in cattle. Determine the biological mechanism, develop diagnostic techniques, and therapeutic procedures. 1.3 Determine factors that influence cattle consumption of pine needles and develop management practices to prevent abortion. Objective II: Broom Snakeweed 2.1 Identify the toxic and abortifacient compounds in broom snakeweed. 2.2 Describe the ecology of broom snakeweed, develop management and control guidelines to reduce incidence of poisoning and abortion in livestock. Objective III: Lupine 3.1 Isolate, identify, and evaluate toxicity and teratogenicity of lupine alkaloids which cause birth defects in calves born to cows that graze these plants. 3.2 Evaluate the role of genotype and environment on lupine alkaloids, and thus the relative toxicity of various species and populations of lupine. 3.3 Determine the physiological mechanism of lupine-induced birth defects and evaluate the maternal and fetal toxicokinetics of alkaloids. 3.4 Evaluate the influence of climate on population cycles of lupine. 3.5 Determine the importance of lupines as nutritional components for cattle during critical times of the year. 3.6 Identify conditions under which cattle graze various lupine species. Objective IV: Veratrum 4.1 Develop models to study the toxicokinetics, including clearance times, and toxicity of steroidal alkaloids in Veratrum californicum. Approach (from AD-416): 1.1 Data on environmental conditions will be collected at each site using local weather stations. ICA levels and environmental conditions will be correlated to determine if any patterns emerge. Soil samples will be collected at each site for future evaluation. 1.2 Samples of maternal and fetal tissues will be collected for histologic analysis and determination of ICA concentrations using existing ELISA�s and GC/MS methods. Proteomic analyses via LC/MS/MS techniques will be done. 1.3 Pen and field studies using cattle in high, medium and low body condition will be done to determine effects on needle consumption and grazing times. Nutrient supplements will be offered to determine if pine needle consumption will be altered. 2.1 The diterpene acid �fingerprint� of broom snakeweed from various populations in Arizona, New Mexico and Utah will be determined by chemical analysis. Subsequent in vitro and in vivo studies will be done to determine abortifacient activity. 2.2 A grazing study will be conducted to determine if various management practices can be implemented to force cattle to graze snakeweed as a biological control. A clipping study will be conducted to further describe the effects of defoliation on snakeweed and the surrounding plant community. 3.1 Alkaloids will be isolated by chemical methods and identified by chromatography, NMR, mass spectrometry, and elemental analysis. Toxicology will be evaluated using a mouse bioassay and cell lines that express nicotinic acetylcholine receptors. 3.2 A chemical fingerprint of Lupinus sulphureus collected from different locations will be generated using chemical methods. Fingerprints will be analyzed via cluster analysis and phylogenetic analysis will be performed using AFLPs (Amplified Fragment Length Polymorphisms) to determine the genetic relationship of the populations. 3.3 Pregnant goats in late gestation will be used to determine the rate of absorption, distribution and elimination of the teratogenic alkaloids. The pharmacokinetic profiles of the alkaloids will be compared between maternal and fetal systems. 3.4 Established transects will be monitored over the next 5 years to determine the influence of weather patterns on lupine density. Correlations of lupine age, class, density, and trends will be made with seasonal precipitation and temperature. 3.5 Consumption of lupines by cattle on rangelands dominated by low quality forages may be related to nutrient content. Twelve yearling heifers in a field study will be supplemented with different levels of protein to compare lupine ingestion. 3.6 Short-duration and high intensity grazing studies in early, mid, and late summer will be used to determine what role grazing pressure has on lupine intake during different seasons of the year. 4.1 A monogastric model (swine) will be used to determine the kinetics (clearance and metabolism) of a well known teratogenic alkaloid from Veratrum. This pilot project will be a model for testing the clearance of other plant toxins from animal tissues to evaluate food safety of animal products. Clearance rates between the monogastric model and small ruminant model will be compared. In collaboration with the Forage and Range Research Laboratory, Logan, UT, scientists at the Poisonous Plant Research Laboratory evaluated replicated plots of various grass species and forage Kochia in Eastern Washington for forage production. Additionally, grazing studies on these plots with cattle were initiated. Chemical analysis of needles, bark, and berries from western juniper trees from 35 locations across the state of Oregon were performed in order to evaluate the risk of western juniper trees to cause late-term abortions in cattle. Metabolites of isocupressic acid were detected in stomach and thoracic fluids from aborted calves. Detection of the metabolites in the fluids from aborted calves will be an important diagnostic tool useful for identification of pine needle abortion cases. Cell culture-based experiments are being used to compare piperidine and quinolizidine alkaloids from poisonous plants to help identify, and further define, the mechanism by which these compounds induce birth defects in livestock species. Additionally, a rat model is being used to characterize the potential of purified plant toxins, including enantiomers of the toxins, to cause birth defects. Accomplishments 01 Method to diagnose field cases of pine-needle induced abortions in cattl In a cooperative effort with the Veterinary Diagnostic Laboratory, Iowa State University, ARS researchers in Logan, UT, examined bovine fetal fluids, including stomach and thoracic fluids, from possible pine needle abortion cases. Tetrahydroagathic acid, a known metabolite of isocupress acid (the abortifacient compound in pine needles), was detected in the aborted bovine fetal fluids from submitted diagnostic cases. Detection o tetrahydroagathic acid could prove to be a valuable diagnostic tool to identify cases of pine-needle induced abortions in cattle. Detection of serum metabolites in mother cows also continues to be a useful diagnosti tool and was used to support pine needle diagnosis in cases from California and Wyoming. 02 Survey of western juniper trees across the state of Oregon. Previous research by ARS researchers in Logan, UT, has demonstrated that western juniper trees can induce late-term abortions in cattle, similar to ponderosa pine trees. However, this risk is not well characterized and i is unclear how much variation there is in the abortifacient compounds in western juniper trees. A previous report has shown that there can be significant variation in the abortifacient compounds in ponderosa pine needles from location to location and over time. Thus, ARS researchers i 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 bette understand the potential abortion risk from western juniper trees. 03 Metabolism of abortifacient compounds from ponderosa pine needles in cattle. Ponderosa pine needles are known to induce abortions in cows whe consumed during the last trimester of pregnancy. The known toxins in the needles that induce abortions in cattle are isocupressic acid and agathi acid, a metabolite of isocupressic acid. Previous research at the Poisonous Plants Research Lab, Logan UT suggested that extended exposure of cattle to pine needles resulted in altered metabolism and/or elimination of the abortifacient compounds. ARS researchers in Logan, UT demonstrated that cattle conditioned to ponderosa pine needles can more efficiently metabolize the abortifacient compound agathic acid than na�v cattle. These results suggest that extended exposure of cattle to pine needles results in a physiological change in the cattle such that the known abortifacient compounds in ponderosa pine needles are metabolized more quickly. 04 An analytical method to measure cyclopamine in biological samples. Cyclopamine, a toxin in the plant Veratrum californicum, has been found be active against several types of cancer. Thus, cyclopamine, and cyclopamine derivatives, have been targeted as potential pharmaceutical treatments for certain cancers. A monoclonal antibody-based enzyme-linke immunosorbent assay (ELISA) was developed by ARS researchers in Logan, U to detect and measure cyclopamine and cyclopamine derivatives in biological samples. The assay was found to be very sensitive, and it was also found to be useful for the detection and measurement of cyclopamine in samples from mice that had been dosed with cyclopamine. The simple ELISA method could be used for the rapid screening of biological samples for the presence, and concentration, of cyclopamine and other cyclopamin derivatives with anticancer potential. 05 Mechanism of poison hemlock induced birth defects. Piperidine alkaloids, present in poison hemlock, cause deformities in developing animals by inhibiting nicotinic acetylcholine receptors (nAChRs) in the developing fetus. It has been hypothesized that piperidine alkaloids act at the fet muscle type nAChRs, but direct evidence is lacking. A recent study by AR scientists in Logan, UT used piperidine alkaloids in a cell culture mode to characterize receptor binding, and a goat model to evaluate fetal movement. The study established that coniine, a piperidine alkaloid, do act at the fetal muscle-type nAChR receptor to inhibit fetal movement, which in turn can result in fetal deformities. A more thorough understanding of the mechanism of action of the plant toxins will aid in the prevention and treatment of birth defects in livestock. 06 A rodent model to identify teratogenic compounds in plants. Mimosa tenuiflora is a perennial tree or shrub native to Brazil, and is also distributed throughout tropical portions of South and Central America an Mexico. The plant causes deformed offspring (teratogenesis) when eaten b pregnant livestock, however, the toxin(s) is not known. Collaborative wo by ARS scientists at Logan, UT was undertaken with scientists from Brazi to identify the teratogenic compounds using a rat model. Various extract and purified compounds from Mimosa were evaluated for their ability to cause fetal deformation and/or to inhibit fetal movement. Although extracts and purified compounds did cause birth defects, clear-cut teratogenic relationships were not established. Consequently, the mechanism by which Mimosa tenuiflora produces birth defects remains unknown. Identification of the compound(s) responsible for causing birth defects will allow for the quantitation of risk for specific plant populations so that better management recommendations can be provided to Brazilian livestock producers to prevent livestock losses.
