Effects of Elevated CO2 on the Swainsonine Chemotypes of Astragalus lentiginosus and Astragalus mollissimus.

Rapid changes in the Earth's atmosphere and climate associated with human activity can have significant impacts on agriculture including livestock production. CO2 concentration has risen from the industrial revolution to the current time, and is expected to continue to rise. Climatic changes alter physiological processes, growth, and development in numerous plant species, potentially changing concentrations of plant secondary compounds. These physiological changes may influence plant population density, growth, fitness, and toxin concentrations and thus influence the risk of toxic plants to grazing livestock. Locoweeds, swainsonine-containing Astragalus species, are one group of plants that may be influenced by climate change. We evaluated how two different swainsonine-containing Astragalus species responded to elevated CO2 concentrations. Measurements of biomass, crude protein, water soluble carbohydrates and swainsonine concentrations were measured in two chemotypes (positive and negative for swainsonine) of each species after growth at CO2 levels near present day and at projected future concentrations. Biomass and water soluble carbohydrate concentrations responded positively while crude protein concentrations responded negatively to elevated CO2 in the two species. Swainsonine concentrations were not strongly affected by elevated CO2 in the two species. In the different chemotypes, biomass responded negatively and crude protein concentrations responded positively in the swainsonine-positive plants compared to the swainsonine-negative plants. Ultimately, changes in CO2 and endophyte status will likely alter multiple physiological responses in toxic plants such as locoweed, but it is difficult to predict how these changes will impact plant herbivore interactions.

Ruminant metabolism of zygacine, the major toxic alkaloid in foothill death camas (Zigadenuspaniculatus).

Death Camas (Zigadenus spp.) are common poisonous plants distributed throughout North America. The toxic alkaloids in foothill death camas are zygadenine and a series of zygadenine esters, with zygacine, the 3-acetyl ester of zygadenine, being the most abundant. Both cattle and sheep can be poisoned by grazing death camas, however, sheep consume death camas more readily and are most often poisoned. We hypothesized that the presence of enzymes, including esterases present in the rumen, liver, and blood of livestock would metabolize zygacine. The objective of this study was to investigate the metabolism of zygacine in sheep and cattle using in-vitro and in-vivo systems. Results from experiments where zygacine was incubated in rumen culture, plasma, liver S9 fractions, and liver microsomes and from the analysis of rumen and sera from sheep and cattle dosed death camas plant material demonstrated that zygacine is metabolized to zygadenine in the rumen, liver and blood of sheep and cattle. The results from this study indicate that diagnosticians should analyze for zygadenine, and not zygacine, in the rumen and sera for the diagnosis of livestock suspected to have been poisoned by foothill death camas.

Acute death as a result of poisoning tropical (Bos taurus indicus) but not temperate (Bos taurus taurus) cattle after oral dosing with Lupinus leucophyllus (velvet lupine).

Due to climate change and increasing summer temperatures, tropical cattle may graze where temperate cattle have grazed, exposing tropical cattle to toxic plants they may be unfamiliar with. This work compared the toxicity of Lupinus leucophyllus (velvet lupine) in temperate and tropical cattle. Orally dosed velvet lupine in tropical cattle caused death. If producers opt to graze tropical cattle, additional care must be taken on rangelands where toxic lupines like velvet lupine grow.

Biomarkers and their potential for detecting livestock plant poisonings in Western North America.

The United States National Cancer Institute defines a biomarker as: "A biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or disease." In Veterinary Medicine, biomarkers associated with plant poisonings of livestock have great utility. Since grazing livestock poisoned by toxic plants are often found dead, biomarkers of plant poisoning allow for a more rapid postmortem diagnosis and response to prevent further deaths. The presence and concentration of toxins in poisonous plants are biomarkers of risk for livestock poisoning that can be measured by the chemical analysis of plant material. More difficult is, the detection of plant toxins or biomarkers in biological samples from intoxicated or deceased animals. The purpose of this article is to review potential biomarkers of plant poisoning in grazing livestock in the Western North America including recently investigated non-invasive sampling techniques. Plants discussed include larkspur, lupine, water hemlock, swainsonine-containing plants, selenium-containing plants, and pyrrolizidine alkaloid containing plants. Other factors such as animal age and sex that affect plant biomarker concentrations in vivo are also discussed.

