Location: Poisonous Plant Research2010 Annual Report
1a. Objectives (from AD-416)
Objective I: Reduce risk of grazing cattle on larkspur-infested rangelands, and increase our understanding of aspects of cattle poisoning by various larkspur species. 1.1 Determine the interaction between ingestion of toxic alkaloids from larkspur and bloat in cattle. 1.2 Determine genetic differences to larkspur toxicity using a small animal model and genetically divergent cattle populations. 1.3 Determine clearance times and toxicokinetics of different toxic and less toxic alkaloid mixtures in small animal models and cattle. 1.4 Determine plant genotype and environmental influences on larkspur alkaloids and evaluate chemotaxonomy,chemical phenology, control strategies, and population dynamics of larkspurs. 1.5 Continue development of management strategies for cattle to safely graze on larkspur-infested ranagelands.
1b. Approach (from AD-416)
1.1 In the first phase, cattle will initially be screened for bloat susceptibility and for susceptibility to larkspur alkaloids. In the second phase, resistant and susceptible cattle will be tested for susceptibility to larkspur-associated bloat. In the third phase, mixtures of MSAL-type alkaloids and consecutive doses of larkspur will be administered at levels seen in field intoxication. 1.2 Strains of mice will be chosen to provide a diverse representation of minor haplotypes across the mouse genome, and lethality of MLA will be determined in each. The second series of experiments will examine the toxicokinetics of MLA in mice. Further work will characterize differences in gene expression between the selected mice strains (i.e., resistant and susceptible) after treatment with MLA. The differences in gene expression between strains will provide candidate genes to determine if susceptibility is heritable, and to identify genetic markers associated with MLA toxicity. 1.3 Initial work will examine the effect of deltaline on the toxicity of MLA by comparing the toxicokinetics and LD50 of alkaloids administered individually to mice vs. the co-administration of alkaloids at various ratios. Toxicokinetic studies will then be conducted to determine the kinetics of MLA, deltaline, or a combination of MLA + deltaline. Various body tissues will be collected and analyzed for alkaloid concentrations. After work on mice is completed, similar studies will be conducted using cattle. 1.4 Larkspur flowering stalks representing D. occidentale and D. barbeyi will be collected throughout their geographical distribution. A chemical fingerprint of alkaloids will be generated from each sample, and samples will be scored for the presence or absence of the MSAL-type alkaloids. Studies will elucidate the biological mechanism(s) responsible for the observed chemical fingerprints. Reciprocal gardens of D. occidentale plants representing both chemical fingerprints (putative hybrids and non-hybrids) will be established. Phylogenetic analyses using individuals representing D. occidentale and D. barbeyi plants containing little or no MSAL type alkaloids (i.e. putative hybrids) will be performed using AFLPs (Amplified Fragment Length Polymorphisms) to determine if tall larkspur plants that do not contain MSAL-type alkaloids are derived hybrids. 1.5 Studies will focus on one particular low larkspur, Delphinium andersonii. Grazing studies of cattle consumption will be conducted at two locations (Idaho, Nevada) for 2 years each from the vegetative to pod stages of growth. Study pastures will be delineated based on low larkspur density. The dependent variables will be daily bite counts of larkspur and other forage components. Bite counts will be taken during all active grazing periods. Larkspur density, biomass, alkaloid concentrations, and nutritional quality (i.e., NDF, IVOMD, CP) will be determined in relation to other forage components.