Impacts
(N/A)
Publications
- Cook, D., Lee, S.T., Pfister, J.A., Stonecipher, C.A., Welch, K.D., Green, B.T., Panter, K.E. 2011. Alkaloid profiling as an approach to differentiate Lupinus garfieldensis, Lupinus sabinianus, and Lupinus sericeus. Phytochemical Analysis. 23(3): 278-84.
- Gardner, D.R., Riet-Correa, F., Panter, K.E. 2011. Alkaloid profiles of Mimosa tenuiflora and associated methods of analysis. In: Riet-Correa, F., Pfister, J., Schild, A.L., Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge, MA: CAB International. 103:600- 5.
- Green, B.T., Lee, S.T., Panter, K.E., Brown, D.R. 2012. Piperidine alkaloids: Human and food animal teratogens. Food and Chemical Toxicology. 50(6): 2049-55.
- Lee, S.T., Panter, K.E., Gardner, D.R., Green, B.T., Welch, K.D., Zhang, J. , Change, C.T. 2012. Development of a monoclonal antibody-based ELISA for the hedgehog inhibitors cyclopamine and cyclopamine-KAAD. Journal of Pharmaceutical and Biomedical Analysis. 66: 282-6.
- Panter, K.E., Stegelmeier, B.L. 2011. Effects of xenobiotics and phytotoxins on reproduction in food animals. Veterinary Clinics of North America. 27(2):429-46.
- Ralphs, M.H., Motteram, E.S., Panter, K.E. 2011. Velvet lupine (Lupinus leucophyllis) population cycles with climate. Western North American Naturalist. 71(3): 396-403.
- Welch, K.D., Gardner, D.R., Panter, K.E., Stegelmeier, B.L., Parsons, C., Pfister, J.A., Cook, D. 2011. Western juniper-induced abortions in beef cattle. International Journal of Poisonous Plant Research. 1(1):72-9.
- Welch, K.D., Panter, K.E., Gardner, D.R., Stegelmeier, B.L. 2012. The good and the bad of poisonous plants: An introduction to the USDA-ARS Poisonous Plant Research Laboratory. Journal of Medical Toxicology. 8(2): 153-9.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Objective I: Pine Needles 1.1 Determine if isocupressic acid (ICA; the abortifacient compound in pine needles) concentration in pine needles is modulated by the environment. 1.2 Identify the matabolites of isocupressic acid in pine needles that cause abortions in cattle. Determine the biological mechanism, develop diagnostic techniques, and therapeutic procedures. 1.3 Determine factors that influence cattle consumption of pine needles and develop management practices to prevent abortion. Objective II: Broom Snakeweed 2.1 Identify the toxic and abortifacient compounds in broom snakeweed. 2.2 Describe the ecology of broom snakeweed, develop management and control guidelines to reduce incidence of poisoning and abortion in livestock. Objective III: Lupine 3.1 Isolate, identify, and evaluate toxicity and teratogenicity of lupine alkaloids which cause birth defects in calves born to cows that graze these plants. 3.2 Evaluate the role of genotype and environment on lupine alkaloids, and thus the relative toxicity of various species and populations of lupine. 3.3 Determine the physiological mechanism of lupine-induced birth defects and evaluate the maternal and fetal toxicokinetics of alkaloids. 3.4 Evaluate the influence of climate on population cycles of lupine. 3.5 Determine the importance of lupines as nutritional components for cattle during critical times of the year. 3.6 Identify conditions under which cattle graze various lupine species. Objective IV: Veratrum 4.1 Develop models to study the toxicokinetics, including clearance times, and toxicity of steroidal alkaloids in Veratrum californicum. Approach (from AD-416) 1.1 Data on environmental conditions will be collected at each site using local weather stations. ICA levels and environmental conditions will be correlated to determine if any patterns emerge. Soil samples will be collected at each site for future evaluation. 1.2 Samples of maternal and fetal tissues will be collected for histologic analysis and determination of ICA concentrations using existing ELISA�s and GC/MS methods. Proteomic analyses via LC/MS/MS techniques will be done. 1.3 Pen and field studies using cattle in high, medium and low body condition will be done to determine effects on needle consumption and grazing times. Nutrient supplements will be offered to determine if pine needle consumption will be altered. 2.1 The diterpene acid �fingerprint� of broom snakeweed from various populations in Arizona, New Mexico and Utah will be determined by chemical analysis. Subsequent in vitro and in vivo studies will be done to determine abortifacient activity. 2.2 A grazing study will be conducted to determine if various management practices can be implemented to force cattle to graze snakeweed as a biological control. A clipping study will be conducted to further describe the effects of defoliation on snakeweed and the surrounding plant community. 3.1 Alkaloids will be isolated by chemical methods and identified by chromatography, NMR, mass spectrometry, and elemental analysis. Toxicology will be evaluated using a mouse bioassay and cell lines that express nicotinic acetylcholine receptors. 3.2 A chemical fingerprint of Lupinus sulphureus collected from different locations will be generated using chemical methods. Fingerprints will be analyzed via cluster analysis and phylogenetic analysis will be performed using AFLPs (Amplified Fragment Length Polymorphisms) to determine the genetic relationship of the populations. 3.3 Pregnant goats in late gestation will be used to determine the rate of absorption, distribution and elimination of the teratogenic alkaloids. The pharmacokinetic profiles of the alkaloids will be compared between maternal and fetal systems. 3.4 Established transects will be monitored over the next 5 years to determine the influence of weather patterns on lupine density. Correlations of lupine age, class, density, and trends will be made with seasonal precipitation and temperature. 3.5 Consumption of lupines by cattle on rangelands dominated by low quality forages may be related to nutrient content. Twelve yearling heifers in a field study will be supplemented with different levels of protein to compare lupine ingestion. 3.6 Short-duration and high intensity grazing studies in early, mid, and late summer will be used to determine what role grazing pressure has on lupine intake during different seasons of the year. 4.1 A monogastric model (swine) will be used to determine the kinetics (clearance and metabolism) of a well known teratogenic alkaloid from Veratrum. This pilot project will be a model for testing the clearance of other plant toxins from animal tissues to evaluate food safety of animal products. Clearance rates between the monogastric model and small ruminant model will be compared. In collaboration with FRRL, Logan, UT., replicated seeding plots were established in 2010 in Eastern Washington using various grass species (including improved and native germplasm) and 3 species of forage Kochia. Additionally, larger demonstration plots were seeded with a mix of crested wheat grass and Emigrant forage Kochia. The hypothesis is that crested wheat grass and forage Kochia will provide alternative forages at the critical time when pregnant cattle begin to eat lupine (July- September). Seeding plots were established on four different ranches on the Scablands and early evaluation demonstrated good to excellent germination on all plots. Further evaluations will occur over the next 5 years, and grazing studies will be done on the larger demonstration plots once the grasses and Kochia become established. The plots will be evaluated in the fall of 2011 and then each spring for the next 5 years. Chemical analysis of needles and bark from various types of pine and juniper trees continues in order to fully understand the abortion risks when late term cattle graze in areas where these types of trees are abundant. Cell culture-based experiments are being used to compare piperidine and quinolizidine alkaloids to help identify the mechanism by which these compounds induce birth defects in livestock species. Current structure-function