Identification of two death camas chemotypes within a population and evaluation of toxicity.

Foothill death camas (Zigadenus paniculatus) is a native, cool-season, bulbous perennial forb found throughout the western U.S. The toxins in death camas are steroidal alkaloids. Zygacine is often the most abundant alkaloid in death camas and is believed to be the primary toxic component. A population of death camas with plants consisting of two different chemical profiles (chemotypes) growing within the same location were identified. The objective of this study was to determine the percentage of a death camas population represented by each of the different chemotypes and to determine if there was a difference in toxicity between the two chemotypes. One third of the population sampled consisted of chemotype 1, while two-thirds of the population consisted of chemotype 2. The zygacine concentration of chemotype 1 was three times higher than chemotype 2. Chemotype 2 contained higher concentrations of several other steroidal alkaloids than chemotype 1. We hypothesized that chemotype 1, which consisted of higher concentrations of zygacine, would be more toxic than chemotype 2. The acute toxicity of each chemotype was determined in mice and sheep. In the mouse LD50 study, the acute toxicity of the chemotype 1 alkaloids (2.3 mg/kg BW) was different than the chemotype 2 alkaloids (3.2 mg/kg BW). However, in the sheep study there were no differences in the adverse effects between chemotypes. Based upon the results of this study, caution should be taken when livestock are grazing death camas, as both chemotypes of death camas appear to pose a similar risk to grazing livestock.

Evaluation of diazepam as a drug treatment for water hemlock (Cicuta species) poisoning in Spanish goats.

Water hemlocks (Cicuta spp.) are toxic members of the Apiaceae plant family. The best drug treatment for the convulsions associated with acute water hemlock poisoning in livestock and humans has not been determined experimentally. This work compared the therapeutic actions of benzodiazepines (diazepam) and barbiturates (phenobarbital) on water hemlock poisoning in a goat model. C. maculata tubers were orally dosed to goats. Experimental groups consisted of; control saline; 20 mg/kg phenobarbital; 1.0 mg/kg diazepam; 10 mg/kg diazepam; and 1.0 mg/kg diazepam administered as needed to moderate convulsions by intravenous (i.v.) infusion. Diazepam provided nearly instant control of convulsions. Clinical signs of poisoning were completely controlled for the duration of the experiment in the goats that received the 10 mg/kg diazepam dose. These results suggest that diazepam is effective at managing the clinical signs of water hemlock poisoning in goats. We speculate that diazepam can be used as a potential treatment for water hemlock poisoning in other livestock species and humans.

Mineral-salt supplementation to ameliorate larkspur poisoning in cattle.

Larkspurs (Delphinium spp.) are native forbs that are poisonous to cattle and cost livestock producers millions of dollars in losses each year. Macro and micro minerals are required for normal functioning of essentially all metabolic processes in ruminants. The role that mineral status may play in larkspur poisoning in cattle is not clear. In this study, we seek to determine the effects a mineral-salt supplement, commonly used by cattle producers, to potentially reduce cattle losses to larkspur. The ability of mineral-salt supplementation to alter susceptibility to larkspur toxicosis was evaluated in a pen study. Animals supplemented with mineral-salt were found to be less susceptible to larkspur poisoning than the non-supplemented animals. A separate group of animals were then grazed on larkspur infested rangelands. One group was supplemented with a mineral-salt mix and the other group did not receive any mineral-salt. Supplementing cattle with the mineral-salt mix did not alter larkspur consumption (P > 0.05). However, overall larkspur consumption was low and averaged 3 ± 1.0% and 2 ± 1.1% for cattle supplemented with mineral and non-supplemented, respectively. Serum was collected from animals once a week during the grazing study. Average and maximum serum concentrations of toxic larkspur alkaloids were numerically higher in mineral-salt supplemented cattle compared with the non-supplemented animals. Results from the pen study suggest that a good mineral supplementation program will provide a protective effect for animals grazing in larkspur-infested ranges. The mineral-salt supplemented steers, in the grazing study, were not observed to consume less larkspur than the non-supplemented animals; however, the mineral-salt supplemented animals had higher concentrations of larkspur alkaloids in their serum indicating they may be able to tolerate higher larkspur consumption. The data also indicate that mineral-salt supplementation must be continuous throughout the time the animals are grazing these rangelands as the positive effects can be lost within 30 d post supplementation.

