Duration: 10/01/2025 to 09/30/2030

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

We propose the continuationof a multi-state project to study poisonous plants, their impact, ecology, and management. Poisonous plants cause problems over a large geographic area and the researchers that are experts in the fields that address the problems are spread over many disciplines, many states, and different groups, ie. state, county, universities, and government agencies. A multistate project will improve communication and research efficiency which are needed to determine the impacts on the rangeland grazing communities and for develoment of management solutions. This group will meet annually to discuss, assess, and prioritize research topics such as as toxicology, diagnostics, toxin detection, range management, as well as plant and fungal ecology and physiology. The group will develop an action plan, including identifying research of highest impact for management, and identifying funding for the highest priority research. The group will coordinate research to provide preliminary information needed to secure grant funding. The group will also bring together research resources including plant samples collected from various locations, and provide periodic written documents reviewing the status of plants, and management for dissemination among collaborating institutions and throughout range communities. Incorporating research from multiple states will contribute to management solutions for local, state-wide, and national poisonous plant problems as well as decreasing duplication of research and increasing dissemination of results. This work can benefit society by helping to more completely understand poisonous plants which in turn may protect the food supply by aiding ranchers whose livestock is impacted.

Statement of Issues and Justification

The livestock industry in the western United States loses over $500,000,000 annually from death losses and abortions due to poisonous plants (Holechek, 2002).  Actual losses due to poisonous plants are much greater due to wasted forage and increased management costs. Poisonous plants are estimated to affect 3-5% of cattle, sheep, goats, and horses in the western US. (Panter et al., 2011).  Direct losses are due to decreased weight, reduced reproduction, failure to thrive, and sometimes death. Indirect costs include medical treatment, increased feed reequirements, altered grazing plans, increased fencing, and decreased forage availability (Panter et al., 2011). Plant poisonings occur worldwide and include 333 million poisonous plant-infested hectares in China (Xing et al. 2001; Lu et al. 2012) and 60 million hectares in Brazil (Low, 2015).  There are hundreds of genera of toxic plants representing thousands of species. Some of these plants produce toxins directly such as larkspur and lupine, while for others, such as locoweeds, the plants contain fungal endophytes that produce the toxic agents. Fungal endophytes of grasses (such as fescue) produce alkaloids that are toxic to grazing animals. Other poisonous plants are toxic due to the accumulation of nitrates or selenium from the soils in which the plants grow.  Poisonous plants continue to cause large losses to the livestock industry through death, reduced production efficiency, reproductive dysfunction, and compromised harvesting of rangeland and pasture forages. New Mexico State University researchers concluded that calf and lamb crops in the western United States are reduced 7% overall from toxic plants, including birth defects, negatively impacting ranchers and rural economies (Holechek, 2002).  Other economic losses are substantial, but difficult to quantify, as significant amounts of nutritious forage are underutilized, and management costs are increased due to the threat of toxic plant-related livestock losses.  These direct and indirect losses from poisonous plants adversely affect the economic viability of individual ranches and nearby rural communities that rely on the livestock industry as a substantial portion of their economic base. Current management grazing strategies could be further refined to reduce livestock losses due to poisoning and to enhance animal welfare.  This in turn ensures proper, safe, and efficient use of available rangeland forage and feeds to provide the production of toxin-free animal products. This project will focus on poisonous plants in the western US that induce toxicoses in cattle, horses, sheep, and goats.

 

Larkspur and lupine are plants that directly produce toxic agents that are important in the western US. Larkspur (Delphinium spp.) poisons cattle, and sometimes horses, throughout western North America due to the toxic alkaloids that they produce (Green et al. 2009). Symptoms include muscle weakness, paralysis, and ultimately death. Grazing recommendations have been developed based upon the relative palatibility and toxicity of the plant resulting in a signficant reduction of losses (Pfister et al. 2002). Several toxic species of larkspur have been identified, as have the alkaloids they produce (Panter et al, 2002). However, not all populations are toxic or have the same alkaloid profile, and toxicity varies with time of the year. This information has also been used to help ranchers limit their losses.

