Progress 02/14/08 to 02/10/13
Outputs
Progress Report Objectives (from AD-416): Objective I: Determine Astragalus and Oxytropis species which contain fungal endophytes that produce swainsonine and describe the plant/ endophyte relationship. 1.1 Identify species that contain the endophyte (Embellisia), determine transfer of the endophyte to successive generations, and determine if the endophyte increases fitness of locoweed plants. 1.2 Describe the distribution of the endophyte and swainsonine as a function of plant part and determine if swainsonine varies as a function of time. 1.3 Determine the effect of the endophyte on palatability of locoweeds. Objective II: Identify environmental conditions that will help predict population outbreaks of major locoweed species (Oxytropis sericea, Astragalus mollissimus, A. lentiginosus). Determine the conditions under which cattle, sheep, and horses graze locoweeds. 2.1 Relate locoweed population outbreaks to weather cycles. 2.2 Determine conditions under which livestock graze various locoweed species. 2.3 Determine influence of nitrogen supplements in livestock diet selection and locoweed poisoning. Objective III: Further describe effects of swainsonine and related polyhydroxy alkaloids on reproduction and body systems among livestock and wildlife species. 3.1 Conduct a comparative study of species differences to determine why mannosidases are inhibited differently. 3.2 Compare the effects of swainsonine on fetotoxicity among breeds of sheep and goats. 3.3 Compare effects of swainsonine on ovarian function among cattle, sheep, and goats. Objective IV: Characterize biomarkers of intoxication and develop better diagnostic and prognostic procedures. 4.1 Develop ELISA for locoweed intoxication. 4.2 Develop biomarkers of poisoning. Objective V: Further describe toxicoses and pathology of animals poisoned by Astragalus species containing nitro-propionic. Objective VI: Further describe the toxicosis, physiologic effects, and pathology of Astragalus and other selenium accumulating plants, and determine absorption, distribution, and elimination (clearance times) of various types and forms of selenium in livestock. 6.1 Describe the etiology and pathogenesis of selenium poisoning and deficiency in livestock and determine safe nutritional levels. 6.2 Determine the effect of selenium-reducing microflora on the selenium pharacokinetics when livestock consume seleniferous plant material. Approach (from AD-416): 1.1 Seed from �endophyte-free� and endophyte-infected locoweed plants will be germinated to determine if the endophyte is transmitted and expressed in the next generation. If so, we will develop endophyte-free and endophyte�infected populations and compare their fitness and competitive ability. 1.2 O. sericea plants will be collected and separated into plant parts and the endophyte measured by PCR. Once the endophyte distribution within the plant is known, we will collect stalks from independent plants at 2 week intervals throughout the growing season to determine endophyte distribution and swainsonine synthesis over time. 1.3 Fungal endophytes will be grown in the laboratory using standard culture techniques, then added to ground alfalfa hay, and presented to individual animals in preference tests. 2.1 Locoweed density will be measured annually in locations throughout the Western US, and correlated with weather data to develop predictive models. 2.2 A series of grazing studies will be conducted in northeastern New Mexico beginning in late summer while grass is green and run through early winter as grasses senesce to determine cattle preference for woolly locoweed. 2.3 Supplemented and nonsupplemented groups of cattle will be grazed to determine if the supplement will reduce locoweed consumption. 3.1 Tissues from several animal species will be analyzed and mannosidase expression compared using immunohistochemistry, Western blotting, real time (RT)-PCR and Northern blots. Enzymatic in vitro assays of mannosidase activity will be compared using a modification of previously developed serum a-mannosidase assays. 3.2 Swainsonine will be fed to hair sheep, wool sheep and goats in increasing doses. Swainsonine absorption and elimination profiles will be developed, fetotoxic effects will be monitored by ultrasound, and maternal histological comparisons will be evaluated. 3.3 Swainsonine will be fed to heifers, ewes, and goats at increasing doses. Ultrasound imaging will be used to evaluate changes in follicular phase and cyst development, histological changes in ovaries will be compared, and the biological activity of anterior pituitary gonadotropins will be assayed. 4.1 Swainsonine-protein conjugates will be synthesized and injected subcutaneously into four sheep and antisera titers determined. Antisera exhibiting high titers that are specific to swainsonine will be developed into ELISA�s. 4.2 Differences in blood proteome from animals poisoned by locoweed plants will be used to identify proteins that can be used as biomarkers, then they will be validated using actual locoweed intake data. 5. A dose response study in sheep and cattle will be conducted and tissues collected for microscopic, ultrastructural and chemical analysis. 6.1 Selenium from plant material will be compared to inorganic forms at increasing doses to determine bioavailability and toxicity in sheep. 6.2 Reproductively mature ewes will be inoculated with selenobacter (Wolinella succinogenes), fed gound seleniferous plant (Astragalus bisulcatus) for eight months to monitor the effects of chronic selenium dosing on estrus cycles, gestation, and initial growth of lambs. This is the final report for project 5428-32000-014-00D. Poisoning by Astragalus and Oxytropis species has been historically divided into three groups based on the toxic syndromes they cause in livestock: locoism caused by the toxin swainsonine, selenium poisoning, and nitrotoxin poisoning. Locoweeds contain the toxic alkaloid swainsonine. A fungal endophyte, Undifilum oxytropis (previously identified as Embellisia), found in locoweed plant species was shown to be responsible for the synthesis of swainsonine. The interaction of the plant and endophyte on swainsonine concentrations has been investigated. Studies have been done comparing differential susceptibility of different livestock species as well as the effect of locoweeds on reproduction. Lastly, studies have been done further characterizing poisoning due to selenium containing plants and the different forms of selenium. In this current year, the influence of elevated CO2 is being evaluated on swainsonine concentrations in locoweed. A reference list of swainsonine-containing Astragalus, Oxytropis, and Ipomoea species is being made. Other plant species contain swainsonine, including some Swainsona (Leguminosae) species in Australia and some Ipomoea (Convolvulaceae), Turbina (Convolvulaceae), and Sida (Malvaceae) species in South America and Africa. Fungal endophytes that produce swainsonine have been isolated and are being characterized from Swainsona canescens and Ipomoea carnea. Studies are being performed to define the active principle in Ipomoea asarifolia using a small animal and large animal model. The influence of post-ingestive feedback as it relates to the consumption of selenium- containing forages is being investigated. Experiments are being performed to investigate the effect of selenium on reproductive rates in grazing livestock in the new replacement project 5428-32630-012-00D. Significant Activities that Support Special Target Populations: Identification and control of poisonous range plants. Northwest Intertribal Agriculture Council Conference. Chief Conventions Center. Fort Hall, ID. Poisonous Plants on the Range. 5th Annual Tri-State Range Meeting. Coleville, UT. Accomplishments 01 Influence of endophyte genotype on swainsonine concentrations in different populations of Oxytropis sericea. Locoism is a toxic syndrome of livestock caused by the ingestion of a subset of legumes belonging to the Astragalus and Oxytropis genera known as �locoweeds�. Locoweeds contain the toxic indolizidine alkaloid swainsonine, which is produced by the endophytic fungi Undifilum species. Previously ARS scientists reported that swainsonine concentrations differ between populations of O. sericea. A study was conducted to determine if the genotype of the plant, endophyte, or an interaction of the two may be responsible for the differences in swainsonine concentration between populations of O. sericea. To investigate this, plants derived from seeds collected at each location were grown in a common garden, Undifilum oxytropis isolates from each location were cultured and grown in a common environment, and a plant genotype by endophyte cross inoculation was performed. Results showed that the genotype of the endophyte is responsible for the differences in swainsonine concentrations observed in the two populations of O. sericea. Due to this research, better predictions can be provided to producers regarding relative risk of animals grazing on locoweeds. 02 The effects of swainsonine, the locoweed toxin, have been documented in various livestock and wildlife species. Horses, goats, cattle and sheep are the most sensitive to swainsonine poisoning while rodents and deer are relatively resistant. The distribution and severity of swainsonine produced lesions are species specific. ARS scientists in Logan, UT have characterized these differences and developed sensitive models to compare swainsonine induced toxicity with those of other glysosidase inhibiting toxins such as calystegines and castanospermine. This information will be used to better define the risk of poisoning and to determine the role swainsonine plays in other plants that contain swainsonine and mixtures of other glycosidase inhibiting alkaloids.