Larkspurs (Delphinium spp.) are native forbs poisonous to cattle and cost livestock producers millions of dollars in losses each year. The role mineral status may play in larkspur poisoning in cattle is unclear. The ability of mineral-salt supplementation to alter susceptibility to larkspur toxicosis was evaluated in a pen and grazing study. In the pen study, animals supplemented with mineral-salt were found to be less susceptible to larkspur poisoning than non-supplemented animals. A separate group of animals grazed on larkspur infested rangelands. One group was supplemented with a mineral-salt mix and the other group did not receive any mineral-salt. Supplementing cattle with the mineral-salt mix did not alter larkspur consumption of grazing cattle. However, overall larkspur consumption was low. Results from the pen study suggest that a good mineral supplementation program will provide a protective effect for animals grazing in larkspur-infested ranges. The mineral-salt supplemented steers, in the grazing study, had higher concentrations of larkspur alkaloids in their blood serum indicating they may be able to tolerate higher larkspur consumption. The data also indicate that mineral-salt supplementation must be continuous throughout the time the animals are grazing as the positive effects can be lost within 30 days after supplementation.

Hepatotoxicity in Cattle Associated with Salvia reflexa Diterpenes, including 7-Hydroxyrhyacophiline, a New Seco-Clerodane Diterpene.

A case of baled alfalfa hay contaminated with multiple weeds induced hepatotoxicity and death in cattle. The hepatotoxic compounds were isolated by bioassay-guided fractionation using a mouse model and identified as salviarin, salvianduline D, rhyacophiline, and 7-hydroxyrhyacophiline. The structure of 7-hydroxyrhyacophiline has not been previously reported. All compounds were found to induce severe acute hepatic necrosis within 24-48 h after a single oral dosage (260-280 mg/kg). The identified diterpenes are known to be found among different Salvia species which led to finding dried plant parts of Salvia reflexa within bales of weedy hay and subsequently a population of S. reflexa was found along the field edges and irrigation ditch banks of the alfalfa hay field. It was thus determined that S. reflexa was responsible for the hepatotoxicity observed in cattle fed the contaminated hay.

Clinical, pathologic, and toxicologic characterization of Salvia reflexa (lance-leaf sage) poisoning in cattle fed contaminated hay.

Salvia reflexa (lance-leaf sage)-contaminated alfalfa hay was fed to ~500 mixed-breed beef cattle. Within hours of exposure, nearly half of the cattle developed lethargy, anorexia, depression, and recumbency, followed by bellowing, colic, and death. Even though the uneaten contaminated hay was removed the first day, nearly 100 animals died within the first 48 h. Three of these cattle were examined postmortem, and tissues and hay samples were collected for microscopic and chemical analysis. Several days later, a smaller number of the clinically poisoned cattle developed neurologic disease with aberrant behavior, aggression, icterus, blindness, exhaustion, and death. A total of 165 cattle were fatally poisoned. Poisoned cattle had swollen, dark, mottled livers that had a prominent nutmeg-like lobular pattern on cut section. Histologically, there was severe centrilobular-to-panlobular hepatic necrosis with marked hepatocellular swelling, degeneration, and necrosis. The surviving cattle developed liver disease characterized by altered serum biochemical analyses and microscopic hepatocellular degeneration and necrosis. In subsequent biopsies and analysis, these lesions resolved within 6-7 mo. After confirming toxicity of the hay in cattle, goats, and mice, followed by a mouse bioassay-guided chemical fractionation process, Salvia reflexa was identified as the contaminant in the hay responsible for the hepatotoxicity. S. reflexa has not been reported previously to cause fatal hepatotoxicity in livestock in North America, to our knowledge.