studies are underway using compounds selective for specific receptor binding sites on the fetal muscle-type receptors comparing alkaloids which do not cause fetal malformations versus alkaloids that do. Initial results suggest that there are differences between the two classes of alkaloids, which could help to determine the molecular mechanism of teratogenesis. These results have led to the generation of the hypothesis that the mechanism behind these types of birth defects is the inhibition of fetal movement due to stimulation followed by desensitization of the receptors. A preliminary chemical analysis of leaves and seeds from Mimosa tenuiflora was performed. An initial evaluation of the teratogenic potential of leaves and seeds, as well as extracts of both, has been made using a rat model in order to identify the teratogenic compound in Mimosa tenuiflora. Significant Activities that Support Special Target Populations Plant samples were analyzed for metabolites of isocupressic acid, which is the abortifacient toxin in pine needles, for ranchers in Wyoming and Oregon as well as a rancher in Northern Spain. This analysis provides the customer with personalized management recommendations regarding grazing. Workshops and presentations targeting rural communities were made to livestock producers, small farmers, and federal agencies. These outreach events focused on the poisoning that occurs via pine needles and lupine ingestion in livestock. Accomplishments 01 Evaluation of intermittent grazing as a method to minimize lupine-induce birth defects. Lupines continue to cause large economic losses to the cattle industry in the northwest. ARS researchers in Logan, UT, analyzed alkaloid in blood from pregnant cows fed lupine plants intermittently during gestation days 30-60 demonstrated that the teratogenic alkaloids clear from the maternal circulation within 48 hours. Fetal movement returned to normal activity shortly thereafter. This suggests that intermittent grazing may be a useful management tool in preventing or reducing the losses from lupine-induced �Crooked Calf Syndrome�. As a follow up study, 15 pregnant black Angus cattle were divided into 3 treatment groups: 1) control; 2) continuous treatment and 3) intermitten treatment. The control cows delivered normal calves. The cows fed lupine continuously from gestation days 30-60 delivered calves with moderate to severe birth defects, while those fed lupine intermittently (10 days on and 5 days off) delivered calves with few minor skeletal defects, and these resolved completely within 4 weeks after birth. These results demonstrate the potential of using an intermittent grazing program to reduce lupine-induced birth defects on ranches where lupine continues to cause economic losses. 02 Grazing trials of lupine throughout the growing season. ARS researchers Logan, UT, conducted intensive grazing trials with cattle in eastern Washington to evaluate the consumption of lupine in different stages of plant development through the growing season. Ten Hereford cows and a bu grazed velvet lupine-infested rangeland in 3 grazing pressure trials eac year over two grazing seasons. Pastures were fenced to limit forage availability to the amount required for the trials. In June, cheatgrass was dry and cattle preferred forbs that were flowering. As availability palatable forbs declined, cows started eating lupine, and this declined its availability declined. In July when annual forbs matured and became fibrous and unpalatable, cattle ate lupine at the beginning of the trial and reverted to dry cheatgrass as lupine became limited. We conclude tha lupine is not palatable early in the growing season in May and early Jun but cattle can be forced to graze it as availability of green cheatgrass and other forbs declines. Intensive grazing systems that force cattle to use all forage may enhance the risk of crooked calf syndrome by forcing cattle to graze lupine throughout the grazing season. Therefore, stockin rates and management programs should be flexible and adjusted to avoid forcing cattle to graze lupines. 03 Management recommendations to minimize the impact of broom snakeweed on plant communities. Broom snakeweed is an invasive native sub-shrub that distributed widely across rangelands of western North America. It often increases to near monocultures following disturbance from overgrazing, fire, or drought. The greatest ecological concern is that broom snakewee displaces desirable forage for livestock or wildlife and greatly reduces biodiversity. Although its populations cycle with climatic patterns, it can be a major factor impeding succession of native or desirable plant communities. It also is toxic and can cause abortions in all species of livestock. Population increases of broom snakeweed are usually higher in wet years, allowing large expanses of even-aged stands to establish and dominate plant communities. ARS researchers in Logan, UT, demonstrated that snakeweed can be controlled by prescribed burning, targeted grazing or spraying with herbicides. Follow up research demonstrated that establishment and maintaining competitive grasses through controlled grazing management can prevent or minimize broom snakeweeds� reinvasion, improve grazing conditions and support critical biodiversity. 04 Toxic alkaloid profiles of poisonous Lupinus species. Many species in th Lupinus genus are poorly defined morphologically, potentially resulting improper taxonomic identification. Correct identification is important because several, but not all, lupine species contain alkaloids that can acutely toxic and/or impair physiological development leading to birth defects in livestock, such as lupine-induced crooked calf syndrome. Consequently, a better method to correctly identify the poisonous Lupinu species is needed. ARS researchers in Logan, UT, characterized the alkaloid profiles of four Lupinus species to aid in discriminating these species from each other. They discovered diagnostic chemical fingerprint These fingerprints can be used as a tool to discriminate these species from each other, as long as one considers locality of the collection. These results will provide livestock producers with valuable information to help reduce economic losses that occur as a result of lupine-induced crooked calf syndrome. 05 Evaluation of western juniper-induced abortions in cattle. Ranchers in Baker County, Oregon have reported atypical late term abortions of 10-15 of their herds. Ponderosa pine and other trees known to cause abortions cattle were not found in the areas in which these abortions occurred. However, there was clear visual evidence that the cattle had been eating the bark of western juniper trees (Juniperus occidentalis). ARS researchers in Logan, UT, analyzed samples of western juniper needles, berries, and bark for labdane acids (the compounds in pine needles that cause abortions in cattle). Analyses indicated that the bark of western juniper trees had a fairly high concentration of labdane acids. Consequently, studies were performed by ARS researchers in Logan, UT, to determine if the bark from western juniper trees could induce abortions cattle. Two cows aborted 4-5 days after the start of the treatment. Both cows had clinical signs consistent with abortion. The remaining 4 cows calved at full term (26-31 days after the start of treatment) and had no clinical signs of abortion. Results from this study indicated that weste juniper trees contain compounds known to cause abortions in cattle and thus consumption of large amounts of bark in the third trimester of gestation may induce abortions. Although the risk of abortion from eatin western juniper bark appears to be less than that of ponderosa pine needles, livestock producers should be aware of this potential. 