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United States Department of Agriculture

Agricultural Research Service

Research Project: LIVESTOCK LOSSES FROM ABORTIFACIENT AND TERATOGENIC PLANTS
2012 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 the Forage and Range Research Laboratory, Logan, UT, scientists at the Poisonous Plant Research Laboratory evaluated replicated plots of various grass species and forage Kochia in Eastern Washington for forage production. Additionally, grazing studies on these plots with cattle were initiated. Chemical analysis of needles, bark, and berries from western juniper trees from 35 locations across the state of Oregon were performed in order to evaluate the risk of western juniper trees to cause late-term abortions in cattle. Metabolites of isocupressic acid were detected in stomach and thoracic fluids from aborted calves. Detection of the metabolites in the fluids from aborted calves will be an important diagnostic tool useful for identification of pine needle abortion cases. Cell culture-based experiments are being used to compare piperidine and quinolizidine alkaloids from poisonous plants to help identify, and further define, the mechanism by which these compounds induce birth defects in livestock species. Additionally, a rat model is being used to characterize the potential of purified plant toxins, including enantiomers of the toxins, to cause birth defects.


4.Accomplishments
1. Method to diagnose field cases of pine-needle induced abortions in cattle. In a cooperative effort with the Veterinary Diagnostic Laboratory, Iowa State University, ARS researchers in Logan, UT, examined bovine fetal fluids, including stomach and thoracic fluids, from possible pine needle abortion cases. Tetrahydroagathic acid, a known metabolite of isocupressic acid (the abortifacient compound in pine needles), was detected in the aborted bovine fetal fluids from submitted diagnostic cases. Detection of tetrahydroagathic acid could prove to be a valuable diagnostic tool to identify cases of pine-needle induced abortions in cattle. Detection of serum metabolites in mother cows also continues to be a useful diagnostic tool and was used to support pine needle diagnosis in cases from California and Wyoming.

2. Survey of western juniper trees across the state of Oregon. Previous research by ARS researchers in Logan, UT, has demonstrated that western juniper trees can induce late-term abortions in cattle, similar to ponderosa pine trees. However, this risk is not well characterized and it is unclear how much variation there is in the abortifacient compounds in western juniper trees. A previous report has shown that there can be significant variation in the abortifacient compounds in ponderosa pine needles from location to location and over time. Thus, ARS researchers in Logan, UT collected samples of bark, needles, and berries from western juniper trees from 35 locations across the state of Oregon in order to determine the variation in abortifacient compounds in western juniper trees. Results indicated that western juniper trees throughout the state of Oregon should be considered a risk to induce late-term abortions in cattle. This knowledge will allow livestock owners, and others, to better understand the potential abortion risk from western juniper trees.

3. Metabolism of abortifacient compounds from ponderosa pine needles in cattle. Ponderosa pine needles are known to induce abortions in cows when consumed during the last trimester of pregnancy. The known toxins in the needles that induce abortions in cattle are isocupressic acid and agathic acid, a metabolite of isocupressic acid. Previous research at the Poisonous Plants Research Lab, Logan UT suggested that extended exposure of cattle to pine needles resulted in altered metabolism and/or elimination of the abortifacient compounds. ARS researchers in Logan, UT demonstrated that cattle conditioned to ponderosa pine needles can more efficiently metabolize the abortifacient compound agathic acid than naïve cattle. These results suggest that extended exposure of cattle to pine needles results in a physiological change in the cattle such that the known abortifacient compounds in ponderosa pine needles are metabolized more quickly.

4. An analytical method to measure cyclopamine in biological samples. Cyclopamine, a toxin in the plant Veratrum californicum, has been found to be active against several types of cancer. Thus, cyclopamine, and cyclopamine derivatives, have been targeted as potential pharmaceutical treatments for certain cancers. A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed by ARS researchers in Logan, UT to detect and measure cyclopamine and cyclopamine derivatives in biological samples. The assay was found to be very sensitive, and it was also found to be useful for the detection and measurement of cyclopamine in samples from mice that had been dosed with cyclopamine. The simple ELISA method could be used for the rapid screening of biological samples for the presence, and concentration, of cyclopamine and other cyclopamine derivatives with anticancer potential.

