2012 Annual Report
1a.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.
1b.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.
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 microbial shifts in selenium metabolizing microbes was investigated by ARS researchers in Logan, UT using artificial rumen fermenters. Orchardgrass containing 50 ppm Se had no negative impacts on laboratory assessments of ruminal fermentation. An increase in selenate metabolizing microbes, however, suggests that high dietary Se may cause ruminal adaptations that 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.
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 or 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.
The toxic principle of Ipomoea asarifolia, a morning glory. The Ipomoea (morning glory) genus contains many species, a number of which contain the same toxin as locoweeds. Additionally, some Ipomoea species contain ergot 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 were 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.
Swainsonine and endophyte interactions in locoweed associated with plant development. It is not known if swainsonine and endophyte amounts in white 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 stages. Swainsonine and endophyte amounts increase as the plant matures and swainsonine amounts were positively associated with endophyte amounts. Due to this research, better predictions can be provided to producers regarding relative risk of animals grazing on locoweeds.
Influence of seed endophyte amount in locoweeds. Two types of plants exist 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 this difference. ARS researchers in Logan, UT demonstrated that these two types of plants could be interchanged by manipulating seed endophyte amounts. ARS researchers demonstrated that plants derived from fungicide treatment of seeds had very little swainsonine and endophyte. Using the methods developed herein and the further understanding of the relationship between swainsonine and the endophyte in different locoweed species may provide information to help manage locoweed infested ranges and render plants non-toxic.
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 were 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 for producers of animals grazing on forages high in selenium.
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.