06 Evaluation of the placental development in ewes with malformed lambs. Cyclopia and a number of other birth defects occur in lambs from ewes th graze Veratrum californicum early in gestation. The mechanism of cyclopamine-induced birth defects has been shown to result from the inhibition of an important signaling pathway. This pathway plays an integral role in cell growth and differentiation. Previous studies by AR researchers in Logan, UT, have demonstrated that lambs with cyclopia wer smaller, under developed, and appeared premature compared to normal lamb of similar gestation age. Preliminary observations suggested this was du to deficiencies in placental development. Consequently, studies were performed by ARS researchers in Logan, UT, to determine if there are deficiencies in placental development in ewes with lambs with cyclopia a other less severe head and face malformations. The results from these studies confirmed that cyclopic lambs are smaller than normal lambs as well as less severely malformed lambs. Additionally, they demonstrated that placental development in ewes with cyclopic lambs is compromised. D to the lack of brain and pituitary gland development in many of the cyclopic lambs, it is quite likely that the lack of normal placental development in ewes with cyclopic lambs is a result of insufficient contribution of the embryo during critical periods of placental development. This information will be useful in further understanding h Veratrum californicum can alter the reproductive capabilities of livesto
Impacts
(N/A)
Publications
- Faeth, S.H., Hayes, C.J., Gardner, D.R. 2010. Asexual endophytes in a native grass: Tradeoffs in mortality, growth, reproduction, and alkaloid production. Microbial Ecology. 60:496-504. DOI: 10.1007/s00248-010-9643-4.
- Ralphs, M.H., Pfister, J.A., Panter, K.E., Lee, S.T., Motteram, E.S. 2011. Influence of grazing pressure on cattle consumption of the teratogenic plant velvet lupine. Professional Animal Scientist. 27(2):101-8.
- Ralphs, M.H., Mcdaniel, K. 2011. Broom snakeweed (Gutierrezia sarothrae): Toxicology, ecology, control, and management. Invasive Plant Science and Management. 4(1):125-32.
- Cook, D., Gardner, D.R., Pfister, J.A., Panter, K.E., Stegelmeier, B.L., Lee, S.T., Welch, K.D., Green, B.T., Davis, T.Z. 2010. Differences in ponderosa pine isocupressic acid concentrations across space and time. Rangelands. 32(2):14-7.
- Cook, D., Lee, S.T., Gardner, D.R., Pfister, J.A., Welch, K.D., Green, B.T. , Davis, T.Z., Panter, K.E. 2011. Lupine-Induced 'Crooked Calf Disease' in Washington and Oregon: Identification of the alkaloid profiles of Lupinus sericeus, Lupinus sulphureus and Lupinus leucophyllus. In: Riet-Correa, F., Pfister, J., Schild, A.L., Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge, MA. CAB International. 97:566- 71.
- Green, B.T., Lee, S.T., Welch, K.D., Panter, K.E., Kem, W. 2011. Determination of the relative toxicity of enantiomers with cell-based assays. In: Riet-Correa, F., Pfister, J., Schild, A.L., Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge, MA. CAB International. 100:581-7.
- Panter, K.E., Gardner, D.R., Stegelmeier, B.L., Welch, K.D., Holstege, D. 2011. Water hemlock poisoning in cattle: Ingestion of immature Cicuta maculata seed as the probable cause. Toxicon. 57(1):157-61. DOI:10.1016/j. toxicon.2010.11.009
- Panter, K.E., Welch, K.D., Lee, S.T., Gardner, D.R., Stegelmeier, B.L., Ralphs, M.H., Davis, T.Z., Green, B.T., Pfister, J.A., Cook, D. 2011. Plants teratogenic to livestock in the United States. In: Riet-Correa, F., Pfister, J., Schild, A.L., Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge, MA. CAB International. 36:236- 42.
- Ralphs, M.H., Waldron, B.L., Panter, K.E. 2011. Cattle preference for forage kochia, crested wheatgrass, and velvet lupine. Professional Animal Scientist. 27:215-218.
- Welch, K.D., Lee, S.T., Gardner, D.R., Panter, K.E., Stegelmeier, B.L., Cook, D. 2011. Dose-Response evaluation of Veratrum californicum in sheep. In: Riet-Correa, F., Pfister, J., Schild, A.L., Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge, MA. CAB International. 37:243-50.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Objective I: Pine Needles 1.1 Determine if isocupressic acid (ICA; the abortifacient compound in pine needles) concentration in pine needles is modulated by the environment. 1.2 Identify the matabolites of isocupressic acid in pine needles that cause abortions in cattle. Determine the biological mechanism, develop diagnostic techniques, and therapeutic procedures. 1.3 Determine factors that influence cattle consumption of pine needles and develop management practices to prevent abortion. Objective II: Broom Snakeweed 2.1 Identify the toxic and abortifacient compounds in broom snakeweed. 2.2 Describe the ecology of broom snakeweed, develop management and control guidelines to reduce incidence of poisoning and abortion in livestock. Objective III: Lupine 3.1 Isolate, identify, and evaluate toxicity and teratogenicity of lupine alkaloids which cause birth defects in calves born to cows that graze these plants. 3.2 Evaluate the role of genotype and environment on lupine alkaloids, and thus the relative toxicity of various species and populations of lupine. 3.3 Determine the physiological mechanism of lupine-induced birth defects and evaluate the maternal and fetal toxicokinetics of alkaloids. 3.4 Evaluate the influence of climate on population cycles of lupine. 3.5 Determine the importance of lupines as nutritional components for cattle during critical times of the year. 3.6 Identify conditions under which cattle graze various lupine species. Objective IV: Veratrum 4.1 Develop models to study the toxicokinetics, including clearance times, and toxicity of steroidal alkaloids in Veratrum californicum. Approach (from AD-416) 1.1 Data on environmental conditions will be collected at each site using local weather stations. ICA levels and environmental conditions will be correlated to determine if any patterns emerge. Soil samples will be collected at each site for future evaluation. 1.2 Samples of maternal and fetal tissues will be collected for histologic analysis and determination of ICA concentrations using existing ELISA�s and GC/MS methods. Proteomic analyses via LC/MS/MS techniques will be done. 1.3 Pen and field studies using cattle in high, medium and low body condition will be done to determine effects on needle consumption and grazing times. Nutrient supplements will be offered to determine if pine needle consumption will be altered. 2.1 The diterpene acid �fingerprint� of broom snakeweed from various populations in Arizona, New Mexico and Utah will be determined by chemical analysis. Subsequent in vitro and in vivo studies will be done to determine abortifacient activity. 2.2 A grazing study will be conducted to determine if various management practices can be implemented to force cattle to graze snakeweed as a biological control. A clipping study will be conducted to further describe the effects of defoliation on snakeweed and the surrounding plant community. 3.1 Alkaloids will be isolated by chemical methods and identified by chromatography, NMR, mass spectrometry, and elemental analysis. Toxicology will be evaluated using a mouse bioassay and cell lines that express nicotinic acetylcholine receptors. 3.2 A chemical fingerprint of Lupinus sulphureus collected from different locations will be generated using chemical methods. Fingerprints will be analyzed via cluster analysis and phylogenetic analysis will be performed using AFLPs (Amplified Fragment Length Polymorphisms) to determine the genetic relationship of the populations. 3.3 Pregnant goats in late gestation will be used to determine the rate of absorption, distribution and elimination of the teratogenic alkaloids. The pharmacokinetic profiles of the alkaloids will be compared between maternal and fetal systems. 3.4 Established transects will be monitored over the next 5 years to determine the influence of weather patterns on lupine density. Correlations of lupine age, class, density, and trends will be made with seasonal precipitation and temperature. 