5. Mechanism of poison hemlock induced birth defects. Piperidine alkaloids, present in poison hemlock, cause deformities in developing animals by inhibiting nicotinic acetylcholine receptors (nAChRs) in the developing fetus. It has been hypothesized that piperidine alkaloids act at the fetal-muscle type nAChRs, but direct evidence is lacking. A recent study by ARS scientists in Logan, UT used piperidine alkaloids in a cell culture model to characterize receptor binding, and a goat model to evaluate fetal movement. The study established that coniine, a piperidine alkaloid, does act at the fetal muscle-type nAChR receptor to inhibit fetal movement, which in turn can result in fetal deformities. A more thorough understanding of the mechanism of action of the plant toxins will aid in the prevention and treatment of birth defects in livestock.

6. A rodent model to identify teratogenic compounds in plants. Mimosa tenuiflora is a perennial tree or shrub native to Brazil, and is also distributed throughout tropical portions of South and Central America and Mexico. The plant causes deformed offspring (teratogenesis) when eaten by pregnant livestock, however, the toxin(s) is not known. Collaborative work by ARS scientists at Logan, UT was undertaken with scientists from Brazil to identify the teratogenic compounds using a rat model. Various extracts and purified compounds from Mimosa were evaluated for their ability to cause fetal deformation and/or to inhibit fetal movement. Although extracts and purified compounds did cause birth defects, clear-cut teratogenic relationships were not established. Consequently, the mechanism by which Mimosa tenuiflora produces birth defects remains unknown. Identification of the compound(s) responsible for causing birth defects will allow for the quantitation of risk for specific plant populations so that better management recommendations can be provided to Brazilian livestock producers to prevent livestock losses.


Review Publications
Cook, D., Lee, S.T., Pfister, J.A., Stonecipher, C.A., Welch, K.D., Green, B.T., Panter, K.E. 2011. Alkaloid profiling as an approach to differentiate Lupinus garfieldensis, Lupinus sabinianus, and Lupinus sericeus. Phytochemical Analysis. 23(3): 278-84.

Gardner, D.R., Riet-Correa, F., Panter, K.E. 2011. Alkaloid profiles of Mimosa tenuiflora and associated methods of analysis. In: Riet-Correa, F., Pfister, J., Schild, A.L., Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge, MA: CAB International. 103:600-5.

Green, B.T., Lee, S.T., Panter, K.E., Brown, D.R. 2012. Piperidine alkaloids: Human and food animal teratogens. Food and Chemical Toxicology. 50(6): 2049-55.

Lee, S.T., Panter, K.E., Gardner, D.R., Green, B.T., Welch, K.D., Zhang, J., Change, C.T. 2012. Development of a monoclonal antibody-based ELISA for the hedgehog inhibitors cyclopamine and cyclopamine-KAAD. Journal of Pharmaceutical and Biomedical Analysis. 66: 282-6.

Panter, K.E., Stegelmeier, B.L. 2011. Effects of xenobiotics and phytotoxins on reproduction in food animals. Veterinary Clinics of North America. 27(2):429-46.

Ralphs, M.H., Motteram, E.S., Panter, K.E. 2011. Velvet lupine (Lupinus leucophyllis) population cycles with climate. Western North American Naturalist. 71(3): 396-403.

Welch, K.D., Gardner, D.R., Panter, K.E., Stegelmeier, B.L., Parsons, C., Pfister, J.A., Cook, D. 2011. Western juniper-induced abortions in beef cattle. International Journal of Poisonous Plant Research. 1(1):72-9.

Welch, K.D., Panter, K.E., Gardner, D.R., Stegelmeier, B.L. 2012. The good and the bad of poisonous plants: An introduction to the USDA-ARS Poisonous Plant Research Laboratory. Journal of Medical Toxicology. 8(2): 153-9.

Last Modified: 10/22/2014
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