Location: Poisonous Plant Research2008 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. 1.1Identify 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.
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
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.
1. Develop method to quantify endophyte species in locoweeds. Locoweed poisoning is one of the most widespread poisonous plant problems in the western United States. Locoweeds species Astragalus and Oxytropis contain the toxic alkaloid swainsonine. Recently a fungal endophyte found in these plant species has been implicated in the synthesis of swainsonine. The objective of this study was to develop a quantitative PCR method to quantify the fungal endophyte in Astragalus mollissimus and Oxytropis sericea, thus enabling a better understanding of the role of the endophyte in swainsonine production and their relationship in different plant parts. A Real-Time (RT) PCR method to quantify the fungal endophyte, Undiffilum oxytropis formally Embellesia, in locoweeds was developed. A RT-PCR method was developed that accurately quantifies the amount of endophyte in a given sample. This method will provide a useful tool to further understand the plant endophyte relationship as it relates to swainsonine production. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P.
2. Transmission of endophyte to 2nd generation. Recently it was discovered that the locoweed toxin swainsonine is produced by a fungal endophyte Undifilum oxytropis. There is a need to determine which Astragalus and Oxytropis species have the endophyte and are toxic. Major locoweed species were surveyed in 2005 to verify they contained the endophyte and produced swainsonine. Seeds were collected from these plants to verify transmission of the endophyte to succeeding generations. Seed from each parent plant was germinated and seedlings were tested for the endophyte by real time PCR and swainsonine concentration by GC/MS. The endophyte was successfully transmitted in 80% of the progeny from maternal plants that had the endophyte and produced swainsonine. The endophyte was suppressed in all progeny from maternal plants in which the endophyte could not be cultured. Determining why and how the endophyte is suppressed, will enable us to predict risk and reduce losses. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P.
3. Locoweeds are responsible for a decrease in reproductive performance. Locoweeds are responsible for a decrease in reproductive performance in livestock resulting in large economic losses to the livestock industry. Toxicity was thought to be similar between animal species and driven by length of time of ingestion. Locoweed (Astragalus lentiginosus) was fed to pregnant sheep and goats at 2 dosages (2.5 and 5 mg swainsonine/ kg BW) to compare clinical toxicoses and fetotoxicity. Two breeds of sheep, hair sheep (St. Croix), and western white faced wool sheep were compared with Spanish goats. The Spanish goats were the most sensitive to the maternal and fetal effects and exhibited severe clinical signs including muscle tremors, ataxia, proprioceptive deficits and fetal death within 2 weeks of dosing. The hair sheep and wool sheep were similar in their clinical response and while they eventually showed severe clinical signs of poisoning, it was much later than the Spanish goats. The proprioceptive deficits in the Spanish goats were very similar to what is observed in horses poisoned on locoweeds. Apparently Spanish goats are very sensitive to the negative effects of locoweeds. This research indicates that Spanish goats should not be allowed to graze locoweeds for even a short period of time. Results of this research will provide better management recommendations to reduce losses from locoweeds and will improve reproductive performance and utilization of locoweed infested rangelands. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P.
4. Locoweeds interfere with normal reproductive processes in livestock. Locoweeds interfere with normal reproductive processes in livestock including estrus behavior, estrous cycle function, pregnancy, etc.Sheep and cattle were dosed with locoweed (Astragalus lentiginosus) at 2.5 and 5 mg swainsonine/kg BW. Ovarian function was evaluated in heifers using ultrasound imaging and in sheep via laparotomy. In heifers ovaries became enlarged and cystic within two weeks after feeding locoweed. The effects were dose and time dependent. Once locoweed feeding stopped the ovaries returned to normal within 30 to 60 days. In sheep ovaries were cystic and CL was hemorrhagic. This research provides a more clear understanding of the negative impact that locoweeds have on early reproductive processes in livestock. Sheep, cattle, and goats respond differently to locoweed and options for ranchers will be better understood. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P.
5. Description of lesions caused by selenite, selenomethionine, and plant associated selenium. Different forms of selenium appear to have different toxicities. A dose response study was conducted to compare the toxicity of selenite, selenomethione, and plant associated selenium (selenium in Woody Aster). The results from this study indicate different forms of selenium are metabolized differently and cause significantly different lesions and clinical signs. The predominant lesions caused by selenite and selenomethionine were pulmonary vasculitis with subsequent pulmonary edema. When ground plant material (woody aster) was used as the selenium source lesions were found predominantly in the cardiac musculature. From this study we documented the importance of knowing the ingested form of selenium when interpreting serum, whole-blood, and tissue data from cases of selenium intoxication. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P.
5. Significant Activities that Support Special Target Populations
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(3):661-669.
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.
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.