2013 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.
This is the final report for 5428-32630-011-00D. 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 clinical signs associated with larkspur intoxication in cattle are enigmatic. Over the past five years, 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 multiple breeds of cattle to larkspur intoxication. An initial genome-wide association study is currently underway in collaboration with Scientists at the US Meat Animal Research Center in Clay Center Nebraska to identify regions of the bovine genome that associate with susceptibility or resistance to intoxication. In addition to studies of the effects of the larkspur toxins in cattle, the effects of plant genotype and environmental influences on plant alkaloids content, chemotaxonomic phylogeny and chemical phenology, and population dynamics of larkspurs was also investigated. For example, populations of D. andersonii, a toxic low-growing larkspur common to arid and semi-arid plant communities across the western U.S., fluctuate dramatically from year to year. However, there is little information available on the life history of Delphinium andersonii. Studies were conducted by ARS researchers in Logan, UT to determine the number of plants emerging from dormancy, plant height, the number of flowers (as measures of plant fitness) and alkaloid content. Plant fitness and other factors vary from year to year depending on precipitation. This information is important to understand major changes in D. andersonii populations over time, and to make recommendations to the livestock industry. Studies by ARS Scientists at Logan Utah have also determined the larkspur alkaloid clearance times with different species of larkspur and breeds of cattle. ARS Scientists at Logan Utah continue development of management strategies for cattle to safely graze on larkspur-infested rangelands in project 5428-32630-012-00D.
Identification of differences between cattle breeds and the clearance of larkspur toxins. Low larkspurs are short (10 to 80 cm tall) members of the genus Delphinium that can produce fatal cattle poisonings. Not all breeds of cattle respond the same to different low larkspur species and plant populations have different toxic potentials to livestock. ARS researchers at Logan, Utah compared the alkaloid toxicokinetic profiles of toxic and less-toxic alkaloids found in the low larkspur D. andersonii in beef and dairy cattle. There was some variation in the clearance times between alkaloids and breeds. For example, in Angus, seven half-lives of 16-deacetylgeyerline would clear 99.2% of the serum alkaloid in 5.4 days. Holsteins cleared larkspur toxins more quickly than Angus cattle as shown by their toxicokinetic data. These results will also be used to establish larkspur breed-specific withdrawal recommendations that will ensure the quality and safety of products from potentially exposed cattle.
Individual variation in the susceptibility to larkspur toxins was assessed. Observations by livestock producers and pilot studies by ARS researchers in Logan, Utah indicate that there is substantial animal-to-animal variation in response of different breeds of cattle to a debilitating dose of larkspur. Further, ARS researchers in Logan, Utah have previously demonstrated that there is variation in the susceptibility to larkspur toxicosis between different strains of mice, when they are used as a model system for cattle. Several potential susceptibility factors that could explain the differences in susceptibility of the different strains of mice to larkspur toxicosis were identified, including the a7 subunit of the nicotinic acetylcholine receptor (nAChR). However, current research has determined that genetically engineered mice lacking the a7 subunit of the nAChR are not more resistant to larkspur toxicosis, indicating that a7 subunit of the nAChR does not play an integral role in the acute toxic effect of larkspur toxins. Other potential genetic markers continue to provide the basis for future experiments to identify genetic factors that correlate with susceptibility to larkspur toxicity in cattle.
The effect of neurotoxic plant alkaloids on muscle function and coordination was characterized. Plants containing neurotoxic alkaloids are commonly found on open rangelands, and livestock can be poisoned when they consume lethal or debilitating amounts of these plants, with negative economic impacts on the livestock industry. A significant clinical sign observed in livestock poisoned by plants that contain neurotoxins is the disruption of coordinated muscle function. Previous to this research there were no studies on the acute effects of these toxins on muscle function and coordination. ARS researchers in Logan, Utah have characterized the motor function and coordination deficiencies that occur upon exposure to a non-lethal dose of the larkspur alkaloids MLA and deltaline, as well as the tobacco alkaloids nicotine and anabasine. These results highlight the fact that animals which survive poisoning episodes from neurotoxic plant alkaloids will fully recover without lasting muscular function and coordination deficits. The results obtained from these studies provide a basic understanding of the acute toxicity and adverse effects, including motor function and coordination, of specific toxic plant alkaloids. This information is used to better design experiments using various livestock species so as to provide management recommendations to livestock producers, extension agents, and government regulatory agencies.
Evaluation of co-administration of multiple poisonous plants in animals. In most cases where livestock are poisoned by plants on rangelands, animals are grazing multiple species of poisonous plants. Two poisonous plants often found growing simultaneously in the same area are death camas and low larkspur. In some years, losses to the livestock industry from low larkspurs exceed $10 million. Death camas also has a significant impact on sheep production and has recently been implicated in several significant cattle losses. Previous risk assessments and management recommendations for these plants, and the rangelands that contain them, have been based only on individual plants. ARS researchers in Logan, Utah recently demonstrated that the co-administration of toxins from death camas and low larkspur have an additive effect (they both contribute to the toxicity), resulting in enhanced toxicity in a rodent model. However, similar experiments indicate that because sheep are resistant to the toxicity of low larkspur, co-exposure to low larkspur has no effect on the toxicity of death camas in sheep. These results provide an increased knowledge and understanding regarding the acute toxicity of death camas and risk of co-exposure of multiple plant toxins. This information is useful in developing livestock management recommendations for ranchers, government action agencies, extension agents, veterinarians, and livestock owners, who can use this information to formulate management recommendations to reduce the risk of losses when grazing cattle on larkspur- and death camas-infested rangelands.
Identification of animals less susceptible to larkspur toxins. ARS scientists in Logan, Utah quantified the responses of cattle to a standardized dose of tall larkspur, and used that response to identify and classify susceptible and resistant individuals. There was a three-fold difference between breeds in their response to a single larkspur dose. All breeds tested in this experiment had individuals either resistant or nearly resistant to larkspur poisoning. These differences are likely due to each individual animal's genetic predisposition for larkspur poisoning which can be predicted from its DNA sequence. These results will be used to identify genetic markers for use by producers to maximize the productivity of cattle on rangelands where larkspur grows.
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Welch, K.D., Green, B.T., Panter, K.E., Pfister, J.A., Gardner, D.R. 2013. The role of the a7 subunit of the nicotinic acetylcholine receptor in the acute toxicosis of methyllycaconitine in mice. Journal of Applied Toxicology. 33(9):1011-6.
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