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United States Department of Agriculture

Agricultural Research Service

Research Project: POISONING OF LIVESTOCK BY VARIOUS LARKSPUR SPECIES (DELPHINIUM)

Location: Poisonous Plant Research

2008 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
This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan Component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P.

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 newly developed physiological measurements. These animals will be used in future work on larkspur-induced bloat. Larkspurs contain two major types of alkaloids, namely N-(Methylsuccinimido)anthranoyllycoctonine (MSAL) and Methylenedioxylycoctonine (MDL) types. MSAL type alkaloids are generally 10-fold more toxic than MDL-type alkaloids. The effect of purified MDL alkaloids on the overall toxicity of larkspur was assessed in a small rodent model. We determined that in mice, the MSAL alkaloids are the primary toxins, however, the MDL alkaloids have an additive effect and can influence the overall toxicity of larkspur plants when present in large quantities. Ten strains of mice have been used to assess the toxicity of larkspur alkaloids; these strains represent different genetic profiles, and this and future information on strain susceptibility will be used to determine if there is a genetic basis for susceptibility, particularly related to such pertinent factors as acetylcholine receptor physiology. There is a need to directly compare the physiological effects of major MSAL and MDL alkaloids in cattle to determine clearance times and other kinetic parameters. Heart rate increased from larkspur poisoning, and reached a maximum of 79.0 ± 5.0 bpm at 17 hours post dosing. Deltaline and MLA were absorbed within 8.8 ± 1.2 and 5.0 ± 0.6 hours, respectively. The half life of elimination for deltaline and MLA was 20.5 ± 4.1 and 8.2 ± 0.6 hours, respectively. Collections representing approximately 40 populations (10 plants per population) of D. occidentale have been made throughout its geographical distribution. Sampling of herbarium specimens has been completed. Chemical fingerprints of herbarium specimens are complete. Potential study sites for grazing studies on D. andersonii were identified based on density and persistence of larkspur populations. A grazing study was conducted with cattle on one site near Picabo, Idaho.


4.Accomplishments
1. Physiological effects of MSAL and MDL alkaloids on cattle. Larkspurs contain two major types of alkaloids, namely MSAL and MDL types. MSAL type alkaloids are generally 10-fold more toxic than MDL-type alkaloids. There is a need to directly compare the physiological effects of major MSAL and MDL alkaloids in cattle to determine clearance times and other kinetic parameters. Absorption and elimination kinetics of two abundant norditerpenoid alkaloids in larkspur, MLA (an MSAL alkaloid) and deltaline (an MDL alkaloid) were measured. Heart rate response of intoxicated cattle over 96 hours was determined. This study documented in five steers that the heart rate reached a maximum of 79.0 ± 5.0 bpm at 17 hours post dosing. Maximum absorption values for deltaline and MLA were 8.8 ± 1.2 and 5.0 ± 0.6 hours, respectively. Half life of elimination values for deltaline and MLA were 20.5 ± 4.1 and 8.2 ± 0.6 hours, respectively. These results indicate that reducing animal stress and movement provides an additional means to alter the outcome of larkspur poisoning in cattle. Moreover, based on the elimination half life it is likely that cattle orally dosed with dried ground larkspur will have eliminated nearly 99% of the MLA and deltaline detectable in serum by seven half-lives or approximately 144 hours. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan Component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P.

2. Test various alkaloids and mixtures in mice. MDL (less toxic) and MSAL (highly toxic) alkaloids occur jointly in larkspurs. Most of the toxicity is attributed to MSAL alkaloids, although MDL alkaloids are often a substantial component of the total alkaloid mix. Thus, the purpose of this study was to determine the effect of MDL alkaloids to potentiate larkspur toxicity in mice (a rodent model). The MDL type alkaloids were found to have an additive affect on the toxicity. When assessing the toxic potential of larkspur plants both the MSAL alkaloid content and total alkaloid content need to be determined. This research enhances the predictability of risk on larkspur ranges. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan Component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P.

3. 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 most poisoning episodes. Localized tall larkspur populations, taxonomically identified as D. occidentale, 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. An initial survey of herbarium specimens (n=>500) representing the distribution of D. occidentale indicates 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. These results will be used to define geographical regions where tall larkspurs are essentially not toxic. Livestock producers will be able to graze cattle in these areas with no risk of loss from tall larkspur poisoning, thus providing valuable spring and summer forage. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan Component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P.

4. 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 a 7 to 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 data analysis 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 practices to reduce that risk. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan Component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P.


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 4 occasions during report period. There was insufficient low larkspur for a grazing study during 2008, but we are planning such a collaborative study in the future if populations rebound.


6.Technology Transfer

Number of Web Sites Managed1
Number of Newspaper Articles and Other Presentations for Non-Science Audiences9
Number of Other Technology Transfer6

Review Publications
Lee, S.T., Molyneux, R.J., Panter, K.E. 2007. Separation Of Enantiomeric Mixtures Of Alkaloids And Their Biological Evaluation. In: Bioactive Natural Products: Detection, Isolation, and Structural Determination, Second Edition, ISBN: 9780849372582, ISBN: 0849372585, Chpt. 7, pp. 209-219.

Welch, K.D., Panter, K.E., Gardner, D.R., Green, B.T., Pfister, J.A., Cook, D., Stegelmeier, B.L. 2008. The effect of 7, 8-Methylenedioxylycoctonine -Type Diterpenoid Alkaloids on the Toxicity of Ethyllycaconitine in Mice. Journal of Animal Science, J. Anim. Sci. 86:2761-2770.

Last Modified: 9/22/2014
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