3.5 Consumption of lupines by cattle on rangelands dominated by low quality forages may be related to nutrient content. Twelve yearling heifers in a field study will be supplemented with different levels of protein to compare lupine ingestion. 3.6 Short-duration and high intensity grazing studies in early, mid, and late summer will be used to determine what role grazing pressure has on lupine intake during different seasons of the year. 4.1 A monogastric model (swine) will be used to determine the kinetics (clearance and metabolism) of a well known teratogenic alkaloid from Veratrum. This pilot project will be a model for testing the clearance of other plant toxins from animal tissues to evaluate food safety of animal products. Clearance rates between the monogastric model and small ruminant model will be compared. Several herbicides were applied to snakeweed at Monticello and Nephi, UT in May and August 2008. Efficacy was evaluated in June 2009 and vegetation response and production was evaluated in 2010. Transects were read in 2009. Eight years of data were summarized and combined with WSU data and correlations were made with seasonal precipitation. Herbicides were evaluated for the control of broom snakeweed, one of the most ubiquitous range weeds in North America. Tordon was the most effective herbicide when applied in the spring and fall. Grazon, Milestone, and Escort were only acceptable when applied in the fall. The concentration of isocupressic acid, the abortifacient compound, in Ponderosa Pine was evaluated throughout the year and at different locations. Isocupressic acid concentrations were shown to vary between locations and between months at some locations. Lupine-infested rangelands are typically of poor nutritional quality during summer months when pregnant cattle most often consume the toxic lupine. Lupine, however, is a high nitrogen feed, and cattle may select lupine to meet their nutritional needs in late summer and during gestation. The consumption of lupine by cattle when supplemented with a high nitrogen feed was evaluated. In these studies the supplementation of nitrogen in the diet did not reduce lupine consumption by pregnant cattle. The influence of climate on population cycles of lupine was evaluated. Lupine populations increase in wet years and die off in drought. Significant Activities that Support Special Target Populations Eastern Washington Cattlemen meeting 1 March 2010. 50 ranchers and land management agency personnel, Ritzville WA. Targeted to small farmers and ranchers. Ranchers will be able to predict lupine population outbreaks and manage cattle to reduce the risk of crooked calves. Accomplishments 01 Broom snakeweed is one of the most ubiquitous range weeds in North Ameri Herbicides were evaluated for its control in the 1970�s but new rangeland herbicides have been developed. ARS scientists at Logan, Utah, compared new herbicides with Tordon to evaluate their efficacy. Tordon w most effective when applied in either spring or fall, while Grazon, Milestone and Escort were acceptable when applied in fall. They also discovered that grass cover and production were greatest in Tordon plots and were correlated with the degree of snakeweed control. These results were important for making informed recommendations for snakeweed control to ranchers and land managers. 02 Evaluate the influence of climate on population cycles of lupine. A catastrophic outbreak of crooked calves occurred in 1997 following 2 extremely wet years, and there appears to be correlation with increased lupine populations and wet years. Since 1996, extended drought condition have caused most lupine populations to die out. Relationships discovered by ARS scientists at Logan, Utah between climatic patterns and lupine populations will enable ranchers and land managers to predict lupine outbreaks and take alternative management measures to avoid crooked calv 03 Evaluate the concentrations of isocupressic acid (ICA) in Ponderosa Pine through the year. Ponderosa Pine (Pinus ponderosa) is distributed throughout the western half of North America, where it is the most widel adapted and ubiquitous conifer. Ponderosa Pine contains ICA, a diterpene acid, which has been shown to be responsible for its abortifacient activity. The objective of this study was to establish a sampling protoc for pine needles and to determine if ICA concentrations change as a function of the environment or if there is location-to-location variatio in ICA content. ARS scientists at Logan, Utah established a sampling protocol for pine needles. They determined that ICA concentrations are n uniform throughout an individual tree. Consequently collecting a composi sample from a tree is most representative of a tree�s ICA content. Additionally, ICA concentrations were shown to vary between locations an between months at some locations. This work may explain partially why th incidence of pine needle abortion may be greater at certain locations. Furthermore, this work provides a foundation for further research to hel understand how ICA concentrations vary between location and time, which may be used to help refine management recommendations. 04 Nitrogen supplementation of cattle grazing lupine-infested rangelands. Lupine infested rangelands are typically of poor nutritional quality during summer months when pregnant cattle most often consume the toxic lupine. Lupine, however, is a high nitrogen feed, and cattle may select lupine to meet their nutritional needs during gestation. ARS scientists Logan, Utah evaluated the consumption of lupine by cattle when supplemented with a high N feed. Three replicates of two treatments (supplemented and not) were used in a grazing study in eastern Washingto with 12 pregnant cows. Cattle grazed on a lupine-infested pasture for 3 weeks during 50-70 days of gestation. Cattle ate considerable lupine during the trials, but there were no differences between the treated and untreated animals. This research indicated that nitrogen supplement did not ameliorate lupine consumption by pregnant cattle. Findings from thes studies will be transferred to cattle producers for eventual application in refining solutions to lupine poisoning in cattle. 05 Effects of teratogenic plants on early embryonic survival. ARS scientist at Logan, Utah treated pregnant Spanish goats with Nicotiana glauca duri various stages of pregnancy (0.10; 10-20; 20-30 and 30-40) to evaluate t question, does lupine ingestion in cows during early gestation cause embryonic loss? Preliminary data, using a goat model suggests that these teratogenic alkaloids do not cause early embryonic loss nor do they caus birth defects when ingested during these early gestational periods. Results from this study will be implemented in grazing recommendations t reduce losses from lupine-induced crooked calf syndrome. 06 Intermittent feeding of teratogenic plants to goats. Intermittent grazin has been suggested as a possible management tool to mitigate the losses from lupine-induced crooked calf syndrome in the northwest. To test this hypothesis, ARS scientists at Logan, Utah fed pregnant Spanish goats the teratogenic plant Nicotiana glauca at intervals of 5 days on treatment a 2 days off from gestation days 30-60. Because the lupine-induced skeleta contractures and cleft palate result from alkaloid-induced fetal inactivity, the theory proposed was that 2 days off treatment would allo fetal movement to return, albeit for only 12-24 hours between treatment periods. Results of the study showed that even though fetal movement as observed with ultrasound did return during the rest periods this was not long enough to prevent the skeletal malformations or cleft palate. This information will be used by cattle producers in grazing strategies to reduce losses from lupine and to improve utilization of critical rangelands.