3. Progress Report
Larkspur (Delphinium spp.) poisoning causes serious economic loss to livestock producers grazing cattle on foothill and mountain rangelands in western North America. Cattle death losses to larkspur are estimated to be 5 to 15% annually in areas where larkspurs are abundant. The occurrence of bloat in cattle grazing tall larkspur may be a significant contributor to animal morbidity and mortality. It is necessary to have quantitative measures of larkspur’s effects in cattle to determine degree of intoxication in order to determine the impact of larkspur-induced bloat. Cattle breeds were screened for susceptibility to the larkspur toxins using physiological measurements. Work on larkspur-induced bloat has largely been redirected to more basic research on cellular mechanisms of intoxication such as the mechanism of action of the toxic alkaloids in various cell lines that differ in receptor types. We developed the testing parameters to determine the role of breed in cattle susceptibility to larkspur poisoning. Further, work is being conducted to measure the susceptibility of Angus and inbred (Line 1) Hereford cattle to larkspur toxicity. Further collections representing populations of D. occidentale have been made throughout its geographical distribution. This work is confirming earlier work that some populations are non-toxic. Putative toxic and non-toxic populations were collected and tested in cattle; results indicate that cattle cannot eat sufficient amounts of the non-toxic populations to be lethally intoxicated. The 3rd year of a grazing study was conducted with cattle on a heavily infested D. andersonii site near Picabo, Idaho. Physiological measurements of intoxication were refined in order to develop protocols to determine the role of breed in cattle susceptibility to larkspur poisoning. Studies were begun to measure the susceptibility of purebred Hereford cattle to larkspur toxicity. A grazing study with D. andersonii was conducted to determine if supplementation would alter consumption of low larkspur. Reciprocal gardens were established and are being evaluated to provide further insight into larkspur hybridization, and to answer questions about the relative roles of environment and genetics in concentrations and composition of larkspur alkaloids. A population of tall larkspur lacking the major toxic alkaloids was dosed to cattle; cattle showed no signs of intoxication from doses similar to what cattle can potentially consume while grazing. A map detailing distribution of toxic and non-toxic tall larkspur populations over several states was refined with further plant collection and analysis.
1. Determination of the clearance times of larkspur alkaloids in Angus cattle. Intoxication of cattle by larkspur alkaloids is highly related to dose and to rate of elimination of individual alkaloids. Further, understanding toxicokinetics of purified alkaloids and establishing clearance times is critical in making management recommendations for producers to reduce losses and for making food safety recommendations. ARS scientists at Logan, UT have shown in Angus cattle that methyllycaconitine (MLA), the major larkspur toxin and deltaline (less toxic alkaloid often found in high concentrations) reaches maximum serum concentrations by 10 hours after dosing and that MLA has a slower half life of elimination compared to deltaline. Our findings correlating serum MLA concentrations with increased heart rate further demonstrates that tachycardia is an early indicator of poisoning. Our results suggest that animals that have been exposed to larkspur be allowed 6-7 days withdrawal time to allow complete clearance of larkspur alkaloids. Establishing clearance times for natural toxins and understanding alkaloid kinetics is part of a complete package of information needed about intoxicated animals to help refine current management recommendations to livestock producers.
2. The larkspur alkaloid methyllycaconitine (MLA) as a potentiator of nicotinic acetylcholine receptors (nAChR). MLA is often described as possessing “curariform-like” activity and is a potent competitive blocker of nicotinic acetylcholine receptors in autonomic neurons and striated muscle. MLA at nanomolar concentrations is a potent and selective competitive antagonist of neuronal a7-nicotinic acetylcholine receptors. However, little research has been done to investigate the pharmacology of MLA at muscle-type receptors. Clinically, this blockade of muscle-type receptors by MLA results in the collapse and death from larkspur poisoning in cattle. Understanding the mechanism of MLA action at the receptor level provides insight into the clinical signs exhibited by cattle poisoned with larkspur. Cell culture-based studies conducted by ARS scientists at Logan, UT showed that MLA at low concentrations potentiates the response of specific receptors to agonists, suggesting that MLA acts to increase the activity of these receptors indirectly through activation of a site on the protein. At high concentrations, MLA does not provoke a response itself after binding to a receptor, but dampens agonist-mediated responses. The results from these mechanistic studies will be used in research in cattle for eventual application in refining solutions such as drug therapy to alleviate larkspur poisoning in cattle.
3. Presence or absence of MSAL-type (toxic) alkaloids in tall larkspur populations. Larkspurs (Delphinium spp.) are poisonous plants found on rangelands in western North America; their toxicity is attributed to the norditerpenoid alkaloids which are divided into two main structural groups; the highly toxic (N-methylsuccinimido) anthranoyllycoctonine type (MSAL-type) and the less toxic 7,8-methylenedioxylycoctonine type (MDL-type). Plants high in the MSAL-type alkaloids are thought to be the most toxic to cattle and the concentrations of these alkaloids have been used as a predictor of plant toxicity. Duncecap larkspur, Delphinium occidentale, occurs throughout much of the Intermountain West and northwestern United States. D. occidentale has two chemotypes, one containing the MSAL-type alkaloids and one lacking the MSAL-type alkaloids. Both chemotypes are unique in their geographical distribution. Recent work conducted by ARS scientists at Logan, UT has determined the relative toxicity of each chemotype in mice and cattle. Plant populations containing MSAL-type alkaloids had an average lethal dose in mice of 2.3 mg/kg BW versus 54.2 mg/kg BW from populations lacking MSAL-type alkaloids. Studies using cattle heart rate and muscle weakness confirmed differences in toxicity. D. occidentale containing the MSAL-type alkaloids poses a distinct risk of being toxic to livestock, whereas D. occidentale containing only the MDL-type alkaloids is much less toxic. These results are being used to define geographical regions where tall larkspurs are essentially not toxic, and to inform livestock producers grazing cattle in these areas that there is little risk of losses from tall larkspur. In addition, reciprocal gardens are being evaluated to determine the relative influences of environment and genetics on larkspur alkaloids in various toxic and non-toxic populations.