Diterpenoids from Gutierrezia sarothrae and G. microcephala: Chemical diversity, chemophenetics and implications to toxicity in grazing livestock.

Broom snakeweed (Gutierrezia sarothrae) and threadleaf snakeweed (G. microcephala) are suffrutescent plants found in many parts of western US rangelands and are possibly toxic to grazing livestock. The toxic components are not known, but it has been suggested that the diterpene acids may be both toxic and abortifacient. One hundred sixty-two samples of snakeweed were collected from 55 locations in Colorado, Oklahoma, New Mexico, Texas, Wyoming and Utah and were taxonomically classified. Samples were analyzed by GC-MS in a chemophenetic analysis and grouped into individual chemotypes based on diterpene acid content. The GC-MS profiles were found to be diverse showing at least eight different chemotypes. From each of the chemotypes the major diterpene acids were isolated and characterized by IR, MS and NMR spectroscopy. Twenty-one diterpenoids were identified and found to be a mix of furano, lactone, di-acid and esters of labdane, ent-labdane and chlerodane acids and alcohols. Only four of the 21 compounds isolated had been previously reported as occurring in G. sarathorea or G. microcephala, while another eight compounds were previously reported from other Gutierrezia or related species. Nine of the isolated diterpenoids have not been previously reported and their structure elucidation is reported.

Analysis of rumen contents and ocular fluid for toxic alkaloids from goats and cows dosed larkspur (Delphinium barbeyi), lupine (Lupinus leucophyllus), and death camas (Zigadenus paniculatus).

Larkspurs, lupines, and death camas can be acutely toxic to livestock and are serious poisonous plant problems in western North America. The toxicity of these plants depends on the composition and concentrations of the toxic alkaloids in the plants. In this study, goats and cows were dosed sub-lethal doses of larkspur, lupine, and death camas. Rumen contents and ocular fluid samples were collected, and simple extraction, sample preparation, and analytical methods were developed for the detection of toxic alkaloids in the rumen contents and ocular fluid samples. Toxic alkaloids were detected in the rumen contents and ocular fluid samples from the goats and cows dosed larkspur, lupine, and death camas. In addition, results from a case report where rumen contents were analyzed from a steer that was suspected to have died due to larkspur are reported. This demonstrates the utility of the methods described for the diagnosis of acute plant poisonings.

Evaluation of noninvasive specimens to diagnose livestock exposure to toxic larkspur (Delphinium spp.).

Larkspurs (Delphinium spp.) are native perennial plants that have a serious toxic potential to cattle on foothill and mountain rangelands in the western United States. Livestock death due to larkspur toxicity is attributed to norditerpenoid alkaloids. Diagnosing plant poisonings in livestock is often challenging. The objective of this study was to evaluate the use of three matrices; earwax, oral fluid, and nasal mucus, as noninvasive specimens to determine livestock exposure to larkspurs. Reversed phase high performance liquid chromatography - high resolution mass spectrometry was used to analyze for norditerpene alkaloids, in all three matrices, in cattle administered a single dose of larkspur. Earwax, oral fluid, and nasal mucus were collected over 6 days post-dosing. Methyllycaconitine (MLA) and deltaline concentrations in earwax ranged from 0.4 ±â€¯0.1 to 0.2 ±â€¯0.06 and 0.6 ±â€¯0.5 to 0.11 ±â€¯0.08 ng/mg, respectively. MLA and deltaline concentrations in oral fluid ranged from 0.08 ±â€¯0.03 to 0.01 ±â€¯0.002 ng/mg and 0.07 ±â€¯0.03 ng/mg to not detected (ND), respectively. MLA and deltaline concentrations in nasal mucus ranged from 0.2 ±â€¯0.13 to 0.03 ±â€¯0.01 ng/mg and 0.2 ±â€¯0.12 ng/mg to ND, respectively. The ability to detect differing norditerpene alkaloid chemotypes from two different Delphinium spp. was also possible in the three matrices. This study demonstrates the potential of earwax, oral fluid, and nasal mucus as noninvasive specimens for chemical analyses to aid in the diagnosis of livestock that may have been exposed to and poisoned by larkspur plants.

