Location: Poisonous Plant Research2009 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, 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.
1. Evaluating the endophyte (Undilfilum oxytropis) in locoweeds. The locoweed toxin, swainsonine, is synthesized by a fungal endophyte (Undifilum oxytropis); there is a need to detect this endophyte, measure its growth in different plant parts and transmission to seed and progeny; and determine the amount of swainsonine it produces. A real-time PCR method was developed to quantify the fungus in plant tissue. Primers amplifying the ITS sequence within the fungal genome were used to detect and quantify the amount of Undifilum oxytropis. This method of analysis was used to quantify the amount of endophyte in 10 plants each of Oxytropis sericea (White locoweed), Astragalus mollissimus (Wooly Locoweed), and Astragalus lentiginosus (Spotted Locoweed). In one O. sericea and one A. lentiginosus plant swainsonine concentrations were near or below the limit of detection. Interestingly, these plants also had lowest amounts of the endophyte when compared to the other specimens. Technology to quantify the endophyte may lead to solutions to the locoweed poisoning problem. It will also allow for future research to understand the biology of the fungus, its growth and transmission, and the synthesis of swainsonine.
2. A comparative toxicity study of the different forms of selenium. Excess consumption of selenium accumulator plants can result in selenium intoxication. The objective of the research was to compare the acute toxicosis caused by selenate and Se-methylselenocysteine, the two most common forms of selenium found in selenium accumulator plants. The absorption and elimination kinetics, respiratory kinetics, and histopathologic changes in lambs dosed with selenate and methylselenocysteine were determined. Severity and time to recovery varied, and were dose dependent. Major histopathologic findings in animals of the high-dose groups included multifocal myocardial necrosis and pulmonary alveolar vasculitis with pulmonary edema and hemorrhage. Results of this research indicate that selenium accumulator plants with the predominant chemical form as Se-methylselenocysteine are more toxic when ingested than plants with the predominant chemical form of selenium as selenate. These findings support previous research that the chemical form of selenium must be known to adequately interpret tissue, blood, and serum selenium concentrations and to determine the risk of toxicity.
3. Comparative effects of Locoweed in sheep and cattle. Locoweed was fed to pregnant western white face ewes (wool sheep), St. Croix ewes (hair sheep) and Spanish goats to compare effects on pregnancy. 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 e.e. abortion occurred much later or not at all. When cattle, sheep and goats were compared, ovarian cysts and altered reproductive cycles occurred. In sheep, laparotomies were performed which showed similar enlarged cyctic ovaries to that observed in cattle with ultrasound. Locoweed induced ovarian dysfunction has not been studied yet in Spanish goats.
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