 

Lupine (Lupinus spp.) are toxic to all livestock species with sheep being affected most often.  Lupines are found in diverse habitats such as mountains and foothills in both wooded and open areas. While most species are toxic, only some contain contain the alkaloids that cause the tetragenic effects, including cleft palates and leg and muscule deformities (Panter et al., 2011). Consumption of lupines causes problems to the offspring of pregnant cattle because of quinolizidine alkaloids in the plant that harm the fetus (Pfister et al, 2016). Factors that influence symptom severity include stage of plant maturity and timing and amount of lupine consumed, as well as stage of pregnancy of the animal.

 

Locoweeds are Astragalus and Oxytropis spp. that are poisonous due to the fungal-produced indolizidine alkaloid swainsonine. Other species of Astragalus may accumulate selenium or nitro compounds (Fox et al., 1998). Locoweeds are the most widespread group of poisonous plants in the western United States (Graham et al., 2009) and also cause significant problems to sheep in China and Inner Mongolia. Consumption of the swainsonine-containing plants induces locoism in grazing animals such as cattle, sheep, and horses. Locoism symptoms include reproductive problems, cellular vacuolization, neurological damage, and lack of coordination (James et al., 1992). In New Mexico in 1985, over 10% of the cow/calf and 40% of the cow stocker operations reported losses of over $20 million from locoism (Torell et al., 2000). The fungal endophytes (Alternaria section Undifilum sp.) of the locoweeds that produce swainsonine are seed transmitted and do not harm their plant host (Pryor et al., 2009; Oldrup et al., 2010).

 

Several other plants are toxic due to the presence of plant-associated fungi that produce swainsonine and other toxins. The fungus, Slafractonia leguminicola, produces both swainsonine, causing locoism, and slaframine, inducing slobbers, in cattle and horses. The fungus also causes a plant disease, black patch, which is found in the southeastern US. In Australia, Alternaria sp. that inhabit Swainsona sp. produce swainsonine, inducing pea struck disease in sheep.

 

Perennial rygrass and tall fescue can contain fungal endophytes that produce alkaloids toxic to grazing mammals. These are problems in the southeastern US, Australia, and New Zealand. The fungi, Neotyphodium coenophialum, produces loline alkaloids, ergovaline, and other toxins that cause high respiration rates, intolerance to heat, poor animal gains, reduced milk production, depressed feed intake, and low conception rates in cattle, and tetragenic effects to horses. The fungal endophyte also has shown benefits to its grass hosts including herbivore defense (Clay 1990), increased heat and drought tolerance (Bacon and White, 2000), and improved plant vigor and resistance to some pathogens (Molyneux et al., 2007). 

 

Effective management of poisonous plants has been difficult to implement and costly despite knowledge of which plants are toxic. Recommendations include restricting access to pastures for grazing, supplementing cattle feed so that they don’t graze on the poisonous plants, spraying herbicides, making sure plants have adequate salt and water, and reducing stress on the animals (Graham et al., 2009; Panter et al., 2011). Behavior modification of cattle and horses has also been attempted as a management option for plants such as locoweeds. Sustainable management programs that can be implemented in multiple states or regions are needed for these difficult problems. 

 

Much more research on poisonous plants is necessary to adequately develop management programs. Rapid identification of plants, understanding the conditions under which the plant is most dangerous to animals, better knowledge of which toxins are present, and the mechanisms of toxicoses would greatly improve management. Understanding locoweed-fungal endophyte interactions and the tall fescue-fungal endophyte interactions can significantly impact plant/microbe interactions, secondary metabolite production and the continuum between mutualistic and commensalistic interactions.