Impacts
(N/A)
Publications
- Bottger, J.A., Creamer, R., Gardner, D.R. 2012. Seasonal changes in Undifilum colonization and swainsonine content of locoweeds. Journal of Chemical Ecology. 38:486-95.
- Cook, D., Grum, D.S., Gardner, D.R., Welch, K.D., Pfister, J.A. 2013. Influence of endophyte genotype on swainsonine concentrations in Oxytropis sericea. Toxicon. 61:105-11.
- Gao, X., Cook, D., Ralphs, M., Yan, L., Gardner, D.R., Lee, S.T., Panter, K.E., Han, B., Zhao, M. 2012. Detection of swainsonine and isolation of the endophyte Undifilum from the major locoweeds in Inner Mongolia. Biochemical Systematics and Ecology. 45: 79-85.
- Grum, D.S., Cook, D., Gardner, D.R., Roper, J.M., Pfister, J.A., Ralphs, M. H. 2012. Influence of seed endophyte amounts on swainsonine concentrations in Astragalus and Oxytropis locoweeds. Journal of Agricultural and Food Chemistry. 66(33): 8083-9.
- Mendonca, F.S., Albuquerque, R.F., Evencio-Neto, J., Freitas, S.H., Doria, R.G., Boabaid, F.M., Driemeier, D., Gardner, D.R., Riet-Correa, F., Colodel, E.M. 2012. Alpha-mannosidosis in goats caused by the swainsonine- containing plant Ipomoea verbascoidea. Journal of Veterinary Diagnostic Investigation. 24(1): 90-5.
- Oldham, M., Ransom, C.V., Ralphs, M.H., Gardner, D.R. 2011. Galegine content in Goatsrue (Galega officinalis) varies by plant part and phenological growth stage. Weed Science. 59(3): 349-52.
- Reyna, R., Cooke, P., Grum, D.S., Cook, D., Creamer, R. 2012. Detection and localization of the endophyte Undifilum oxytropis in locoweed tissues. Botany. 90(12): 1229-36.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): Objective I: Determine Astragalus and Oxytropis species which contain fungal endophytes that produce swainsonine and describe the plant/endophyte relationship. 1.1 Identify species that contain the endophyte (Embellisia), determine transfer of the endophyte to successive generations, and determine if the endophyte increases fitness of locoweed plants. 1.2 Describe the distribution of the endophyte and swainsonine as a function of plant part and determine if swainsonine varies as a function of time. 1.3 Determine the effect of the endophyte on palatability of locoweeds. Objective II: Identify environmental conditions that will help predict population outbreaks of major locoweed species (Oxytropis sericea, Astragalus mollissimus, A. lentiginosus). Determine the conditions under which cattle, sheep, and horses graze locoweeds. 2.1 Relate locoweed population outbreaks to weather cycles. 2.2 Determine conditions under which livestock graze various locoweed species. 2.3 Determine influence of nitrogen supplements in livestock diet selection and locoweed poisoning. Objective III: Further describe effects of swainsonine and related polyhydroxy alkaloids on reproduction and body systems among livestock and wildlife species. 3.1 Conduct a comparative study of species differences to determine why mannosidases are inhibited differently. 3.2 Compare the effects of swainsonine on fetotoxicity among breeds of sheep and goats. 3.3 Compare effects of swainsonine on ovarian function among cattle, sheep, and goats. Objective IV: Characterize biomarkers of intoxication and develop better diagnostic and prognostic procedures. 4.1 Develop ELISA for locoweed intoxication. 4.2 Develop biomarkers of poisoning. Objective V: Further describe toxicoses and pathology of animals poisoned by Astragalus species containing nitro-propionic. Objective VI: Further describe the toxicosis, physiologic effects, and pathology of Astragalus and other selenium accumulating plants, and determine absorption, distribution, and elimination (clearance times) of various types and forms of selenium in livestock. 6.1 Describe the etiology and pathogenesis of selenium poisoning and deficiency in livestock and determine safe nutritional levels. 6.2 Determine the effect of selenium-reducing microflora on the selenium pharacokinetics when livestock consume seleniferous plant material. Approach (from AD-416): 1.1 Seed from �endophyte-free� and endophyte-infected locoweed plants will be germinated to determine if the endophyte is transmitted and expressed in the next generation. If so, we will develop endophyte-free and endophyte�infected populations and compare their fitness and competitive ability. 1.2 O. sericea plants will be collected and separated into plant parts and the endophyte measured by PCR. Once the endophyte distribution within the plant is known, we will collect stalks from independent plants at 2 week intervals throughout the growing season to determine endophyte distribution and swainsonine synthesis over time. 1.3 Fungal endophytes will be grown in the laboratory using standard culture techniques, then added to ground alfalfa hay, and presented to individual animals in preference tests. 2.1 Locoweed density will be measured annually in locations throughout the Western US, and correlated with weather data to develop predictive models. 2.2 A series of grazing studies will be conducted in northeastern New Mexico beginning in late summer while grass is green and run through early winter as grasses senesce to determine cattle preference for woolly locoweed. 2.3 Supplemented and nonsupplemented groups of cattle will be grazed to determine if the supplement will reduce locoweed consumption. 3.1 Tissues from several animal species will be analyzed and mannosidase expression compared using immunohistochemistry, Western blotting, real time (RT)-PCR and Northern blots. Enzymatic in vitro assays of mannosidase activity will be compared using a modification of previously developed serum a-mannosidase assays. 3.2 Swainsonine will be fed to hair sheep, wool sheep and goats in increasing doses. Swainsonine absorption and elimination profiles will be developed, fetotoxic effects will be monitored by ultrasound, and maternal histological comparisons will be evaluated. 3.3 Swainsonine will be fed to heifers, ewes, and goats at increasing doses. Ultrasound imaging will be used to evaluate changes in follicular phase and cyst development, histological changes in ovaries will be compared, and the biological activity of anterior pituitary gonadotropins will be assayed. 4.1 Swainsonine-protein conjugates will be synthesized and injected subcutaneously into four sheep and antisera titers determined. Antisera exhibiting high titers that are specific to swainsonine will be developed into ELISA�s. 4.2 Differences in blood proteome from animals poisoned by locoweed plants will be used to identify proteins that can be used as biomarkers, then they will be validated using actual locoweed intake data. 5. A dose response study in sheep and cattle will be conducted and tissues collected for microscopic, ultrastructural and chemical analysis. 6.1 Selenium from plant material will be compared to inorganic forms at increasing doses to determine bioavailability and toxicity in sheep. 