An Evaluation of Hair, Oral Fluid, Earwax, and Nasal Mucus as Noninvasive Specimens to Determine Livestock Exposure to Teratogenic Lupine Species.

The livestock industry in the western United States loses an estimated $500 million annually from livestock production losses due to poisonous plants. Poisoning of livestock by plants often goes undiagnosed because there is a lack of appropriate or available specimens for analysis. The Lupinus species represent an important toxic plant in western North America that can be toxic and/or teratogenic to livestock species due to the quinolizidine alkaloids. The objective of this study was to evaluate the potential of using earwax, hair, oral fluid, and nasal mucus as noninvasive specimens to determine livestock exposure to the teratogenic Lupinus species. Quinolizidine alkaloids were detected in these four matrices in cattle that were administered a single dose of Lupinus leucophyllus. In addition, quinolizidine alkaloids from lupine were detected in the earwax of cattle that grazed on lupine-infested rangelands. This study demonstrates the potential of earwax, hair, oral fluid, and nasal mucus as noninvasive specimens for chemical analyses to aid in the diagnosis of livestock that may have been exposed to and poisoned by plants.

Genetic Relationships among Different Chemotypes of Lupinus sulphureus.

Lupines (Lupinus spp.) are a common plant legume species found on western U.S. rangelands. Lupinus spp. may contain quinolizidine and/or piperidine alkaloids that can be toxic and/or teratogenic to grazing livestock. Alkaloid profiles may vary between and within a species. The objectives of this study were to (1) further explore the characteristic alkaloid profiles of Lupinus sulphureus using field collections and (2) explore the phylogenetic relationship of the different populations and chemotypes of L. sulphureus using the amplified fragment length polymorphism method of DNA fingerprinting, thus providing possible explanations to the phenomena of multiple chemotypes within a species. A total of 49 accessions of L. sulphureus were classified into seven chemotypes. The DNA profiles showed that one L. sulphureus chemotype, chemotype A, is genetically divergent from the other chemotypes of L. sulphureus, suggesting that it represents an unresolved lupine taxon, possibly a new lupine species. Additionally, the different chemotypes of L. sulphureus represented different genetic groups, as shown by Bayesian cluster analysis and principle component analysis.

The Effect of Co-Administration of Death Camas (Zigadenus spp.) and Low Larkspur (Delphinium spp.) in Cattle.

In many rangeland settings, there is more than one potential poisonous plant. Two poisonous plants that are often found growing simultaneously in the same location in North American rangelands are death camas (Zigadenus spp.) and low larkspur (Delphinium spp.). The objective of this study was to determine if co-administration of death camas would exacerbate the toxicity of low larkspur in cattle. Cattle dosed with 2.0 g of death camas/kg BW showed slight frothing and lethargy, whereas cattle dosed with both death camas and low larkspur showed increased clinical signs of poisoning. Although qualitative differences in clinical signs of intoxication in cattle co-treated with death camas and low larkspur were observed, there were not any significant quantitative differences in heart rate or exercise-induced muscle fatigue. Co-treatment with death camas and low larkspur did not affect the serum zygacine kinetics, however, there was a difference in the larkspur alkaloid kinetics in the co-exposure group. Overall, the results from this study suggest that co-exposure to death camas and low larkspur is not significantly more toxic to cattle than exposure to the plants individually. The results from this study increase our knowledge and understanding regarding the acute toxicity of death camas and low larkspur in cattle.