 

      We propose establishment of a multi-state project to study poisonous plants, their impact, ecology, and management. Poisonous plants cause problems over a large geographic area and the researchers that are experts in the fields that address the problems are spread over many disciplines, many states, and over different groups, ie. state, county, universities, and government agencies. A multistate project will improve communication and research efficiency which are needed to determine the impacts on the rangeland grazing communities and for develoment of management solutions. This group will meet annually to discuss, assess, and prioritize research topics such as as toxicology, diagnostics, toxin detection, range management, as well as plant and fungal ecology and physiology. The group will develop an action plan to determine who will accomplish which aspects of the research, including identifying research of highest impact for management, and who will work together to seek funding for the highest priority research. The group will coordinate research to provide preliminary information needed to secure grant funding. The group will also bring together research resources including plant samples collected from various locations, and provide periodic written documents reviewing the status of plants, and management both for dissemination among collaborating institutions and throughout range communities. Incorporating research from multiple states will contribute to management solutions for local, state-wide, and national poisonous plant problems as well as decreasing duplication of research and increasing dissemination of results. This work can benefit society by helping to more completely understand poisonous plants which in turn may ultimately protect the food supply by aiding ranchers whose livestock is impacted.

Related, Current and Previous Work

There are no current ongoing multistate projects to address poisonous plants. The International Symposium on Poisonous Plants is an international group that meets every four years. The W2193 program members have met together for the past 5 years. This group unites experts on toxic plants with those that specialize on the veterinary aspects of toxicoses to better develop management tools.

Objectives

1. 1. Bring together a group of university, government, extension, and industry-based individuals to assess the current status of poisonous plants in the US and set priorities for research.
   
2. 2. Identify poisonous plants and their toxins
   
3. 3. Characterize the toxicoses induced by consumption of toxic plants
   
4. 4. Determine the ecology and physiology of poisonous plants in the western US
   
5. 5. Develop diagnostic and other management tools for poisonous plants
   
6. 6. Develop and coordinate management of toxicoses.
   

Methods

1. 1. Bring together a group of university, government, extension, and industry-based individuals to assess the current status of poisonous plants and set priorities for research. Individuals working on different aspects of poisonous plants, including their toxicoses, their ecology, and their management, work together already or are planning potential projects. Those committed to joining the group include various disciplines including range science, weed science, animal science, toxicology, plant science, and molecular biology from universities and government agencies. The committee will meet annually to discuss the status of poisonous plants in the US and present the latest developments in research. At the annual meetings, the group will also discuss gaps in the knowledge related to poisonous plants and set priorities for research. The annual meeting will provide a forum for distribution of current and ongoing research that may not yet be available by other means which in turn will allow for discussion, exchange of ideas, and identification of issues at the forefront of the problems with poisonous plants. The objectives presented below were named as likely research topics by potential committee members.

    

   2. Identify poisonous plants and their toxins.

   Proper identification of poisonous plants is essential, as is characterization of their toxin(s). Knowledge of how a toxin is produced and how it causes disease is an esential first step in understanding the toxic potential and developing tools for management.

   Researchers at New Mexico State University (NMSU) and the USDA Poinsous Plant Research Lab (PPRL) are pursuing a collaborative project to determine which Astragalus and Oxytropis species throughout the western US contain swainsonine as well characterize the associated fungal endophytes. Recent research has resulted in a reference list of species that contain swainsonine as well as the identification of three Alternaria species associated with three locoweed species. More work is needed to further define which plant species contain swainsonine as well characterize the associated fungal endophytes both morphologically and phylogenetically. Work will be done in collaboration of Rebecca Creamer and Daniel Cook. Hypothesis: Identification of new swainsonine producing endophytes will facilitate the understanding of plant-fungal relationships. Rationale: Locoweeds are Astragalus, Swainsona and Oxytropis species that are  known to contain swainsonine, the toxic principle produced by the plant endophytic fungus Alternaria section Undifilum. Swainsona is a large genus of flowering plants native to Australia, and the genus Astragalus is considered the largest and the most diverse genus among all flowering plants consisted of 2,500 to 3,000 species worldwide 373 species which of present in the United States. Recently, fungal DNA extraction from Swainsona and Astragalus unraveled the presence of undefined Alternaria spp. Morphological and molecular identification techniques must be applied to identify and characterize these new species of Undifilum to aid plant-fungal relationship. Experimental Design: New species of fungi have been isolated and characterized morphologically. Fungal DNA has been extracted and tested using PCR with primer sets to both conserved and variable regions. Current work is focusing on developing new primers to variable intergenic regions from the SWN gene cluster. All amplicons will be analyzed using Illumina sequencing technology. Finally, phylogenetic trees will be generated using Geneious Prime.  