6.2 Reproductively mature ewes will be inoculated with selenobacter (Wolinella succinogenes), fed gound seleniferous plant (Astragalus bisulcatus) for eight months to monitor the effects of chronic selenium dosing on estrus cycles, gestation, and initial growth of lambs. Poisoning by Astragalus and Oxytropis species has been historically divided into three groups based on the toxic syndromes they cause in livestock: locoism caused by the toxin swainsonine, selenium poisoning, and nitrotoxin poisoning. Locoweeds contain the toxic alkaloid swainsonine. A fungal endophyte, Undifilum oxytropis (previously identified as Embellisia), found in locoweed plant species was shown to be responsible for the synthesis of swainsonine. The influence of environment and genotype on swainsonine concentrations in locoweeds is being evaluated. A reference list of swainsonine-containing Astragalus and Oxytropis species is being made. Other plant species contain swainsonine, including some Swainsona (Leguminosae) species in Australia and some Ipomoea (Convolvulaceae), Turbina (Convolvulaceae), and Sida (Malvaceae) species in South America and Africa. Fungal endophytes that produce swainsonine have been isolated and are being characterized from Swainsona canescens and Ipomoea carnea. Studies are being performed to define the active principle in Ipomoea asarifolia. The influence of post- ingestive feedback as it relates to the consumption of selenium- containing forages is being investigated. Experiments are being performed to investigate the effect of selenium on reproductive rates in grazing livestock. Accomplishments 01 Defining the clinical signs associated with selenium poisoning. Selenium concentrations in plants above 10 ppm are reported to be toxic to livestock. ARS scientists in Logan, UT fed sheep rations containing 0 to 60 ppm selenium for 90 days. There were no observable signs of toxicosis although food intake decreased as the concentration of selenium in the ration increased. Sheep fed rations above 30 ppm Se had decreased reproductive rates. Whole blood, serum, hair and liver biopsy samples we analyzed to better understand selenium absorption, metabolism and clearance. Understanding selenium elimination rates and selenium effects on reproduction rates provides useful information for food safety and fo producers of animals grazing on forages high in selenium. 02 Effect of selenium on rumen microflora. Some animals appear to be more tolerant to selenium toxicosis than others when grazing Se-accumulating forages. The effect of a high-Se diet on rumen fermentation and microbia shifts in selenium metabolizing microbes was investigated by ARS researchers in Logan, UT using artificial rumen fermenters. Orchardgras containing 50 ppm Se had no negative impacts on laboratory assessments o ruminal fermentation. An increase in selenate metabolizing microbes, however, suggests that high dietary Se may cause ruminal adaptations tha render selenium less bioavailable. Such an adaptation of rumen microbes helps to explain why some animals are more Se-tolerant than others. This information will be used to better manage livestock when grazing on seleniferous forages. 03 Palatability of selenium-containing forage. Rangeland plants that uptake selenium in moderate to high concentrations are reputed to be very unpalatable to livestock, yet free-grazing livestock are periodically poisoned when grazing on ranges with high Se-containing plants. Studies were conducted by ARS scientists in Logan, UT to determine if cattle and sheep could discriminate Se concentrations in forages in the absence of post-ingestive feedback, and to determine the inherent animal preference for Se-containing forages. Neither cattle nor sheep initially selected o rejected forages based on Se concentrations (0 � 4000 ppm) when offered various Se-containing plants in preference tests. However, cattle were totally averted to the high selenium plant after initial exposure. Understanding the relative palatability of selenium containing forage relative to other forages provides useful information to producers of animals grazing on forages high in selenium. 04 The toxic principle of Ipomoea asarifolia, a morning glory. The Ipomoea (morning glory) genus contains many species, a number of which contain t same toxin as locoweeds. Additionally, some Ipomoea species contain ergo alkaloids. Consumption of these Ipomoea species is reputed to cause muscular tremors and behavioral alterations in domestic livestock. The effects of various Ipomoea asarifolia concentrations (0, 5, 10, 15, & 20 of the diet for 60 days were examined by ARS researchers in Logan, UT using a mouse model in collaboration with Brazilian scientists. Mice wer evaluated periodically for physiologic and behavioral signs of intoxication. No physiologic and behavioral signs were observed in mice. Defining the active principle of a poisonous plant is key to making management-based decisions for livestock managers. 05 Swainsonine and endophyte interactions in locoweed associated with plant development. It is not known if swainsonine and endophyte amounts in whi point loco (Oxytropis sericea) are influenced by geographic location, portion of the plant being measured (crown, floral, and leaves), and the developmental stage of the plant. ARS researchers in Logan, UT analyzed swainsonine and endophyte amounts in white loco at four different locations, in different plant parts, and at different developmental stag Swainsonine and endophyte amounts increase as the plant matures and swainsonine amounts were positively associated with endophyte amounts. D to this research, better predictions can be provided to producers regarding relative risk of animals grazing on locoweeds. 06 Influence of seed endophyte amount in locoweeds. Two types of plants exi within toxic populations of locoweeds: one that is toxic and contains swainsonine and endophyte and one that is much less toxic and contains very little or no swainsonine and endophyte. It is not known whether the plant genotype or endophyte amounts in the seeds are responsible for thi difference. ARS researchers in Logan, UT demonstrated that these two typ of plants could be interchanged by manipulating seed endophyte amounts. ARS researchers demonstrated that plants derived from fungicide treatmen of seeds had very little swainsonine and endophyte. Using the methods developed herein and the further understanding of the relationship betwe swainsonine and the endophyte in different locoweed species may provide information to help manage locoweed infested ranges and render plants no toxic.
Impacts
(N/A)
Publications
- Cook, D., Shi, L., Gardner, D.R., Pfister, J.A., Grum, D.S., Welch, K.D., Ralphs, M.H. 2012. Influence of phenological stage on swainsonine concentrations and endophyte amounts in Oxytropis sericea. Journal of Chemical Ecology. 38(2): 195-203.
- Davis, T.Z., Stegelmeier, B.L., Panter, K.E., Cook, D., Gardner, D.R., Hall, J.O. 2012. Toxicokinetics and pathology of plant-associated acute selenium toxicosis in steers. Journal of Veterinary Diagnostic Investigation. 24(2): 319-27.
- Furlan, S., Panter, K.E., Pfister, J.A., Stegelmeier, B.L. 2011. Fetotoxicity of Astragalus lentiginosus (locoweed) in Spanish goats. International Journal of Poisonous Plant Research. 1(1):35-40.
- Gao, X., Cook, D., Ralphs, M.H., Yan, L., Gardner, D.R., Lee, S.T., Panter, K.E., Han, B., Zhao, M. 2012. Detection of swainsonine and isolation of the endophyte Undifilum form the major locoweeds in Inner Mongolia. Biochemical Systematics and Ecology. 45: 79-85.
- Panter, K.E., Welch, K.D., Gardner, D.R. 2011. Toxic plants: Effects on reproduction and fetal and embryonic development in livestock. In: Gupta, R.C., editor. Reproductive and Developmental Toxicology. San Diego, CA: Academic Press. p. 689-704.
- Ralphs, M.H., Stegelmeier, B.L. 2011. Locoweed toxicity, ecology, control and management. International Journal of Poisonous Plant Research. 1(1):47- 64.