Impacts
(N/A)
Publications
- Gardner, D.R., Panter, K.E., Stegelmeier, B.L. 2010. Implication of Agathic Acid from Utah Juniper Bark as an Abortifacient Compound in Cattle. Journal of Applied Toxicology, 30:115-119, 2010. DOI 10:1002/jat.1476
- Green, B.T., Lee, S.T., Panter, K.E., Welch, K.D., Cook, D., Pfister, J.A., Kem, W.R. 2010. Actions of Piperidine Alkaloid Teratogens at Fetal Nicotinic Acetylcholine Receptors. Neurotoxicology and Teratology. 32:383- 390. Published online at: http://dx.doi.org/10.1016/j.ntt.2010.01.011
- Thacker, E., Ralphs, M.H., Monaco, T.A. 2009. Seeding Cool-Season Grasses to Suppress Broom Snakeweed (Gutierrezia sarothrae), Downy Brome (Bromus tectorum), and Weedy Forbs. Journal of Invasive Plant Science and Management, 2:237-246.
- Rogosic, J., Pfister, J.A., Provenza, F.D., Pavlicevic, J. 2008. The Effect of Polyethylene Glycol on Intake of Mediterranean Shrubs by Sheep and Goats. Journal of Animal Science 86:3491-3496. doi:10.2527/jas.2007- 0828.
- Green, B.T., Brown, D.R. 2006. Differential effects of clathrin and actin inhibitors on internalization of Escherichia coli and Salmonella choleraesuis in porcine jejunal Peyer's patches. Veterinary Microbiology, 113:117-122.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Objective I: Pine Needles 1.1 Determine if isocupressic acid (ICA; the abortifacient compound in pine needles) concentration in pine needles is modulated by the environment. 1.2 Identify the matabolites of isocupressic acid in pine needles that cause abortions in cattle. Determine the biological mechanism, develop diagnostic techniques, and therapeutic procedures. 1.3 Determine factors that influence cattle consumption of pine needles and develop management practices to prevent abortion. Objective II: Broom Snakeweed 2.1 Identify the toxic and abortifacient compounds in broom snakeweed. 2.2 Describe the ecology of broom snakeweed, develop management and control guidelines to reduce incidence of poisoning and abortion in livestock. Objective III: Lupine 3.1 Isolate, identify, and evaluate toxicity and teratogenicity of lupine alkaloids which cause birth defects in calves born to cows that graze these plants. 3.2 Evaluate the role of genotype and environment on lupine alkaloids, and thus the relative toxicity of various species and populations of lupine. 3.3 Determine the physiological mechanism of lupine-induced birth defects and evaluate the maternal and fetal toxicokinetics of alkaloids. 3.4 Evaluate the influence of climate on population cycles of lupine. 3.5 Determine the importance of lupines as nutritional components for cattle during critical times of the year. 3.6 Identify conditions under which cattle graze various lupine species. Objective IV: Veratrum 4.1 Develop models to study the toxicokinetics, including clearance times, and toxicity of steroidal alkaloids in Veratrum californicum. Approach (from AD-416) 1.1 Data on environmental conditions will be collected at each site using local weather stations. ICA levels and environmental conditions will be correlated to determine if any patterns emerge. Soil samples will be collected at each site for future evaluation. 1.2 Samples of maternal and fetal tissues will be collected for histologic analysis and determination of ICA concentrations using existing ELISA�s and GC/MS methods. Proteomic analyses via LC/MS/MS techniques will be done. 1.3 Pen and field studies using cattle in high, medium and low body condition will be done to determine effects on needle consumption and grazing times. Nutrient supplements will be offered to determine if pine needle consumption will be altered. 2.1 The diterpene acid �fingerprint� of broom snakeweed from various populations in Arizona, New Mexico and Utah will be determined by chemical analysis. Subsequent in vitro and in vivo studies will be done to determine abortifacient activity. 2.2 A grazing study will be conducted to determine if various management practices can be implemented to force cattle to graze snakeweed as a biological control. A clipping study will be conducted to further describe the effects of defoliation on snakeweed and the surrounding plant community. 3.1 Alkaloids will be isolated by chemical methods and identified by chromatography, NMR, mass spectrometry, and elemental analysis. Toxicology will be evaluated using a mouse bioassay and cell lines that express nicotinic acetylcholine receptors. 3.2 A chemical fingerprint of Lupinus sulphureus collected from different locations will be generated using chemical methods. Fingerprints will be analyzed via cluster analysis and phylogenetic analysis will be performed using AFLPs (Amplified Fragment Length Polymorphisms) to determine the genetic relationship of the populations. 3.3 Pregnant goats in late gestation will be used to determine the rate of absorption, distribution and elimination of the teratogenic alkaloids. The pharmacokinetic profiles of the alkaloids will be compared between maternal and fetal systems. 3.4 Established transects will be monitored over the next 5 years to determine the influence of weather patterns on lupine density. Correlations of lupine age, class, density, and trends will be made with seasonal precipitation and temperature. 3.5 Consumption of lupines by cattle on rangelands dominated by low quality forages may be related to nutrient content. Twelve yearling heifers in a field study will be supplemented with different levels of protein to compare lupine ingestion. 3.6 Short-duration and high intensity grazing studies in early, mid, and late summer will be used to determine what role grazing pressure has on lupine intake during different seasons of the year. 4.1 A monogastric model (swine) will be used to determine the kinetics (clearance and metabolism) of a well known teratogenic alkaloid from Veratrum. This pilot project will be a model for testing the clearance of other plant toxins from animal tissues to evaluate food safety of animal products. Clearance rates between the monogastric model and small ruminant model will be compared. Significant Activities that Support Special Target Populations Grazing of ponderosa pine needles, broom snakeweed, specific juniper species, lodgepole pine and Monterey cypress by pregnant cows results in abortion/premature parturition, retained placentas, endometritis, death in cows, and dead calves. Isocupressic acid (ICA) has been identified as the toxin in pine needles, lodgepole pine and Monterey cypress responsible for the abortion and other associated complications. ICA has been shown to be metabolized in the rumen and in the liver. Initial collections to evaluate if ICA concentrations change as a function of the environment have been made. An ELISA assay has been developed for ICA and ICA metabolites and has been used to detect these compounds in cow tissues. Pen studies showed that cattle on low protein diets consume lower amounts of fresh pine needles. Analytical methods to profile labdane diterpene in broom snakeweed plants have been investigated. Both gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry methods have been established to profile the diterpene acids. These methods will be used to examine differences in broom snakeweed populations and examine metabolism of diterpene acids to possible abortifacient compounds in the rumen or hepatic system. Several herbicides were applied to broom snakeweed at Monticello and Nephi UT in May and August 2008. Plots were evaluated in June 2009. Picloram was most effective, Picloram + 2,4-D1/4, Aminopyralid and Metsulfuron were acceptable. In the western United States lupines cause sporadic and often large losses to cattle from �crooked calf syndrome�. When pregnant cattle graze lupine containing select piperdine and quinolizidine alkaloids during days 40-100 of gestation calves with congenital deformities such as crooked legs, neck and back and cleft palate can be born. A pharmacodynamic comparison of the alkaloids anabasine, anabaseine, anagyrine, ammodendrine, and coniine and their enantiomers in two cell lines and in a murine model was performed and the alkaloids and enantiomers were rank ordered in terms of potency (toxicity). In three grazing trials, cattle were reluctant to graze lupine in May and June when cheatgrass and forbs were green and succulent. In July, lupine was preferred, because cheatgrass was dry and annual forbs had matured. Increasing grazing pressure can force cattle to graze lupine in any season as desirable forages are depleted. Technology Transfer Number of Web Sites managed: 1 Number of Other Technology Transfer: 10
Impacts
(N/A)
Publications
- Cook, D., Lee, S.T., Gardner, D.R., Pfister, J.A., Welch, K.D., Green, B.T. , Davis, T.Z., Panter, K.E. 2009. The Alkaloid Profiles of Lupinus sulphureus. Journal of Agriculture and Food Chemistry, 57(4):1646-1653.
