Location: Poisonous Plant Research2010 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.
3. Progress Report
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.
1. 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 swainsonine 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 of 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.
2. Effects of locoweed on fetal survival in goats. Locoweeds adversely affect most aspects of reproduction in livestock. One of these effects is 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 manage livestock grazing on locoweed infected ranges.
3. 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 pathology. This research describes the mobility of the locoweed toxin across the placenta and the results are used by livestock producers and veterinarians to better manage livestock on rangelands infested with locoweeds.
5. 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.
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