- Santos, F.M., Latorre, A.O., Hueza, I.M., Sanches, D.S., Lippi, L.L., Gardner, D.R., Spinosa, H.S. 2011. Increased antitumor efficacy by the combined administration of swainsonine and cisplatin in vivo. Phytomedicine. 18(12):1096-1101.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Objective I: Determine Astragalus and Oxytropis species which contain fungal endophytes that produce swainsonine and describe the plant/endophyte relationship. 1.1 Identify species that contain the endophyte (Embellisia), determine transfer of the endophyte to successive generations, and determine if the endophyte increases fitness of locoweed plants. 1.2 Describe the distribution of the endophyte and swainsonine as a function of plant part and determine if swainsonine varies as a function of time. 1.3 Determine the effect of the endophyte on palatability of locoweeds. Objective II: Identify environmental conditions that will help predict population outbreaks of major locoweed species (Oxytropis sericea, Astragalus mollissimus, A. lentiginosus). Determine the conditions under which cattle, sheep, and horses graze locoweeds. 2.1 Relate locoweed population outbreaks to weather cycles. 2.2 Determine conditions under which livestock graze various locoweed species. 2.3 Determine influence of nitrogen supplements in livestock diet selection and locoweed poisoning. Objective III: Further describe effects of swainsonine and related polyhydroxy alkaloids on reproduction and body systems among livestock and wildlife species. 3.1 Conduct a comparative study of species differences to determine why mannosidases are inhibited differently. 3.2 Compare the effects of swainsonine on fetotoxicity among breeds of sheep and goats. 3.3 Compare effects of swainsonine on ovarian function among cattle, sheep, and goats. Objective IV: Characterize biomarkers of intoxication and develop better diagnostic and prognostic procedures. 4.1 Develop ELISA for locoweed intoxication. 4.2 Develop biomarkers of poisoning. Objective V: Further describe toxicoses and pathology of animals poisoned by Astragalus species containing nitro-propionic. Objective VI: Further describe the toxicosis, physiologic effects, and pathology of Astragalus and other selenium accumulating plants, and determine absorption, distribution, and elimination (clearance times) of various types and forms of selenium in livestock. 6.1 Describe the etiology and pathogenesis of selenium poisoning and deficiency in livestock and determine safe nutritional levels. 6.2 Determine the effect of selenium-reducing microflora on the selenium pharacokinetics when livestock consume seleniferous plant material. Approach (from AD-416) 1.1 Seed from �endophyte-free� and endophyte-infected locoweed plants will be germinated to determine if the endophyte is transmitted and expressed in the next generation. If so, we will develop endophyte-free and endophyte�infected populations and compare their fitness and competitive ability. 1.2 O. sericea plants will be collected and separated into plant parts and the endophyte measured by PCR. Once the endophyte distribution within the plant is known, we will collect stalks from independent plants at 2 week intervals throughout the growing season to determine endophyte distribution and swainsonine synthesis over time. 1.3 Fungal endophytes will be grown in the laboratory using standard culture techniques, then added to ground alfalfa hay, and presented to individual animals in preference tests. 2.1 Locoweed density will be measured annually in locations throughout the Western US, and correlated with weather data to develop predictive models. 2.2 A series of grazing studies will be conducted in northeastern New Mexico beginning in late summer while grass is green and run through early winter as grasses senesce to determine cattle preference for woolly locoweed. 2.3 Supplemented and nonsupplemented groups of cattle will be grazed to determine if the supplement will reduce locoweed consumption. 3.1 Tissues from several animal species will be analyzed and mannosidase expression compared using immunohistochemistry, Western blotting, real time (RT)-PCR and Northern blots. Enzymatic in vitro assays of mannosidase activity will be compared using a modification of previously developed serum a-mannosidase assays. 3.2 Swainsonine will be fed to hair sheep, wool sheep and goats in increasing doses. Swainsonine absorption and elimination profiles will be developed, fetotoxic effects will be monitored by ultrasound, and maternal histological comparisons will be evaluated. 3.3 Swainsonine will be fed to heifers, ewes, and goats at increasing doses. Ultrasound imaging will be used to evaluate changes in follicular phase and cyst development, histological changes in ovaries will be compared, and the biological activity of anterior pituitary gonadotropins will be assayed. 4.1 Swainsonine-protein conjugates will be synthesized and injected subcutaneously into four sheep and antisera titers determined. Antisera exhibiting high titers that are specific to swainsonine will be developed into ELISA�s. 4.2 Differences in blood proteome from animals poisoned by locoweed plants will be used to identify proteins that can be used as biomarkers, then they will be validated using actual locoweed intake data. 5. A dose response study in sheep and cattle will be conducted and tissues collected for microscopic, ultrastructural and chemical analysis. 6.1 Selenium from plant material will be compared to inorganic forms at increasing doses to determine bioavailability and toxicity in sheep. 6.2 Reproductively mature ewes will be inoculated with selenobacter (Wolinella succinogenes), fed gound seleniferous plant (Astragalus bisulcatus) for eight months to monitor the effects of chronic selenium dosing on estrus cycles, gestation, and initial growth of lambs. Poisoning by Astragalus and Oxytropis species has been historically divided into three groups based on the toxic syndromes they cause in livestock: locoism caused by the toxin swainsonine, selenium poisoning, and nitrotoxin poisoning. Locoweeds contain the toxic alkaloid swainsonine. A fungal endophyte, Undifilum oxytropis, found in locoweed plant species was shown to be responsible for the synthesis of swainsonine. Two types of Oxytropis and Astragalus locoweeds were identified from field collections, plants that accumulate high concentrations of swainsonine and plants that accumulate low or non- detectable swainsonine concentrations. The plants with high swainsonine concentrations had relatively high amounts of endophyte while plants with low or non-detectable swainsonine concentrations had low or non- detectable endophyte amounts. The group of Oxytropis and Astragalus locoweeds containing low amounts of swainsonine can be inoculated with the endophyte resulting in a plant with high amounts of endophyte and swainsonine. These results suggest that the endophyte amount must reach some critical threshold before swainsonine is present at meaningful concentrations rather than some type of suppression of the endophyte. Furthermore, in plants colonized by the endophyte, their progeny inherit the endophyte and the ability to produce swainsonine. However, in plants where the endophyte has not effectively colonized the plant, endophyte colonization and the production of swainsonine are diminished significantly in their progeny. Additionally other plant species have been documented to contain swainsonine, including some Swainsona (Leguminosae) species in Australia and some Ipomoea (Convolvulaceae), Turbina (Convolvulaceae), and Sida (Malvaceae) species in South America and Africa. Fungal endophytes that produce swainsonine have been isolated from Swainsona canescens and Ipomoea carnea. Additionally, the toxicokinetics and pathology of steers poisoned by grazing forage contaminated with elevated selenium was investigated. Furthermore experiments have been initiated to investigate the effects of swainsonine on the concentration effect curves of agonists in cultured cells. Preliminary data suggests that swainsonine changes the concentration effect curves of a number of agonists. Significant Activities that Support Special Target Populations Workshops and presentations have been given in Colorado, Idaho, Utah and Nevada to extension personnel, producers and the general public. These presentations targeted small ranchers and farmers in rural areas to reduce risk of locoweed poisoning and decrease losses from ingestion of seleniferous forages. Accomplishments 01 Selenium toxicity in cattle. Although selenium is an essential mineral, livestock that eat poisonous selenium-accumulating plants can die. ARS researchers in Logan, UT, investigating acute selenium toxicity determin that 3% of yearling steers died after grazing rangelands infested with a selenium accumulating poisonous plant called western aster. Several stee were necropsied and severe damage to muscle tissue of the heart was associated with selenium toxicity. Additionally, some poisoned steers developed congestive heart failure weeks after the toxic exposure becaus the high levels of selenium are slowly eliminated requiring a relatively long period for selenium to clear from the animal�s system. Selenium elimination rates from animals provided useful information for food safe and for producers of animals grazing on forages high in selenium. 02 Swainsonine endophyte interaction in Astragalus and Oxytropis spp. Astragalus lentiginosus and Astragalus mollissimus are two major locowee species in the southwestern United States causing significant losses. Swainsonine was detected in all parts of both these species with greater amounts found in the above ground parts. Likewise, the fungal endophyte, Undifilum oxytropis, responsible for the synthesis of swainsonine can be detected in both these species. Two chemotypes of plants were detected i population of both these species: those that contain swainsonine (> 0.1% chemotype 1) and those that contain little or no detectable swainsonine 0.01%; chemotype 2). Chemotype 1 plants in both species had quantitative higher amounts of endophyte compared to chemotype 2 plants. Understandin the relationship between swainsonine and the endophyte responsible for i production in natural plant populations may provide information to help render locoweeds non-toxic. 03 Survey of locoweed species in the Western United States. The locoweed endophyte is vertically transmitted to seed. Progeny resulting from pare plants that contained high amounts swainsonine and corresponding high amounts of endophyte inherited both high and low amounts of swainsonine and corresponding amounts of endophyte. Progeny resulting from parent plants containing low amounts of swainsonine and corresponding low amoun of endophyte inherited a similar profile of swainsonine and endophyte as the parent. Understanding how swainsonine and the endophyte responsible for its production are inherited may provide information to help render locoweeds non-toxic. 04 Locoweed poisoning in Spanish goats. Locoweeds (plant species of Astragalus and Oxytropis containing swainsonine) cause large economic losses to the livestock industry throughout the world from reproductive losses. ARS researchers in Logan, UT, fed locoweed to pregnant Spanish goats resulting in fetal death and abortions. Clinical signs including muscle tremors and aberrant behavior were also observed in the mother go It was concluded that similar reproductive and clinical signs that are observed in Spanish goats are also observed in other livestock. It shoul be noted that Spanish goats like horses are one of the more sensitive livestock species to locoweed poisoning with clinical signs and reproductive effects developing earlier. In summary, the research data suggests a rank order of sensitivity to be Spanish goats > horses > catt > sheep.
