Location: Poisonous Plant Research2011 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 and the clinical signs associated with larkspur intoxication in cattle are enigmatic. Quantitative measures of larkspur intoxication in cattle have been developed to assess the degree of intoxication and are currently being used to assess the susceptibility or resistance of different breeds of cattle to larkspur intoxication. Toxicokinetic and screening of breeds has continued with the aim of increasing the numbers of evaluation breeds tested for gene expression (Angus, Line 1 Hereford, Holstein, and Jersey cattle). Work on larkspur-induced bloat has been redirected to a receptor-based approach through the use of a cell-culture model. Results from these experiments suggest that methyllycaconitine in addition to being a selective and potent antagonist at alpha-7 nicotinic acetylcholine receptors can bind to fetal-muscle type nicotinic acetylcholine receptors to form a heteroliganded receptor state with one molecule of methyllycaconitine and one molecule of agonist. The heteroliganded state then results in the activation of the receptor. In these experiments methyllycaconitine produced sinistral and upward shifts in the concentration-effect relationships of all agonists tested. Moreover, experiments with the carbamate acetylcholinesterase inhibitor neostigmine that has been used experimentally to reverse larkspur intoxication in cattle was shown to possess agonistic effect that were also potentiated by methyllycaconitine providing additional evidence to the utility of neostigmine as a clinical treatment of larkspur intoxication in cattle.
1. Blood concentrations of toxic alkaloids from low larkspurs in beef cattle. Cattle poisoning from eating low larkspur plants causes serious economic loss to livestock producers on foothill and mountain rangelands in western North America. ARS researchers in Logan, UT, conducted controlled studies in cattle orally dosed with low larkspur. They determined how long it would take beef cattle to clear the toxic alkaloids from their blood so that an accurate risk assessment can be made. They discovered that clinical poisoning is likely to be the most severe approximately 18 hours after exposure and those animals should be closely monitored for at least 36 hours beyond initial exposure. Additionally, a withdrawal time of approximately seven days is required to clear over 99% of the toxic alkaloids from the bodies of poisoned cattle that have ingested low larkspur. These findings will have a positive economic impact on ranchers in the Western U.S. since cattle death losses to larkspur are estimated to be 5 to 15% annually.
2. Blood alkaloid concentrations in beef cattle ingesting tall larkspur. Cattle are poisoned from eating tall larkspur and this causes serious economic losses to livestock producers in western North America. ARS researchers in Logan, UT, investigated differences in the toxic effects of larkspurs from Montana and Utah. This was done to determine if differences in toxicity between larkspur from Montana and Utah were due to differences in uptake or elimination of the plant toxins. They discovered that alkaloids such as deltaline modulate the blood concentrations of other more toxic alkaloids and result in increased toxicity in beef cattle. These alkaloids in larkspur can vary from region to region in western North America making it imperative that livestock producers know which species of poisonous larkspur grow on their rangeland. Livestock producers can then make management decisions based on the alkaloid composition of the plants found on their rangeland. These findings will have a positive economic impact on ranchers in the Western US since cattle death losses to larkspur are estimated to be up to 20% annually.
3. Biological and molecular effects of methyllycaconitine, the main toxin in larkspur. Methyllycaconitine, a toxic constituent of tall larkspur is classified as a blocker of nicotinic acetylcholine receptors in the brain. This classification is incongruent with clinical observations of muscle weakness in poisoned cattle. ARS researchers in Logan, UT, characterized the affects of the methyllycaconitine on the actions of three neurological receptors in cultured muscle cells. Methyllycaconitine alone was without effect and at low concentrations increased the response of cultured muscle cells to four chemicals that elicit different responses. This work is aimed at determining the molecular mechanism behind larkspur poisoning and suggests that methyllycaconitine is not simply acting as a blocker of muscle and nerve activity in cattle. A better understanding of the molecular mechanism of methyllycaconitine action in intoxicated beef cattle will lead to improved treatments for poisoned livestock and a reduction in the number of dead animals on mountain rangelands.
4. Additive nature of toxic low larkspur and death camas. Rangelands in the western United States contain multiple poisonous plants. A consequence of this is that one poisonous plant can exacerbate the toxicity of another poisonous plant. Two poisonous plants often found growing together are low larkspur and death camas. ARS researchers in Logan, UT, using a mouse model, determined that there is an additive effect when alkaloids from both low larkspur and death camas are administered. This suggests that beef cattle grazing both larkspur and death camas can eat less of each plant in combination compared to consuming each plant individually. The results from this study increase knowledge and understanding regarding the acute toxicity of death camas and low larkspur. As combined intoxications are most likely common, this information will be useful in further developing management recommendations for ranchers and in designing additional experiments to study the toxicity of death camas to livestock. These findings will have a positive economic impact on ranchers in the Western US where annual cattle death losses to larkspurs exceed 5%.
5. Role of MDL-type alkaloids in tall larkspur toxicity. Larkspur contain norditerpenoid alkaloids which are structurally delineated as 7, 8-methylenedioxylycoctonine (MDL-type) and N-(methylsuccinimido) anthranoyllycoctonine (MSAL)-type alkaloids. The toxicity of many tall larkspur species has been attributed to their high concentration of MSAL-type alkaloids, which are typically 20 times more toxic than the MDL-type alkaloids. However, the less toxic MDL-type alkaloids are often more abundant than MSAL-type alkaloids in most larkspur populations. ARS researchers in Logan, UT, dosed beef cattle with plant material from two different populations of tall larkspur containing either exclusively MDL- or MSAL-type alkaloids. These two plant populations were combined to create mixtures with ratios of 0.3:1, 1:1, 5:1, and 10:1 MDL- to MSAL-type alkaloids. The dose that elicited similar clinical signs of poisoning in mice and cattle was determined for each mixture based on the MSAL-type alkaloid content. As the ratio of less toxic MDL- to MSAL-type alkaloids increased, the amount of MSAL-type alkaloids required to elicit clinical signs decreased. These results indicate that the less toxic MDL-type alkaloids in tall larkspur exacerbate the toxicity of the plant. Consequently, both the amount of MSAL-type alkaloids and the amount of total alkaloids should be fully characterized to more accurately determine the relative toxicity of tall larkspur plant material. These findings will have a positive economic impact on ranchers in the Western U.S. since cattle death losses to larkspur are estimated to be 5 to 15% annually.
6. Analysis of toxic low larkspur alkaloids in animal sera samples. The toxicokinetics of the toxic alkaloids of low larkspur in cattle has not been well defined as the low larkspur contains significant concentration of other MSAL alkaloids in addition to methyllycaconitine. ARS researchers in Logan, UT, developed methods to detect and measure a number of these different MSAL type toxic alkaloids in the blood of cattle that have consumed low larkspur. The method has been applied to a toxicokinetic study of the low larkspur alkaloids in cattle. The information will be used to help define the different toxic effects of low larkspur in comparison to tall larkspur. These findings will have a positive economic impact on ranchers in the Western U.S. since cattle death losses to larkspur are estimated to be 5 to 15% annually.
Green, B.T., Welch, K.D., Cook, D., Gardner, D.R. 2011. Potentiation of the actions of acetylcholine, epibatidine, and nicotine by methyllycaconitine at fetal muscle-type nicotinic acetylcholine receptors. European Journal of Pharmacology. 662(1-3):15-21.