    

   Research at the USDA PPRL has characterized the alkaloid composition of many larkspur species. Alkaloid composition may vary qualitatively and quantitatively between different species as well as populations of larkspur. This information has been used to make better management decisions on some larkpur infested pastures. Nonetheless, more research is needed to characterize the alkaloid composition of other larkspur species in the Western United States.  Work will be led by Daniel Cook. Hypothesis: The alkaloid profiles will differ with Delphinium species. Rationale: Concentrations of the MSAL (highly toxic) and non-MSAL-type (less toxic) alkaloids differ qualitatively and quantitatively among and within Delphinium species (Gardner et al. 2002; Cook et al. 2009a; 2015; 2017).  Management recommendations for cattle grazing on rangelands containing larkspur are based primarily upon the concentration of the MSAL-type alkaloids (Pfister et al. 1999; 2002).  Specific information on the alkaloid composition in regard to the MSAL and non-MSAL-type alkaloids is lacking for many Delphinium species.  The objective of this research is to define the alkaloid composition of the more than 40 species of Delphinium that have not been investigated.  The alkaloid profiles found in Delphinium herbarium specimens are representative of field collections suggesting that the larkspur alkaloids do not deteriorate over time (Cook et al. 2009a; 2015; 2017).  Experimental Design:  Herbarium specimens will be sampled from collaborating herbaria with an aim to sample the representative geographic distribution of each Delphinium species based upon the USDA plants database (https://www.plants.usda.gov/java/).  The number of specimens surveyed for each species will be a minimum of 4 to 6 specimens with greater numbers based upon the geographic distribution of each species.  Approximately 25-50 mg of plant material will be removed from each specimen.  Information from each specimen including species, state, county, and voucher number will be recorded from each specimen.  Alkaloid profiles will be evaluated for the presence or absence of the non-MSAL and MSAL alkaloids by electrospray Mass Spectrometry.

    

   Slafractonia leguminicola, a fungal pathogen, produces two toxins, swainsonine and slaframine. Investigators at NMSU, USDA PPRL, and Univerisity of KY have cooperated to characterize the fungus, its production of toxins, and its impact on plants. They are currently investigating the biosynthesis of slaframine. Work to be led by Rebecca Creamer. Hypothesis: RNA-mediated down regulation and gene knockouts in the fungus Slafractonia leguminicola will result in reduction of both slaframine and swainsonine transport. Rationale:   The fungus Slafractonia leguminicola causes black patch disease of red clover plants and other legumes. This mold produces two toxins, slaframine and swainsonine, that are harmful to livestock grazing of legume hay or pasture infested with the fungus, causing slobbers syndrome (by slaframine) and locoism (by swainsonine). The mechanism by which the fungus porduces slaframine is poorly understood. This project is to study the biochemical synthetic pathway for slaframine. This research will lead to a better understanding of the interaction of plant and fungus on slaframine and subsequent effects on animals that consume the fungus. Through increased knowledge of this interaction, we may be able to develop strategies to minimize swainsonine or slaframine poisoning in these poisonous plants. This work can benefit society by helping to more completely understand the plant-pathogen interaction which will eventually safeguard the food supply by aiding ranchers whose livestock suffer from slobbers and locoism. Experimental Design:  We will generate a silencing constructs pSilent-swnK, -swnKp1, and -swnKp2 using pSilent-1 vector developed Nakayashiki et al., which will include inverted repeat transgenes (IRT) of the genes. In addition, we will produce knockouts of the same regions, swnK, swnKp1, and swnKp2 using CRISPR-Cas9 technology developed for Aspergillus.  We will transform S. leguminicola protoplasts, and test for transformants. 100mg of dry mycelia from each culture will be weighed and extracted for swainsonine and slaframine and also the toxin concentration of the media will be measured according to a previously published method. Liquid chromatography–Mass spectrometry (LCMS) analysis of samples will be done at the USDA Poisonous Plant Research Laboratory, Logan, UT. Swainsonine and slaframine levels will be compared using paired Student t-test analyses.