- Lee, S.T., Gardner, D.R., Chang, C.T., Panter, K.E., Molyneux, R.J. 2008. Separation and Measurement of Plant Alkaloid Enantiomers by RP-HPLC Analysis of their Fmoc-Alanine Analogs. Phytochemical Analysis, 19:395-402. DOI: 10.1002/pca.1064.
- Lee, S.T., Panter, K.E., Gay, C., Pfister, J.A., Ralphs, M.H., Gardner, D. R., Stegelmeier, B.L., Motteram, E., Cook, D., Welch, K.D., Green, B.T., Davis, T.Z. 2009. Lupine Induced "Crooked Calf Disease": The Last 20 Years. Rangelands, 31(1):13-18. http://www.bioone.org/doi/pdf/10.2111/1551-501X- 31.1.45?
- Lee, S.T., Panter, K.E., Pfister, J.A., Gardner, D.R., Welch, K.D. 2008. The Effect of Body Condition on Serum Concentrations of Two Teratogenic Alkaloids (Anagyrine and Ammodendrine) From Lupines (Lupinus spp.) That Cause Crooked Calf Disease. Journal of Animal Science. 86:2771-2778 doi:10. 2527/jas.2007-0610.
- Panter, K.E., Motteram, E., Cook, D., Lee, S.T., Ralphs, M.H., Platt, T.E., Gay, C.C. 2009. Cooked Calf Syndrome: Managing Lupines on Rangelands of the Channel Scablands of East-Central Washington State. Rangelands 31(1) :10-15. http://www.bioone.org/doi/pdf/10.2111/1551-501X-31.1.45?
- Pfister, J.A., Panter, K.E. 2009. Introduction to the USDA-Agricultural Research Service Poisonous Plant Research Laboratory. Rangelands (Special Issue). 31(1):3-4 http://www.bioone.org/doi/pdf/10.2111/1551-501X-31.1.45?
- Rader, E.P., Cederna, P.S., Mcclellan, W.T., Caterson, S.A., Panter, K.E., Yu, D., Buchman, S.R., Larkin, L.M., Faulkner, J.A., Weinzweig, J. 2008. Effect of Cleft Palate Repair on the Susceptibility to Contraction-Induced Injury of Single Permeabilized Muscle Fibers from Congenitally-Clefted Goat Palates. Cleft Palate-Craniofacial Journal. 45(2):113-120. DOI:10. 1597/06-171.1
- Welch, K.D., Davis, T.Z., Panter, K.E., Pfister, J.A., Green, B.T. 2009. The Effect of Poisonous Range Plants on Abortions in Livestock. Rangelands, 31(1):28-34. http://www.bioone.org/doi/pdf/10.2111/1551-501X-31.1.45?
- Welch, K.D., Panter, K.E., Lee, S.T., Gardner, D.R., Stegelmeier, B.L., Cook, D. 2009. Cyclopamine-induced Synophthalmia in Sheep: Defining a Critical Window and Toxicokinetic Evaluation. Journal of Applied Toxicology, 29:414-421. (www.interscience.wiley.com) DOI 10.1002/jat.1427
- Thacker, E., Ralphs, M.H., Monaco, T.A. 2009. A Comparison of Inter- and Intraspecific Interference on Broom Snakeweed (Gutierrezia sarothrae) Seedling Growth. Journal of Invasive Plant Science and Management. 2(1):36- 44. DOI 10.1614/PSM-08-099.1
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) Objective I: Pine Needles 1.1 Determine if isocupressic acid (ICA; the abortifacient compound in pine needles) concentration in pine needles is modulated by the environment. 1.2 Identify the matabolites of isocupressic acid in pine needles that cause abortions in cattle. Determine the biological mechanism, develop diagnostic techniques, and therapeutic procedures. 1.3 Determine factors that influence cattle consumption of pine needles and develop management practices to prevent abortion. Objective II: Broom Snakeweed 2.1 Identify the toxic and abortifacient compounds in broom snakeweed. 2.2 Describe the ecology of broom snakeweed, develop management and control guidelines to reduce incidence of poisoning and abortion in livestock. Objective III: Lupine 3.1 Isolate, identify, and evaluate toxicity and teratogenicity of lupine alkaloids which cause birth defects in calves born to cows that graze these plants. 3.2 Evaluate the role of genotype and environment on lupine alkaloids, and thus the relative toxicity of various species and populations of lupine. 3.3 Determine the physiological mechanism of lupine-induced birth defects and evaluate the maternal and fetal toxicokinetics of alkaloids. 3.4 Evaluate the influence of climate on population cycles of lupine. 3.5 Determine the importance of lupines as nutritional components for cattle during critical times of the year. 3.6 Identify conditions under which cattle graze various lupine species. Objective IV: Veratrum 4.1 Develop models to study the toxicokinetics, including clearance times, and toxicity of steroidal alkaloids in Veratrum californicum. Approach (from AD-416) 1.1 Data on environmental conditions will be collected at each site using local weather stations. ICA levels and environmental conditions will be correlated to determine if any patterns emerge. Soil samples will be collected at each site for future evaluation. 1.2 Samples of maternal and fetal tissues will be collected for histologic analysis and determination of ICA concentrations using existing ELISA�s and GC/MS methods. Proteomic analyses via LC/MS/MS techniques will be done. 1.3 Pen and field studies using cattle in high, medium and low body condition will be done to determine effects on needle consumption and grazing times. Nutrient supplements will be offered to determine if pine needle consumption will be altered. 2.1 The diterpene acid �fingerprint� of broom snakeweed from various populations in Arizona, New Mexico and Utah will be determined by chemical analysis. Subsequent in vitro and in vivo studies will be done to determine abortifacient activity. 2.2 A grazing study will be conducted to determine if various management practices can be implemented to force cattle to graze snakeweed as a biological control. A clipping study will be conducted to further describe the effects of defoliation on snakeweed and the surrounding plant community. 3.1 Alkaloids will be isolated by chemical methods and identified by chromatography, NMR, mass spectrometry, and elemental analysis. Toxicology will be evaluated using a mouse bioassay and cell lines that express nicotinic acetylcholine receptors. 3.2 A chemical fingerprint of Lupinus sulphureus collected from different locations will be generated using chemical methods. Fingerprints will be analyzed via cluster analysis and phylogenetic analysis will be performed using AFLPs (Amplified Fragment Length Polymorphisms) to determine the genetic relationship of the populations. 3.3 Pregnant goats in late gestation will be used to determine the rate of absorption, distribution and elimination of the teratogenic alkaloids. The pharmacokinetic profiles of the alkaloids will be compared between maternal and fetal systems. 