Impacts
(N/A)
Publications
- Graham, D., Creamer, R., Cook, D., Stegelmeier, B.L., Welch, K.D., Pfister, J.A., Panter, K.E., Cibils, A., Ralphs, M.H., Encinias, M., Mc Daniel, K., Thompson, D., Gardner, K. 2009. Solutions to locoweed poisoning in New Mexico and the Western United States. Rangelands. 31(6):3-8.
- Gardner, D.R., Cook, D. 2011. A comparison of alternate sample preparation procedures for the analysis of swainsonine using LC-MS/MS. Phytochemical Analysis. 22(2):95-188.
- Cook, D., Gardner, D.R., Grum, D.S., Pfister, J.A., Ralphs, M.H., Welch, K. D., Green, B.T. 2011. Swainsonine and endophyte relationships in Astragalus mollissimus and Astragalus lentiginosus. Journal of Agricultural and Food Chemistry. 59(4):1281-7.
- Ralphs, M.H., Cook, D., Gardner, D.R., Grum, D.S. 2011. Transmission of the locoweed endophyte to the next generation of plants. Fungal Ecology. 4(4):251-5.
- Gotardo, A.T., Pfister, J.A., Ferreira, M.B., Gorniak, S.L. 2011. Effects of prepartum ingestion of Ipomoea carnea on postpartum maternal and neonate behavior in goats. Birth Defects Research Part B: Developmental and Reproductive Toxicology. 92(2):131-8.
- Dantas, A.F., Riet-Correa, F., Gardner, D.R., Medeiros, R.M., Barros, S.S., Anjos, B.L., Lucena, R.B. 2009. Swainsonine-induced lysosomal storage disease in goats caused by the ingestion of Turbina cordata in Northeastern Brazil. Toxicon. 49:111-6.
- Davis, T.Z., Hall, J.O. 2011. Selenium. In: Gupta, R.C., editor. Reproductive and Developmental Toxicology. San Diego, CA. Academic Press. p. 61-8.
- Panter, K.E., Welch, K.D., Gardner, D.R. 2011. Toxic plants. In: Gupta, R. C., editor. Reproductive and Developmental Toxicology. San Diego, CA. Academic Press. p. 689-705.
- Stegelmeier, B.L., Davis, T.Z., Welch, K.D., Green, B.T., Gardner, D.R., Lee, S.T., Ralphs, M.H., Pfister, J.A., Cook, D., Panter, K.E. 2011. The comparative pathology of locoweed poisoning in horses and other livestock. In: Riet-Correa, F., Pfister, J., Schild, A.L., Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge, MA. CAB International. 48:309-10.
- Gotardo, A.T., Pfister, J.A., Barbosa-Ferreira, M., Gorniak, S.L. 2011. Neonate behavior in goats is affected by maternal ingestion of Ipomoea carnea. In: Riet-Correa, F., Pfister, J., Schild, A.L., Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge, MA. CAB International. 47:302-8.
- Davis, T.Z., Stegelmeier, B.L., Green, B.T., Welch, K.D., Panter, K.E., Hall, J.O. 2011. Acute toxicity of selenium compounds commonly found in selenium-accumulator plants. In: Riet-Correa, F., Pfister, J., Schild, A.L. , Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge, MA. CAB International. 91:525-31.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Objective I: Determine Astragalus and Oxytropis species which contain fungal endophytes that produce swainsonine and describe the plant/endophyte relationship. 1.1 Identify species that contain the endophyte (Embellisia), determine transfer of the endophyte to successive generations, and determine if the endophyte increases fitness of locoweed plants. 1.2 Describe the distribution of the endophyte and swainsonine as a function of plant part and determine if swainsonine varies as a function of time. 1.3 Determine the effect of the endophyte on palatability of locoweeds. Objective II: Identify environmental conditions that will help predict population outbreaks of major locoweed species (Oxytropis sericea, Astragalus mollissimus, A. lentiginosus). Determine the conditions under which cattle, sheep, and horses graze locoweeds. 2.1 Relate locoweed population outbreaks to weather cycles. 2.2 Determine conditions under which livestock graze various locoweed species. 2.3 Determine influence of nitrogen supplements in livestock diet selection and locoweed poisoning. Objective III: Further describe effects of swainsonine and related polyhydroxy alkaloids on reproduction and body systems among livestock and wildlife species. 3.1 Conduct a comparative study of species differences to determine why mannosidases are inhibited differently. 3.2 Compare the effects of swainsonine on fetotoxicity among breeds of sheep and goats. 3.3 Compare effects of swainsonine on ovarian function among cattle, sheep, and goats. Objective IV: Characterize biomarkers of intoxication and develop better diagnostic and prognostic procedures. 4.1 Develop ELISA for locoweed intoxication. 4.2 Develop biomarkers of poisoning. Objective V: Further describe toxicoses and pathology of animals poisoned by Astragalus species containing nitro-propionic. Objective VI: Further describe the toxicosis, physiologic effects, and pathology of Astragalus and other selenium accumulating plants, and determine absorption, distribution, and elimination (clearance times) of various types and forms of selenium in livestock. 6.1 Describe the etiology and pathogenesis of selenium poisoning and deficiency in livestock and determine safe nutritional levels. 6.2 Determine the effect of selenium-reducing microflora on the selenium pharacokinetics when livestock consume seleniferous plant material. Approach (from AD-416) 1.1 Seed from �endophyte-free� and endophyte-infected locoweed plants will be germinated to determine if the endophyte is transmitted and expressed in the next generation. If so, we will develop endophyte-free and endophyte�infected populations and compare their fitness and competitive ability. 1.2 O. sericea plants will be collected and separated into plant parts and the endophyte measured by PCR. Once the endophyte distribution within the plant is known, we will collect stalks from independent plants at 2 week intervals throughout the growing season to determine endophyte distribution and swainsonine synthesis over time. 1.3 Fungal endophytes will be grown in the laboratory using standard culture techniques, then added to ground alfalfa hay, and presented to individual animals in preference tests. 2.1 Locoweed density will be measured annually in locations throughout the Western US, and correlated with weather data to develop predictive models. 2.2 A series of grazing studies will be conducted in northeastern New Mexico beginning in late summer while grass is green and run through early winter as grasses senesce to determine cattle preference for woolly locoweed. 2.3 Supplemented and nonsupplemented groups of cattle will be grazed to determine if the supplement will reduce locoweed consumption. 3.1 Tissues from several animal species will be analyzed and mannosidase expression compared using immunohistochemistry, Western blotting, real time (RT)-PCR and Northern blots. Enzymatic in vitro assays of mannosidase activity will be compared using a modification of previously developed serum a-mannosidase assays. 3.2 Swainsonine will be fed to hair sheep, wool sheep and goats in increasing doses. Swainsonine absorption and elimination profiles will be developed, fetotoxic effects will be monitored by ultrasound, and maternal histological comparisons will be evaluated. 3.3 Swainsonine will be fed to heifers, ewes, and goats at increasing doses. Ultrasound imaging will be used to evaluate changes in follicular phase and cyst development, histological changes in ovaries will be compared, and the biological activity of anterior pituitary gonadotropins will be assayed. 