   Ipomoea sp. are toxic plants many of which contain a Chaetothyriales fungus that produces the toxin swainsonine. Feeding on this plant causes toxicoses to animals including goats. Prior research established the microscopic location of the fungus within plants and seeds. Cooperative research between NMSU and USDA PPRL is ongoing to characterize the relationships among endophytes from different Ipomoea sp. and locations. Work to be done in collaboration of Rebecca Creamer and Daniel Cook. Hypothesis: Chaetothyriales seed endophyte/leaf epiphyte sp. will differ by Ipomoea sp. and location. Rationale: Ipomoea canescens has yielded a Chaetothyriales fungus. However the diversity in species of the fungus has not been determined. It is likely that different species of Ipomoea will yield different species of fungi. Since Ipomoea canescens is found in a variety of locations with different environmental conditions, it is possible that there will be diversity among the the Chaetothyriales from plants in different locations. Experimental design: Seeds from Ipomoea canescens collected from different locations and from different Ipomoea sp. will be tested by isolating total nucleic acid using a kit (Zymogen – seed). The extractions will be characterized for fungal identity using PCR and primers to gpd (gylceraldehyde phosphate dehydrogenase), ITS region, swnKS, and 3 intergenic regions in the SWN cluster. All amplicons will be analyzed using Illumina sequencing technology. Finally, phylogenetic trees will be generated using Geneious Prime.  

    

   3. Characterize the toxicoses induced by consumption of toxic plants

   Understanding toxicoses requires knowledge of the type of toxin, body condition, age, type, and sex of the animal, abd animal history. Research is being pursued by the USDA ARS PPRL to investigate the relative susceptiblity of native versus naïve animals to the toxic effects of larkspur.  Research is being led by Clint Stonecipher.  Hypothesis: Animals native to larkspur-infested rangelands have been selected for resistance to the toxic effects of larkspur and are less likely to be poisoned than cattle naïve to larkspur rangelands.  Rationale: Ranchers that graze cattle on rangelands with large populations of toxic larkspur often have yearly herd mortalities up to 10%. The proper selection of replacement animals for grazing on larkspur containing rangelands is important. For example, ranchers have often reported that the greatest larkspur losses occur with replacement animals, and once the initial losses are over, larkspur poisoning is much less of a problem. A similar observation has been reported for regional fescue toxicosis in cattle, where “fescue native” animals are more resistant to the toxic effects (Johnson et al., 2015). Identifying replacement animals that are less likely to be poisoned by larkspur will be highly beneficial to livestock producers.  Experimental Design: A grazing study will be conducted on larkspur-infested rangeland in southeastern Idaho. Six pastures will be established using electric fence encompassing an area with an abundant duncecap larkspur (Delphinium occidentale) population. Six native cattle with extensive experience grazing on larkspur-infested rangelands will be randomly assigned to one of three pastures with two animals per pasture; six naïve cattle will be assigned to one of three pastures with two animals per pasture. Cattle will graze for a four-week period when larkspur has reached the flower stage of phenological development. Bite counts will be recorded by forage class (grass, forbs, and larkspur) on each animal to determine diet. Each animal will be observed for five minutes before moving to the next animal. Animals will be observed during active grazing bouts throughout the day. Animals will be monitored and any clinical signs of intoxication from larkspur will be recorded for each individual animal. A timeline will be recorded of initial poisoning, any signs/symptoms of intoxication, any treatments, and when the animal recovers. Forage will be clipped prior to grazing and every week during the study to determine forage availability and forage quality. Larkspur plants will be collected on a weekly basis to quantitate the larkspur alkaloid concentration during the grazing study. Blood will be collected from animals on a weekly basis to determine alkaloid concentrations in the serum. The study will be repeated a second year with a new set of animals for two years of data. Bite count data will be analyzed using a linear mixed model (PROC MIXED) in SAS. Daily means will be calculated for each animal’s diet selection. The model will include treatment (native vs. naïve animals), date, the treatment x date interaction, pasture nested within treatment, animals nested within treatment and pasture, and date x pasture within treatment. Pasture, animal, and date will be random factors in the model. Means and SE will be calculated for forage availability, nutritional analysis of forage, and the alkaloid concentration in larkspur.