3.4 Established transects will be monitored over the next 5 years to determine the influence of weather patterns on lupine density. Correlations of lupine age, class, density, and trends will be made with seasonal precipitation and temperature. 3.5 Consumption of lupines by cattle on rangelands dominated by low quality forages may be related to nutrient content. Twelve yearling heifers in a field study will be supplemented with different levels of protein to compare lupine ingestion. 3.6 Short-duration and high intensity grazing studies in early, mid, and late summer will be used to determine what role grazing pressure has on lupine intake during different seasons of the year. 4.1 A monogastric model (swine) will be used to determine the kinetics (clearance and metabolism) of a well known teratogenic alkaloid from Veratrum. This pilot project will be a model for testing the clearance of other plant toxins from animal tissues to evaluate food safety of animal products. Clearance rates between the monogastric model and small ruminant model will be compared. Significant Activities that Support Special Target Populations This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P. In pen studies, cattle showed preference to different lupines containing different profiles of alkaloids. From this study, six different lupine plants growing in the same region were ranked ordered in terms of cattle preference. Lupine seasonal grazing studies showed that cattle did not prefer lupine early in the growing season, but were forced to graze it as other forage became limited. However, lupine was preferred forage late in the growing season when other forage was dry and dormant. Enantiomeric alkaloids that cause birth defects (teratogens) were isolated and their toxicities determined in both mouse and cell line bioassays. The enanatiomeric pairs were found to have the same relative toxicities in both the mouse and cell line assays. These alkaloids were used in a pilot study in which the alkaloids were administered to the fetus via osmotic mini-pumps. Preliminary data on the maternal-fetal transfer of teratogenic alkaloids indicates the fetus circulation profile mimics the material profile but is delayed. Utah Juniper bark and needles containing primarily agathic acid as the diterpene acid were dosed to cattle. The cow serum metabolites of agathic acid were the same as the cow serum metabolites as isocupressic acid, the primary abortifacient in Ponderosa Pine needles. In addition, pregnant cows that were dosed with Utah Juniper aborted. This study determined agathic acid is an abortifacient. Vegetative response to defoliation showed snakeweed mortality increased, blue bunch wheat grass cover declined, and crested wheat grass increased. In order to better understand the mechanism of action of isocupressic- induced abortions, an ELISA was used to determine the distribution of isocupressic acid and its metabolites in cows. Veratrum californicum has been shown to be teratogenic in sheep. A detailed analysis of the kinetic profile of cyclopamine in sheep was initiated. Technology Transfer Number of Web Sites managed: 1 Number of Other Technology Transfer: 4
Impacts
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Publications
- Lee, S.T., Cook, D., Panter, K.E., Gardner, D.R., Ralphs, M.H., Motteram, E.S., Pfister, J.A., Gay, C. 2007. Lupine induced "Crooked Calf Disease" in Washington and Oregon: Identification of the alkaloid profiles in Lupinus sulphureus, Lupinus leucophyllus, and Lupinus sericeus. Journal of Agricultural and Food Chemistry, Web release 11/27/2007 Article DOI: 10. 1021/jf0723110 Journal releae Volume 55, Number 26, pp. 10649-10655.
- Weinzweig, J., Panter, K.E., Patel, J., Smith, D.M., Spangenberger, A., Freeman, B. 2008. The Fetal Cleft palate: V. Elucidation of the Mechanism of Palatal Clefting in the Congenital Caprine Model. Plastic and Reconstructive Surgery, pp.1328-1334
- Pfister, J.A., Lee, S.T., Panter, K.E., Motteram, E.S., Gay, C. 2008. Effects of Experience and Lactation on Lupine Consumption by Cattle. Rangeland Ecology and Management 61:240-244 March 2008
- Hanes, M.C., Weinzweig, J., Panter, K.E., Mcclellan, T., Caterson, S.A., Buchman, S.R., Faulkner, J.A., Yu, D., Cederba, P.S., Larkin, L.M. 2008. The Effect of Cleft Palate Repair on Contractile Properties of Single Permeabilized Muscle Fibers From Congenitally Cleft Goats Palates. Annals of Plastic Surgery, Vol. 60 No. 2, pp. 188-193.
- Hanes, M.C., Weinzweig, J., Kuzon, W.M., Panter, K.E., Buchman, S.R., Faulkner, J.A., Yu, D., Cederna, P.S., Larkin, L.M. 2007. Contractile properties of single permeabilized muscle fibers from congenital cleft palates and normal palates of Spanish goats. Plastic and Reconstructive Surgery, 119:1685 - 1694.
- Rader, E.P., Cederna, P.S., Weinzweig, J., Panter, K.E., Yu, D., Buchman, S.R., Larkin, L.M., Faulkner, J.A. 2007. Contraction-induced injury to single permeabilized muscle fibers from normal and congenitally-clefted goat palates. Cleft Palate-Craniofacial Journal, Vol. 44 No. 2 pp. 216-222.
- Gorniak, S.L., Pfister, J.A., Lanzoniz, E.C., Raspantini, E.R. 2008. A note on averting goats to a toxic but palatable plant, Leucaena leucocephala. Applied Animal Behaviour Science, Vol. 111, No. 3-4, pp. 306- 401.
- Ralphs, M.H., Panter, K.E., Gay, C., Motteram, E., Lee, S.T. 2007. Cattle grazeing velvet lupine (lupinus leucophyllus): influence of associated forages, alkaloid levels and population cycles. Poisonous Plant Global Research and Solutions, Chpt. 67, pp. 401-406
- Thacker, E., Ralphs, M.H., Call, C., Benson, B., Green, S. 2008. Invasion of Broom Snakeweed (Gutierrezia sarothrae) Following Disturbance: Evaluating Change in a State-and-Transition Model. Rangeland Ecology and Management, Vol. 61, Issue 3, pp. 263-268. DOI:10:2111/07.043.1
- Ralphs, M.H., Wiedmeier, R.D., Banks, J.E. 2007. Decreasing forage allowance can force cattle to graze broom snakeweed (gutierrezia sarothrae) as a potential biological control. Rangeland Ecology and Management, 60:487-497.
- Panter, K.E., Gardner, D.R., Lee, S.T., Pfister, J.A., Ralphs, M.H., Stegelmeier, B.L., James, L.F. 2007. Important poisonous plants of the United States. Veterinary Toxicology: Basic and Clinical Principles, Chpt. 66 pp. 825-872.
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