A swainsonine survey of North American Astragalus and Oxytropis taxa implicated as locoweeds.

Swainsonine, an indolizidine alkaloid with significant physiological activity, is an α-mannosidase and mannosidase II inhibitor that causes lysosomal storage disease and alters glycoprotein processing. Swainsonine is found in a number of plant species worldwide, and causes severe toxicosis in livestock grazing these plants, leading to a chronic wasting disease characterized by weight loss, depression, altered behavior, decreased libido, infertility, and death. Swainsonine has been detected in 19 Astragalus and 2 Oxytropis species in North America by thin layer chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry and a jack bean α-mannosidase inhibition assay. In addition, 5 species in North America are presumed to contain swainsonine based upon reports from field cases. Many of these plant species have not been analyzed for swainsonine using modern instrumentation such as gas or liquid chromatography coupled with mass spectrometry. To provide clarification, 22 Astragalus species representing 93 taxa and 4 Oxytropis species representing 18 taxa were screened for swainsonine using both liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Swainsonine was detected in 48 Astragalus taxa representing 13 species and 5 Oxytropis taxa representing 4 species. Forty of the fifty-three swainsonine-positive taxa had not been determined to contain swainsonine previously using liquid or gas chromatography coupled with mass spectrometry. The list of swainsonine-containing taxa reported here will serve as a reference for risk assessment and diagnostic purposes.

A survey of tremetone, dehydrotremetone, and structurally related compounds in Isocoma spp. (goldenbush) in the southwestern United States.

Isocoma pluriflora, a plant prevalent on land used for livestock production and native to Arizona, New Mexico, West Texas, and Northern Mexico, is poisonous and causes trembles in livestock. Tremetone and dehydrotremetone have been suggested as the toxic compounds in I. pluriflora. In this study several different Isocoma spp., including I. pluriflora, I. tenuisecta, I. azteca, I. acradenia, and I. rusbyi, that are native to land used for grazing livestock in the southwestern United States were analyzed by high performance liquid chromatography (HPLC) for tremetone, dehydrotremetone, and other structurally related compounds. This is the first report of tremetone, dehydrotremetone, and 3-oxyangeloyltremetone in I. tenuisecta, I. azteca, I. acradenia, I. rusbyi, and several other Isocoma spp. In addition, this is the first report of 4-hydroxy-3-(3-methyl-2-butenyl)acetophenone and 7-isopentenyloxycoumarin in Isocoma spp.

The relative toxicity of Delphinium stachydeum in mice and cattle.

Larkspurs (Delphinium spp.) are poisonous plants on rangelands throughout the Western United States and Canada. Larkspur-induced poisoning in cattle is due to norditerpene alkaloids that are represented by two main structural groups of norditerpene alkaloids, the N-(methylsuccinimido) anthranoyllycoctonine type (MSAL-type) and the non-MSAL type. Information on the alkaloid composition and resulting toxicity in mice and cattle is lacking for a number of Delphinium species, including Delphinium stachydeum. The objective of this study was to determine the alkaloid composition of D. stachydeum and to characterize its relative toxicity in mice and cattle compared to two reference species Delphinium barbeyi and Delphinium occidentale. D. stachydeum contains the non-MSAL-type alkaloids but not the MSAL-type alkaloids. D. stachydeum was less toxic than D. barbeyi and D. occidentale in the mouse model. D. stachydeum was less toxic than the MSAL-containing D. barbeyi but much more toxic than the non-MSAL-containing D. occidentale in cattle as measured by heart rate and time of exercise. These results indicate that predictions of Delphinium toxicity can't be accurately made based solely on results from the mouse model or the absence of the MSAL-type alkaloids in the plant.