4. Is the toxicity of larkspur alkaloids potentiated when animals also consume toxic alkaloids from death camas (Zygadenus paniculatus)? In most cases where livestock are poisoned by plants in a rangeland setting, there is more than one potential poisonous plant in that area. One common poisonous plant found simultaneously in the same ranges as death camas is low larkspur (Delphinium nuttallianum). Consequently, the objective of this research was to determine if co-administration of low larkspur will exacerbate the toxicity of death camas in mice. ARS scientists at Logan, UT determined the lethal dose of total alkaloid extracts from Utah and Nevada death camas collections were 2.8 and 2.2 mg/kg, respectively. Zygacine is a major alkaloid in death camas, and the lethal dose was 2.0 mg/kg given intravenously, and 132 mg/kg given orally. Zygacine was eliminated from blood rapidly. The lethal dose in mice of the primary larkspur toxin, methyllycaconitine (MLA) was 4.6 mg/kg. In comparison, the lethal dose of a 1:1 mixture of MLA and zygacine was 2.9 mg/kg. The clinical signs in mice treated with this mixture were very similar to those of mice treated with zygacine, including the time of onset and death. These results suggest that there is not a synergistic effect of co-administering these two alkaloids intravenously in mice, however, there may be a slight additive effect. Results from this study increase understanding about the acute toxicity of death camas alkaloids when given in conjunction with larkspur alkaloids. As combined intoxications are probably common in livestock, this information will be useful in developing recommendations for ranchers and in designing additional experiments to study the toxicity of death camas and larkspur to livestock.
5. Consumption of Delphinium andersonii by cattle. The 3rd in a series of grazing studies was conducted by ARS scientists at Logan, UT to evaluate the potential influence of supplementation on cattle consumption of low larkspur (D. andersonii) in Idaho. Previous studies have shown that cattle consume low larkspur in relation to availability, and that yearlings consume more low larkspur than do adult cattle. D. andersonii plants were abundant albeit small and immature at the beginning of the study. Unfortunately, the plants did not grow and mature but slowly died during the course of the grazing study, probably from the extremely cold and wet spring weather, and cattle selected few of these immature plants. They have also initiated a series of studies that are based on the oral dosing of cattle with dried ground D. andersonii. The aim of these studies is to determine the toxicity of D. andersonii in cattle relative to other species of larkspur and to determine the toxicokinetics of the alkaloids found in D. andersonii. There has been no previous work on consumption of D. andersonii by cattle, nor on toxicokinetics when the plant is eaten by cattle, even though the plant is a major component of rangeland vegetation in semi-arid portions of the western U.S. This work will help to refine recommendations to livestock producers so that cattle losses may be reduced through grazing management. Further, it is important to establish clearance times for natural toxins for future food safety considerations, and to understand alkaloid kinetics in order to make recommendations to livestock producers about clearance times in intoxicated animals.
5. Significant Activities that Support Special Target Populations
Numberous workshops and outreach efforts to small farmers and livestock producers were accomplished in 2010. Collaborative studies with the Shoshone-Bannock ranchers in Idaho and the Navajo ranchers on the Arizona Strip are planned for 2011. Target Groups: Evaluated Shoshone-Bannock Reservation (Ft. Hall, Idaho) pastures for low larkspur and as possible study sites. Met with Reservation personnel on several occasions during report period. Provided grazing management recommendations that Reservation personnel credit with reducing risk and cattle losses. There was insufficient low larkspur for a grazing study during 2010, but we are planning such a collaborative study in the future if populations rebound.