2009 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.
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 were screened for susceptibility to the larkspur toxins using physiological measurements. In addition, some bloat work has 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 has been conducted to measure the susceptibility of Angus 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 2nd 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 Angus cattle to larkspur toxicity. Purebred Line 1 Hereford cattle will be purchased for similar tests. A grazing study with D. andersonii was conducted to determine if yearling heifers differed from adult cows in consumption of low larkspur. Reciprocal gardens were established 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.
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 voluntary, striated muscle. MLA at nanomolar concentrations is a potent and selective competitive antagonist of neuronal a7-nicotinic acetylcholine receptors. However, relatively little research has been done to investigate the pharmacology of MLA at muscle-type nAChRs. Clinically, it is the blocking of muscle-type nAChRs by MLA which results in the morbidity and mortality of larkspur poisoning in cattle. Novel results from cell culture based studies suggest that MLA at low concentrations is acting as a potentiator of the acetylcholine response and at high concentrations acts as a noncompetitive antagonist. Further work is ongoing to better determine the mechanism of MLA action at cellular level. Findings from these mechanistic studies will be transferred into cattle research for eventual application in refining solutions to larkspur poisoning in cattle.
Presence or absence of MSAL-type (toxic) alkaloids in tall larkspur populations. Tall Larkspurs (Delphinium spp.) contain norditerpene alkaloids that cause neuromuscular paralysis. Norditerpene alkaloids have been isolated, characterized chemically, and structure-activity relationships performed. The MSAL-type norditerpene alkaloids have the highest toxicity and are thought to be responsible for poisoning episodes. Localized tall larkspur populations, taxonomically identified as D. occidentale, have been observed that do not contain or contain very little of the MSAL-type alkaloids and are postulated to be derived hybrids of D. occidentale and D. barbeyi. However, it is not known if these localized populations are sporadic or if they are more widespread geographically. Further survey work on the distribution of D. occidentale confirms that there is a defined geographical region that does not contain or contains very little of the MSAL alkaloids as well as a geographical region that does contain the MSAL type alkaloids. In addition, reciprocal gardens have been established to answer questions about the relative influences of environment and genetics on larkspur alkaloids in various populations. These results are being used to define geographical regions where tall larkspurs are essentially not toxic, and to understand why some populations contain or lack toxic alkaloids. Livestock producers will be able to graze cattle in these areas with no risk of losses from tall larkspur, thus providing valuable spring and summer forage.
Consumption of Delphinium andersonii by cattle. Two grazing studies have been conducted to evaluate aspects of cattle consumption of low larkspur (D. andersonii) in Idaho. These studies investigated the timing and amount consumed by cattle in relation to plant availability and toxicity. These studies have shown that cattle consume low larkspur in relation to availability, and that yearlings in general consume more low larkspur than do adult cattle. Further, cattle consume low larkspur in a cyclic manner, with periods of high, but non-fatal, consumption followed by several days of detoxification and reduced consumption. Periods of high, non-fatal consumption are typically ended by episodic bouts of severe muscular tremors and sternal and lateral recumbency. Any exercise, such as walking to water or feed, exacerbates these clinical signs. There has been no previous work on consumption of D. andersonii prior to these studies, and 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.
Low larkspur density related to weather. Cattle losses to low larkspur are directly related to plant density, as density is a major determining factor in whether or not cattle can ingest a lethal dose while grazing. This long-term study examined the influence of weather variables on low larkspur density at 4 sites in Utah, Idaho, and Colorado over an 8 year period. These locations have historically had substantial cattle losses from low larkspur, and are located near NRCS SNOTEL weather stations. Density measurements were taken yearly for 8 years at 4 sites: Yampa and Collbran, Colorado; Huntington, Utah, and Ashton, Idaho. Weather variables (e.g., temperature, precipitation, snowfall) were related to plant density using a multiple regression approach. The final collection was completed in July, 2008 and manuscript preparation is ongoing. This information will be useful to help scientists and livestock producers understand relationships between winter and spring weather variables and low larkspur density. Understanding the weather conditions that influence greater low larkspur densities will help livestock producers recognize high risk years, and thus alter cattle management to reduce that risk.
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 food safety recommendations. ARS scientists in Logan, UT have shown in Angus cattle that 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 (t1/2) of 20.5 hours when compared to deltaline at 8.2 hours. The longer MLA clearance suggests that a withdrawal time of 7 days be used to allow poisoned animals to clear these toxins. This finding correlating serum MLA concentrations with increased heart rate further demonstrates that tachycardia is an early indicator of poisoning. It is suggested that animals that have been exposed to larkspur be allowed 6-7 days withdrawal time to allow complete clearance of larkspur alkaloids. It is important to establish clearance times for natural toxins for future food safety considerations, and to understand alkaloid kinetics as part of a complete package of information for use in management recommendations about intoxicated animals. Understanding alkaloid clearance times will help refine current management recommendations to livestock producers.
5.Significant Activities that Support Special Target Populations
Evaluated Shoshone-Bannock Reservation (Ft. Hall, Idaho) pastures for low larkspur and as possible study sites. Meet with Reservation personnel on several occasions during report period. There was insufficient low larkspur for a grazing study during 2009, but we are planning such a collaborative study in the future if populations rebound.
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Cook, D., Gardner, D.R., Pfister, J.A., Welch, K.D., Green, B.T., Lee, S.T. 2009. The Biogeographical Distribution of Duncecap Larkspur (Delphinium occidentale) Chemotypes and Their Potential Toxicity. Journal of Chemical Ecology 35:6443-652
Gardner, D.R., Pfister, J.A. 2008. HPLC/MS Analysis of Toxic Norditerpenoid Alkaloids: Refinement of toxicity assessment of low larkspurs (Delphinium spp.). Phytochemical Analysis 20:104-113.
Green, B.T., Gardner, D.R., Pfister, J.A., Cook, D. 2009. Larkspur - Poison Weed (100 Years of Delphinium Research). Rangelands, 31(1):22-27. http://www.bioone.org/doi/pdf/10.2111/1551-501X-31.1.45?
Green, B.T., Pfister, J.A., Cook, D., Welch, K.D., Stegelmeier, B.L., Lee, S.T., Gardner, D.R., Knoppel, E.L., Panter, K.E. 2009. Effects of Larkspur (Delphinium barbeyi) on Heart Rate and Electrically Evoked Electromyographic Response of the External Anal Sphincter in Cattle. American Journal of Veterinary Research. 70(4):539-546
Green, B.T., Welch, K.D., Gardner, D.R., Stegelmeier, B.L., Davis, T.Z., Cook, D., Lee, S.T., Pfister, J.A., Panter, K.E. 2009. Serum Elimination Profiles of Methyllycaconitine and Deltaline in Cattle Following Oral Administration of Larkspur (Delphinium barbeyi). American Journal of Veterinary Research, 70(7):926-931.
Welch, K.D., Green, B.T., Panter, K.E., Gardner, D.R., Pfister, J.A., Cook, D., Stegelmeier, B.L. 2009. Investigation of the Susceptibility of Various Strains of Mice to Methyllycaconitine Toxicosis. Journal of Animal Science, 87:1558-1564. doi:102527/jas.2008-1577