4.1 Swainsonine-protein conjugates will be synthesized and injected subcutaneously into four sheep and antisera titers determined. Antisera exhibiting high titers that are specific to swainsonine will be developed into ELISA�s. 4.2 Differences in blood proteome from animals poisoned by locoweed plants will be used to identify proteins that can be used as biomarkers, then they will be validated using actual locoweed intake data. 5. A dose response study in sheep and cattle will be conducted and tissues collected for microscopic, ultrastructural and chemical analysis. 6.1 Selenium from plant material will be compared to inorganic forms at increasing doses to determine bioavailability and toxicity in sheep. 6.2 Reproductively mature ewes will be inoculated with selenobacter (Wolinella succinogenes), fed gound seleniferous plant (Astragalus bisulcatus) for eight months to monitor the effects of chronic selenium dosing on estrus cycles, gestation, and initial growth of lambs. Poisoning by Astragalus and Oxytropis species has been historically divided into three groups based on the toxic syndromes they cause in livestock. Locoweeds contain the toxic alkaloid swainsonine. Recently, a fungal endophyte (Undifilum oxytropis) was shown to be responsible for the synthesis of swainsonine. There was no difference in growth or vigor between endophyte free (E-) and endophyte infected (E+) seedlings. Different endophyte haplotypes have been identified, but E+ and E- plants are not restricted to specific endophyte haplotypes. Two groups of Oxytropis sericea were identified: one that accumulated high concentrations of swainsonine and had higher amounts of endophyte; and another where swainsonine was not detected or concentrations that were near the detection threshold and little endophyte was found. In addition, swainsonine and endophyte amounts were shown to not be uniformly distributed among stalks within the same plant. Cell-culture based models have been developed to investigate the actions of swainsonine on ligand-gated ion channels. Studies are ongoing to further characterize the model. Horses and goats are highly sensitive to the reproductive effects of locoweed poisoning. Additional histologic and morphometric studies characterizing these changes will continue. Linkers were synthesized and reacted with swainsonine to form swainsonine haptens, however products of reactions were not clean and satisfactory swainsonine haptens have not been isolated. Sera from cattle, sheep, and horses poisoned with locoweed were analyzed for proteomic changes that could serve as a biomarker of locoweed poisoning. Markers of glycosylation have not been successful. Studies that described nitrotoxin induced lesions in horses were conducted and histology on the tissues was performed. We found that nitrotoxins are neurotoxic and seem to specifically alter many large neurons of the basal ganglia. Additional histologic and immunohistologic studies continue. Data comparing differences in elimination kinetics and ultrastructural lesions in sheep dosed with various forms of selenium (organic and inorganic) were compiled and prepared in publication format and submitted for publication. Cattle that consumed selenium accumulator plants were monitored and serum, liver and muscle were collected monthly to determine the metabolism and elimination of selenium and possible long-term negative effects. Significant Activities that Support Special Target Populations Workshops and presentations have been given in Arizona, Colorado, Idaho, and Utah to extension personnel, producers and the general public, targeting small ranchers and farmers in rural areas to reduce risk of locoweed poisoning and decrease losses from ingestion of seleniferous forages. A presentation titled �Toxicity of Selenium Accumulator Plants in Livestock� was give to approximately 30 individuals representing mining companies, The Idaho Department of Environmental Quality, and The US Environmental Pollution Agency at the Fort Hall Shoshone-Bannock Reservation in Fort Hall, Idaho. The presentation was on selenium accumulator plants and their toxic properties. An update on the losses of livestock on reclaimed mine sites in southeastern Idaho, due to selenium accumulator plants was also given. Following the presentation there was a 1.5 hour discussion on procedures that should be followed when livestock are poisoned by selenium accumulator plants and precautions that can be taken to avoid poisoning. These procedures are currently being evaluated by the mining companies and governmental agencies. Accomplishments 01 Improved extraction procedures for analysis of swainsonine in locoweed samples. The published extraction procedure for the analysis of the locoweed toxin, swainsonine, is a detailed procedure involving solvent extraction followed by ion-exchange solid phase extraction and is time consuming when preparing large number of samples (e.g. >100). New procedures were developed by ARS scientists at Logan, UT and compared to the published method to simplify the extraction of samples for swainsoni analysis. They demonstrated that a simple one step solvent extraction procedure would be a more efficient method for preparing large number of samples for swainsonine screening. No differences were detected between the new procedure and the previously published method in a detailed comparison. The new method will allow rapid analysis of a large number samples to be completed in a timely manner and this will greatly enhance current research efforts in the area of locoweed/endophyte/swainsonine interactions and international locoweed plant sample surveys. 02 Effects of locoweed on fetal survival in goats. Locoweeds adversely affect most aspects of reproduction in livestock. One of these effects early embryonic resorption, fetal loss or abortion. After comparing sensitivity to locoweed poisoning between livestock species, ARS scientists in Logan, UT concluded that goats and horses are highly sensitive followed by sheep and cattle. Additionally, these results indicated that goats exhibit major propioceptive deficits much like the clinical signs observed in locoweed-poisoned horses. This research further defines the effects of locoweed on livestock and the results are used by livestock producers, extension agents and veterinarians to manag livestock grazing on locoweed infected ranges. 03 Detection of the loco toxin (swainsonine) in the fetal compartment. ARS scientists at Logan, UT fed pregnant goats locoweed, and amniotic and allantoic fluids were collected from the uterus and analyzed for swainsonine activity using a competitive binding assay. They discovered swainsonine was present in both fluids suggesting that the loco toxin crosses the placental barrier causing direct fetal toxicosis and patholo This research describes the mobility of the locoweed toxin across the placenta and the results are used by livestock producers and veterinaria to better manage livestock on rangelands infested with locoweeds.
