2013 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.
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.
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.
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.
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.