Location: Poisonous Plant Research2011 Annual Report
1a. Objectives (from AD-416)
Objective I: Pine Needles 1.1 Determine if isocupressic acid (ICA; the abortifacient compound in pine needles) concentration in pine needles is modulated by the environment. 1.2 Identify the matabolites of isocupressic acid in pine needles that cause abortions in cattle. Determine the biological mechanism, develop diagnostic techniques, and therapeutic procedures. 1.3 Determine factors that influence cattle consumption of pine needles and develop management practices to prevent abortion. Objective II: Broom Snakeweed 2.1 Identify the toxic and abortifacient compounds in broom snakeweed. 2.2 Describe the ecology of broom snakeweed, develop management and control guidelines to reduce incidence of poisoning and abortion in livestock. Objective III: Lupine 3.1 Isolate, identify, and evaluate toxicity and teratogenicity of lupine alkaloids which cause birth defects in calves born to cows that graze these plants. 3.2 Evaluate the role of genotype and environment on lupine alkaloids, and thus the relative toxicity of various species and populations of lupine. 3.3 Determine the physiological mechanism of lupine-induced birth defects and evaluate the maternal and fetal toxicokinetics of alkaloids. 3.4 Evaluate the influence of climate on population cycles of lupine. 3.5 Determine the importance of lupines as nutritional components for cattle during critical times of the year. 3.6 Identify conditions under which cattle graze various lupine species. Objective IV: Veratrum 4.1 Develop models to study the toxicokinetics, including clearance times, and toxicity of steroidal alkaloids in Veratrum californicum.
1b. Approach (from AD-416)
1.1 Data on environmental conditions will be collected at each site using local weather stations. ICA levels and environmental conditions will be correlated to determine if any patterns emerge. Soil samples will be collected at each site for future evaluation. 1.2 Samples of maternal and fetal tissues will be collected for histologic analysis and determination of ICA concentrations using existing ELISA’s and GC/MS methods. Proteomic analyses via LC/MS/MS techniques will be done. 1.3 Pen and field studies using cattle in high, medium and low body condition will be done to determine effects on needle consumption and grazing times. Nutrient supplements will be offered to determine if pine needle consumption will be altered. 2.1 The diterpene acid “fingerprint” of broom snakeweed from various populations in Arizona, New Mexico and Utah will be determined by chemical analysis. Subsequent in vitro and in vivo studies will be done to determine abortifacient activity. 2.2 A grazing study will be conducted to determine if various management practices can be implemented to force cattle to graze snakeweed as a biological control. A clipping study will be conducted to further describe the effects of defoliation on snakeweed and the surrounding plant community. 3.1 Alkaloids will be isolated by chemical methods and identified by chromatography, NMR, mass spectrometry, and elemental analysis. Toxicology will be evaluated using a mouse bioassay and cell lines that express nicotinic acetylcholine receptors. 3.2 A chemical fingerprint of Lupinus sulphureus collected from different locations will be generated using chemical methods. Fingerprints will be analyzed via cluster analysis and phylogenetic analysis will be performed using AFLPs (Amplified Fragment Length Polymorphisms) to determine the genetic relationship of the populations. 3.3 Pregnant goats in late gestation will be used to determine the rate of absorption, distribution and elimination of the teratogenic alkaloids. The pharmacokinetic profiles of the alkaloids will be compared between maternal and fetal systems. 3.4 Established transects will be monitored over the next 5 years to determine the influence of weather patterns on lupine density. Correlations of lupine age, class, density, and trends will be made with seasonal precipitation and temperature. 3.5 Consumption of lupines by cattle on rangelands dominated by low quality forages may be related to nutrient content. Twelve yearling heifers in a field study will be supplemented with different levels of protein to compare lupine ingestion. 3.6 Short-duration and high intensity grazing studies in early, mid, and late summer will be used to determine what role grazing pressure has on lupine intake during different seasons of the year. 4.1 A monogastric model (swine) will be used to determine the kinetics (clearance and metabolism) of a well known teratogenic alkaloid from Veratrum. This pilot project will be a model for testing the clearance of other plant toxins from animal tissues to evaluate food safety of animal products. Clearance rates between the monogastric model and small ruminant model will be compared.