Impacts
(N/A)
Publications
- Cook, D., Gardner, D.R., Ralphs, M.H., Pfister, J.A., Welch, K.D., Green, B.T. 2009. Swainsonine Concentrations and Endophyte Amounts of Undifilum oxytropis in Different Plant Parts of Oxytropis sericea. Journal of Chemical Ecology, Vol. 35(10):1272-1278. (www.springerlink.com) DOI 0. 1007/s10886-009-9710-9
- Pfister, J.A., Gardner, D.R., Cheney, C.C., Panter, K.E., Hall, J.0. 2010. The Capability of Several Toxic Plants to Condition Taste Aversions in Sheep. Small Ruminant Research, 90(1):114-119. doi:10.1016/j.smallrumres. 2010.02.009
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Objective I: Determine Astragalus and Oxytropis species which contain fungal endophytes that produce swainsonine and describe the plant/endophyte relationship. 1.1 Identify species that contain the endophyte (Embellisia), determine transfer of the endophyte to successive generations, and determine if the endophyte increases fitness of locoweed plants. 1.2 Describe the distribution of the endophyte and swainsonine as a function of plant part and determine if swainsonine varies as a function of time. 1.3 Determine the effect of the endophyte on palatability of locoweeds. Objective II: Identify environmental conditions that will help predict population outbreaks of major locoweed species (Oxytropis sericea, Astragalus mollissimus, A. lentiginosus). Determine the conditions under which cattle, sheep, and horses graze locoweeds. 2.1 Relate locoweed population outbreaks to weather cycles. 2.2 Determine conditions under which livestock graze various locoweed species. 2.3 Determine influence of nitrogen supplements in livestock diet selection and locoweed poisoning. Objective III: Further describe effects of swainsonine and related polyhydroxy alkaloids on reproduction and body systems among livestock and wildlife species. 3.1 Conduct a comparative study of species differences to determine why mannosidases are inhibited differently. 3.2 Compare the effects of swainsonine on fetotoxicity among breeds of sheep and goats. 3.3 Compare effects of swainsonine on ovarian function among cattle, sheep, and goats. Objective IV: Characterize biomarkers of intoxication and develop better diagnostic and prognostic procedures. 4.1 Develop ELISA for locoweed intoxication. 4.2 Develop biomarkers of poisoning. Objective V: Further describe toxicoses and pathology of animals poisoned by Astragalus species containing nitro-propionic. Objective VI: Further describe the toxicosis, physiologic effects, and pathology of Astragalus and other selenium accumulating plants, and determine absorption, distribution, and elimination (clearance times) of various types and forms of selenium in livestock. 6.1 Describe the etiology and pathogenesis of selenium poisoning and deficiency in livestock and determine safe nutritional levels. 6.2 Determine the effect of selenium-reducing microflora on the selenium pharacokinetics when livestock consume seleniferous plant material. Approach (from AD-416) 1.1 Seed from �endophyte-free� and endophyte-infected locoweed plants will be germinated to determine if the endophyte is transmitted and expressed in the next generation. If so, we will develop endophyte-free and endophyte�infected populations and compare their fitness and competitive ability. 1.2 O. sericea plants will be collected and separated into plant parts and the endophyte measured by PCR. Once the endophyte distribution within the plant is known, we will collect stalks from independent plants at 2 week intervals throughout the growing season to determine endophyte distribution and swainsonine synthesis over time. 1.3 Fungal endophytes will be grown in the laboratory using standard culture techniques, then added to ground alfalfa hay, and presented to individual animals in preference tests. 2.1 Locoweed density will be measured annually in locations throughout the Western US, and correlated with weather data to develop predictive models. 2.2 A series of grazing studies will be conducted in northeastern New Mexico beginning in late summer while grass is green and run through early winter as grasses senesce to determine cattle preference for woolly locoweed. 2.3 Supplemented and nonsupplemented groups of cattle will be grazed to determine if the supplement will reduce locoweed consumption. 3.1 Tissues from several animal species will be analyzed and mannosidase expression compared using immunohistochemistry, Western blotting, real time (RT)-PCR and Northern blots. Enzymatic in vitro assays of mannosidase activity will be compared using a modification of previously developed serum a-mannosidase assays. 3.2 Swainsonine will be fed to hair sheep, wool sheep and goats in increasing doses. Swainsonine absorption and elimination profiles will be developed, fetotoxic effects will be monitored by ultrasound, and maternal histological comparisons will be evaluated. 3.3 Swainsonine will be fed to heifers, ewes, and goats at increasing doses. Ultrasound imaging will be used to evaluate changes in follicular phase and cyst development, histological changes in ovaries will be compared, and the biological activity of anterior pituitary gonadotropins will be assayed. 4.1 Swainsonine-protein conjugates will be synthesized and injected subcutaneously into four sheep and antisera titers determined. Antisera exhibiting high titers that are specific to swainsonine will be developed into ELISA�s. 4.2 Differences in blood proteome from animals poisoned by locoweed plants will be used to identify proteins that can be used as biomarkers, then they will be validated using actual locoweed intake data. 5. A dose response study in sheep and cattle will be conducted and tissues collected for microscopic, ultrastructural and chemical analysis. 6.1 Selenium from plant material will be compared to inorganic forms at increasing doses to determine bioavailability and toxicity in sheep. 6.2 Reproductively mature ewes will be inoculated with selenobacter (Wolinella succinogenes), fed gound seleniferous plant (Astragalus bisulcatus) for eight months to monitor the effects of chronic selenium dosing on estrus cycles, gestation, and initial growth of lambs. Significant Activities that Support Special Target Populations Poisoning by Astragalus and Oxytropis species has been historically divided into three groups based on the toxic syndromes they cause in livestock, 1) locoism caused by the toxin swainsonine, 2) nitrotoxin poisoning, and 3) selenium poisoning. Locoweed poisoning is one of the most widespread poisonous plant problems in the western United States. Locoweeds are Astragalus and Oxytropis species that contain the toxic alkaloid swainsonine. Recently, a fungal endophyte, Undifilum oxytropis, was shown to be responsible for the synthesis of swainsoine. Research is being pursued to generate endophyte free plants via application of fungicides and by removal of the seed coat (the endophyte has been shown to reside in the seed coat). The distribution of the endophyte and swainsonine are being investigated in Astragalus and Oxytropis species. Preliminary results suggest that the endophyte is found primarily in the crown and above ground parts of the plant while swainsonine is found primarily in above ground parts of the plants. Locoweed was fed to pregnant western white face ewes (wool sheep), St. Croix ewes (hair sheep) and Spanish goats to compare effects on pregnancy. The Spanish goats were most sensitive resulting in relatively early abortions and proprioceptive deficits in treated does. Hair sheep and wool sheep were similar in their response i.e. abortion occurred much later or not at all. Cows, sheep and goats were fed locoweed to study reproductive effects. Ultrasound imaging and hormone analysis in heifers demonstrated ovarian cysts and altered reproductive cycles. In sheep, laparotomies were performed and similar ovarian cysts were observed in sheep. Spanish goats have not been completed. Sera from cattle, sheep, and horses poisoned with locoweed were analyzed for proteomic changes that could serve as a biomarker of locoweed poisoning. Some alterations in protein profiles were identified, but it is not known at this time if these changes are indicative of locoweed poisoning. Studies have been conducted in horses and histology was performed to describe nitrotoxin induced lesions. Histological analyses of lesions in lambs dosed with various forms of selenium (organic and inorganic) were completed and publications are being prepared. Samples have been analyzed to determine the absorption and elimination kinetics of various forms of selenium dosed to sheep under different conditions. Additionally, protocols were partially developed for the speciation of different forms of selenium by HPLC-ICP-MS. Technology Transfer Number of Web Sites managed: 1 Number of Other Technology Transfer: 8
Impacts
(N/A)
Publications
- Cook, D., Gardner, D.R., Welch, K.D., Roper, J.M., Ralphs, M.H., Green, B. T. 2009. Quantitative PCR Method to Measure the Fungal Endopyte in Locoweeds. Journal of Agricultural and Food Chemistry. 57:6050-6054
- Cook, D., Ralphs, M.H., Welch, K.D., Stegelmeier, B.L. 2009. Locoweed Poisoning in Cattle. Rangelands, 31(1):16-21. http://www.bioone. org/doi/pdf/10.2111/1551-501X-31.1.45?
- Ralphs, M.H. 2009. Response of Broom Snakeweed (Gutierrezia sarothrae) and Cool-Season Grasses to Defoliation. Journal of Invasive Plant Science and Management. 2(1):28-35. DOI. 10.1614/PSM-08-075.1
- Ralphs, M.H., Banks, J. 2009. Cattle Grazing as a Biological Control for Broom Snakeweed: Vegetation Response. Rangeland Ecology and Management, 62:38-43.
- Ralphs, M.H., Jensen, D.T. 2007. Population Cycles of Poisonous Plants. Poisonous Plant Global Research and Solutions. Ch 73. pp. 432-437.
- Ping, L., Child, D., Meng-Li, Z., Gardner, D.R., Gui-Fen, L., Guo-Dong, H. 2009. Culture and Identification of Endophytic Fungi from Oxytropis glabra DC. ACTA Ecologica Sinica. 20(1):53-58
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) Objective I: Determine Astragalus and Oxytropis species which contain fungal endophytes that produce swainsonine and describe the plant/endophyte relationship. 1.1Identify species that contain the endophyte (Embellisia), determine transfer of the endophyte to successive generations, and determine if the endophyte increases fitness of locoweed plants. 1.2 Describe the distribution of the endophyte and swainsonine as a function of plant part and determine if swainsonine varies as a function of time. 1.3 Determine the effect of the endophyte on palatability of locoweeds. Objective II: Identify environmental conditions that will help predict population outbreaks of major locoweed species (Oxytropis sericea, Astragalus mollissimus, A. lentiginosus). Determine the conditions under which cattle, sheep, and horses graze locoweeds. 2.1 Relate locoweed population outbreaks to weather cycles. 2.2 Determine conditions under which livestock graze various locoweed species. 2.3 Determine influence of nitrogen supplements in livestock diet selection and locoweed poisoning. Objective III: Further describe effects of swainsonine and related polyhydroxy alkaloids on reproduction and body systems among livestock and wildlife species. 3.1 Conduct a comparative study of species differences to determine why mannosidases are inhibited differently. 3.2 Compare the effects of swainsonine on fetotoxicity among breeds of sheep and goats. 3.3 Compare effects of swainsonine on ovarian function among cattle, sheep, and goats. Objective IV: Characterize biomarkers of intoxication and develop better diagnostic and prognostic procedures. 4.1 Develop ELISA for locoweed intoxication. 4.2 Develop biomarkers of poisoning. Objective V: Further describe toxicoses and pathology of animals poisoned by Astragalus species containing nitro-propionic. Objective VI: Further describe the toxicosis, physiologic effects, and pathology of Astragalus and other selenium accumulating plants, and determine absorption, distribution, and elimination (clearance times) of various types and forms of selenium in livestock. 6.1 Describe the etiology and pathogenesis of selenium poisoning and deficiency in livestock and determine safe nutritional levels. 6.2 Determine the effect of selenium-reducing microflora on the selenium pharacokinetics when livestock consume seleniferous plant material. Approach (from AD-416) 1.1 Seed from �endophyte-free� and endophyte-infected locoweed plants will be germinated to determine if the endophyte is transmitted and expressed in the next generation. If so, we will develop endophyte-free and endophyte�infected populations and compare their fitness and competitive ability. 1.2 O. sericea plants will be collected and separated into plant parts and the endophyte measured by PCR. Once the endophyte distribution within the plant is known, we will collect stalks from independent plants at 2 week intervals throughout the growing season to determine endophyte distribution and swainsonine synthesis over time. 1.3 Fungal endophytes will be grown in the laboratory using standard culture techniques, then added to ground alfalfa hay, and presented to individual animals in preference tests. 2.1 Locoweed density will be measured annually in locations throughout the Western US, and correlated with weather data to develop predictive models. 2.2 A series of grazing studies will be conducted in northeastern New Mexico beginning in late summer while grass is green and run through early winter as grasses senesce to determine cattle preference for woolly locoweed. 2.3 Supplemented and nonsupplemented groups of cattle will be grazed to determine if the supplement will reduce locoweed consumption. 3.1 Tissues from several animal species will be analyzed and mannosidase expression compared using immunohistochemistry, Western blotting, real time (RT)-PCR and Northern blots. Enzymatic in vitro assays of mannosidase activity will be compared using a modification of previously developed serum a-mannosidase assays. 3.2 Swainsonine will be fed to hair sheep, wool sheep and goats in increasing doses. Swainsonine absorption and elimination profiles will be developed, fetotoxic effects will be monitored by ultrasound, and maternal histological comparisons will be evaluated. 3.3 Swainsonine will be fed to heifers, ewes, and goats at increasing doses. Ultrasound imaging will be used to evaluate changes in follicular phase and cyst development, histological changes in ovaries will be compared, and the biological activity of anterior pituitary gonadotropins will be assayed. 4.1 Swainsonine-protein conjugates will be synthesized and injected subcutaneously into four sheep and antisera titers determined. Antisera exhibiting high titers that are specific to swainsonine will be developed into ELISA�s. 4.2 Differences in blood proteome from animals poisoned by locoweed plants will be used to identify proteins that can be used as biomarkers, then they will be validated using actual locoweed intake data. 5. A dose response study in sheep and cattle will be conducted and tissues collected for microscopic, ultrastructural and chemical analysis. 6.1 Selenium from plant material will be compared to inorganic forms at increasing doses to determine bioavailability and toxicity in sheep. 6.2 Reproductively mature ewes will be inoculated with selenobacter (Wolinella succinogenes), fed gound seleniferous plant (Astragalus bisulcatus) for eight months to monitor the effects of chronic selenium dosing on estrus cycles, gestation, and initial growth of lambs. 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. Locoweeds (Astragalus and Oxytropis species that contain swainsonine) are wide spread on rangelands of the western United States and in many other parts of the world. Swainsonine is also found in other plant species, including Swainsona spp in Australia, Ipomoea spp in Brazil and Africa. Recently, an endophyte was discovered in the locoweed species and was shown to be responsible for swainsonine production. PCR methods have been developed to quantify the endophyte in Astragalus mollissimus and Oxytropis sericea. Locoweeds cause poisoning in all classes of livestock but horses and Spanish goats are especially sensitive. In addition to behavioral changes after prolonged ingestion of locoweeds, reproductive processes are inhibited and fetal toxicity and death occurs, neonatal/maternal behavior is altered and abortions and occasional birth defects result. Selenium is often taken up by certain plant species and may be toxic if eaten in excess. Some Astragalus species act as selenium pumps, bringing selenium from lower soil profiles to the surface where shallow rooted species may take it up. While selenium is an essential micro nutrient, if ingested in excess lesions of the skin, wool, hair, hooves etc may occur. Absorption, distribution, retention and elimination of selenium depends on the form in which it is ingested. Plant forms may be organic or inorganic. The organic forms are more bioavailable and the kinetic profiles much different than the inorganic forms. Both forms are toxic but clinical effects and pathology can be very different depending on the chemical form of the selenium. There are Astragalus species that accumulate nitro toxins. Studies have shown that 3-nitro propanol is toxic to horses and occasional field cases have been reported. Technology Transfer Number of Web Sites managed: 1 Number of Other Technology Transfer: 2
Impacts
(N/A)
Publications
- Ralphs, M.H., Monaco, T.A., Valdez, J.R., Graham, D. 2007. Seeding cool- season grasses to suppress white locoweed (Oxytropis sericea) reestablishment and increase forage production. Weed Technology, 21:661- 669.
- Ralphs, M.H., Creamer, R., Baucom, D., Gardner, D.R., Welch, S.L., Graham, J.D., Hart, C., Cook, D., Stegelmeier, B.L. 2007. Relationship between the endophyte embellisia spp. and the toxic alkaloid swainsonine in major locoweed species (Astragalus and Oxytropis). Journal of Chemical Ecology, Vol. 34 No. 1 pp.32-28 (2008). Published online 12/01/2007 DOI 10. 1007/s10886-007-9399-6
- Barbosa, R.C., Riet-Correa, F., Lima, E.F., Medeiros, R.M., Guedes, K.M., Gardner, D.R., Molyneux, R.J., De Melo, L.E. 2007. Experimental swainsonine poisoning in goats ingesting Ipomoea sericophylla and Ipomoea riedelii (Convolvulaceae). Pesquisa Veterinaria Brasileira, 27(10):409-414.
- Barbosa, R.C., Riet-Correa, F., Medeiros, R.M., Lima, E.F., Gimeno, J.E., Barros, S.S., Molyneux, R.J., Gardner, D.R. 2006. Intoxication by ipomoea sericophylla and ipomoea riedelii in goats in the state of paraiba, northeastern brazil. Toxicon. Volume:47:371-379.
- Hueza, I.M., Guerra, J., Haraguchi, M., Gardner, D.R., Asano, N., Ikeda, K. , Gorniak, S.L. 2007. Assessment of the perinatal effects of maternal ingestion of Ipomoea carnea in rats. Experimental and Toxicologic Pathology, 58:439-446.
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