3. Progress Report
In collaboration with FRRL, Logan, UT., replicated seeding plots were established in 2010 in Eastern Washington using various grass species (including improved and native germplasm) and 3 species of forage Kochia. Additionally, larger demonstration plots were seeded with a mix of crested wheat grass and Emigrant forage Kochia. The hypothesis is that crested wheat grass and forage Kochia will provide alternative forages at the critical time when pregnant cattle begin to eat lupine (July-September). Seeding plots were established on four different ranches on the Scablands and early evaluation demonstrated good to excellent germination on all plots. Further evaluations will occur over the next 5 years, and grazing studies will be done on the larger demonstration plots once the grasses and Kochia become established. The plots will be evaluated in the fall of 2011 and then each spring for the next 5 years. Chemical analysis of needles and bark from various types of pine and juniper trees continues in order to fully understand the abortion risks when late term cattle graze in areas where these types of trees are abundant. Cell culture-based experiments are being used to compare piperidine and quinolizidine alkaloids to help identify the mechanism by which these compounds induce birth defects in livestock species. Current structure-function studies are underway using compounds selective for specific receptor binding sites on the fetal muscle-type receptors comparing alkaloids which do not cause fetal malformations versus alkaloids that do. Initial results suggest that there are differences between the two classes of alkaloids, which could help to determine the molecular mechanism of teratogenesis. These results have led to the generation of the hypothesis that the mechanism behind these types of birth defects is the inhibition of fetal movement due to stimulation followed by desensitization of the receptors. A preliminary chemical analysis of leaves and seeds from Mimosa tenuiflora was performed. An initial evaluation of the teratogenic potential of leaves and seeds, as well as extracts of both, has been made using a rat model in order to identify the teratogenic compound in Mimosa tenuiflora.
1. Evaluation of intermittent grazing as a method to minimize lupine-induced birth defects. Lupines continue to cause large economic losses to the cattle industry in the northwest. ARS researchers in Logan, UT, analyzed alkaloid in blood from pregnant cows fed lupine plants intermittently during gestation days 30-60 demonstrated that the teratogenic alkaloids clear from the maternal circulation within 48 hours. Fetal movement returned to normal activity shortly thereafter. This suggests that intermittent grazing may be a useful management tool in preventing or reducing the losses from lupine-induced “Crooked Calf Syndrome”. As a follow up study, 15 pregnant black Angus cattle were divided into 3 treatment groups: 1) control; 2) continuous treatment and 3) intermittent treatment. The control cows delivered normal calves. The cows fed lupine continuously from gestation days 30-60 delivered calves with moderate to severe birth defects, while those fed lupine intermittently (10 days on and 5 days off) delivered calves with few minor skeletal defects, and these resolved completely within 4 weeks after birth. These results demonstrate the potential of using an intermittent grazing program to reduce lupine-induced birth defects on ranches where lupine continues to cause economic losses.
2. Grazing trials of lupine throughout the growing season. ARS researchers in Logan, UT, conducted intensive grazing trials with cattle in eastern Washington to evaluate the consumption of lupine in different stages of plant development through the growing season. Ten Hereford cows and a bull grazed velvet lupine-infested rangeland in 3 grazing pressure trials each year over two grazing seasons. Pastures were fenced to limit forage availability to the amount required for the trials. In June, cheatgrass was dry and cattle preferred forbs that were flowering. As availability of palatable forbs declined, cows started eating lupine, and this declined as its availability declined. In July when annual forbs matured and became fibrous and unpalatable, cattle ate lupine at the beginning of the trial and reverted to dry cheatgrass as lupine became limited. We conclude that lupine is not palatable early in the growing season in May and early June, but cattle can be forced to graze it as availability of green cheatgrass and other forbs declines. Intensive grazing systems that force cattle to use all forage may enhance the risk of crooked calf syndrome by forcing cattle to graze lupine throughout the grazing season. Therefore, stocking rates and management programs should be flexible and adjusted to avoid forcing cattle to graze lupines.
3. Management recommendations to minimize the impact of broom snakeweed on plant communities. Broom snakeweed is an invasive native sub-shrub that is distributed widely across rangelands of western North America. It often increases to near monocultures following disturbance from overgrazing, fire, or drought. The greatest ecological concern is that broom snakeweed displaces desirable forage for livestock or wildlife and greatly reduces biodiversity. Although its populations cycle with climatic patterns, it can be a major factor impeding succession of native or desirable plant communities. It also is toxic and can cause abortions in all species of livestock. Population increases of broom snakeweed are usually higher in wet years, allowing large expanses of even-aged stands to establish and dominate plant communities. ARS researchers in Logan, UT, demonstrated that snakeweed can be controlled by prescribed burning, targeted grazing or spraying with herbicides. Follow up research demonstrated that establishment and maintaining competitive grasses through controlled grazing management can prevent or minimize broom snakeweeds’ reinvasion, improve grazing conditions and support critical biodiversity.
4. Toxic alkaloid profiles of poisonous Lupinus species. Many species in the Lupinus genus are poorly defined morphologically, potentially resulting in improper taxonomic identification. Correct identification is important because several, but not all, lupine species contain alkaloids that can be acutely toxic and/or impair physiological development leading to birth defects in livestock, such as lupine-induced crooked calf syndrome. Consequently, a better method to correctly identify the poisonous Lupinus species is needed. ARS researchers in Logan, UT, characterized the alkaloid profiles of four Lupinus species to aid in discriminating these species from each other. They discovered diagnostic chemical fingerprints. These fingerprints can be used as a tool to discriminate these species from each other, as long as one considers locality of the collection. These results will provide livestock producers with valuable information to help reduce economic losses that occur as a result of lupine-induced crooked calf syndrome.
5. Evaluation of western juniper-induced abortions in cattle. Ranchers in Baker County, Oregon have reported atypical late term abortions of 10-15% of their herds. Ponderosa pine and other trees known to cause abortions in cattle were not found in the areas in which these abortions occurred. However, there was clear visual evidence that the cattle had been eating the bark of western juniper trees (Juniperus occidentalis). ARS researchers in Logan, UT, analyzed samples of western juniper needles, berries, and bark for labdane acids (the compounds in pine needles that cause abortions in cattle). Analyses indicated that the bark of western juniper trees had a fairly high concentration of labdane acids. Consequently, studies were performed by ARS researchers in Logan, UT, to determine if the bark from western juniper trees could induce abortions in cattle. Two cows aborted 4-5 days after the start of the treatment. Both cows had clinical signs consistent with abortion. The remaining 4 cows calved at full term (26-31 days after the start of treatment) and had no clinical signs of abortion. Results from this study indicated that western juniper trees contain compounds known to cause abortions in cattle and thus consumption of large amounts of bark in the third trimester of gestation may induce abortions. Although the risk of abortion from eating western juniper bark appears to be less than that of ponderosa pine needles, livestock producers should be aware of this potential.
6. Evaluation of the placental development in ewes with malformed lambs. Cyclopia and a number of other birth defects occur in lambs from ewes that graze Veratrum californicum early in gestation. The mechanism of cyclopamine-induced birth defects has been shown to result from the inhibition of an important signaling pathway. This pathway plays an integral role in cell growth and differentiation. Previous studies by ARS researchers in Logan, UT, have demonstrated that lambs with cyclopia were smaller, under developed, and appeared premature compared to normal lambs of similar gestation age. Preliminary observations suggested this was due to deficiencies in placental development. Consequently, studies were performed by ARS researchers in Logan, UT, to determine if there are deficiencies in placental development in ewes with lambs with cyclopia and other less severe head and face malformations. The results from these studies confirmed that cyclopic lambs are smaller than normal lambs as well as less severely malformed lambs. Additionally, they demonstrated that placental development in ewes with cyclopic lambs is compromised. Due to the lack of brain and pituitary gland development in many of the cyclopic lambs, it is quite likely that the lack of normal placental development in ewes with cyclopic lambs is a result of insufficient contribution of the embryo during critical periods of placental development. This information will be useful in further understanding how Veratrum californicum can alter the reproductive capabilities of livestock.
Faeth, S.H., Hayes, C.J., Gardner, D.R. 2010. Asexual endophytes in a native grass: Tradeoffs in mortality, growth, reproduction, and alkaloid production. Microbial Ecology. 60:496-504. DOI: 10.1007/s00248-010-9643-4.