    

    

    

   4. Determine the ecology and physiology of poisonous plants in the western US.

   The toxic potential of a plant may be influenced by environmental factors as well as genotype. Research being pursued by Montana State University and the USDA PPRLhas investigated the relative role of the locoweed endophyte in contributing to plant fitness. Work by Rebecca Creamer, Tracy Sterling, Chris Schardl, and Daniel Cook looks at the role that ecology plays in seed transmitted pathogens. Hypothesis:  Expressed genes differ among E+ and E- locoweed plants from different locations. Rationale:  Locoweed plants have long been associated with Alternaria section Undifilum fungi. The role of the endophyte could be commensal, not helping or harming the plant and the genotype of the endophyte may differ between different collection locations. Experimental design:  Locoweed plants collected from two locations in Colorado were assessed for fungal endophyte by testing for swainsonine. Similarly locoweed plants E+ and E- were planted into a common garden. Total nucleic acid from E+ and E- plants were extracted and subjected to next gen sequencing to determine if there was variability in fungus by location. RNAseq will be conducted to determine if the plant expressed sequences differ between E+ and E- status.

    

   5. Develop diagnostic and other management tools for poisonous plants

   Current diagnostic and management tools are lacking for many toxic plants. Investigators at the USDA PPRL are devloping tools to aid in diagnostics.  For example, methods are being developed to determine if animals were exposed to a toxic plant by evaluating different matrices such as rumen contents, ocular fluid, and ear wax. These tools may aid in detemining the plant animals may have been exposed too.  Work to be led by Stephen Lee and Clint Stonecipher.

   Hypothesis: The toxins from poisonous plants are excreted in the earwax and hair of livestock in sufficient concentrations to be detected by analytical methods.  Rationale: Poisonous plant-induced death losses often go undiagnosed because there is a lack of appropriate, or available, specimens for analysis. Guidelines have been developed to assist in collection and preparation of several tissue specimens for diagnosis of plant poisoning including GI contents, serum, eyeballs etc. (Stegelmeier et al., 2009, Lee et al., 2020). However, earwax and hair have been neglected as potential specimens in determining livestock consumption of poisonous plants. Initial work on the evaluation of noninvasive specimens, including earwax, hair, oral fluid, and nasal mucus, in livestock that have ingested lupine and larkspur plant material suggests that earwax and hair are specimens that may prove to be valuable tools in the assessment of livestock poisoned by plants (Lee et al., 2019, Stonecipher et al., 2019). A benefit of these specimens is that they can easily be collected by untrained personnel without the need for any special equipment.  Experimental Design: Cattle and sheep will be administered specific doses of death camas and locoweed. The doses of each plant will be based on previous experiments at the lab. These doses will cause little to no clinical signs of poisoning. Ear wax will be collected by wiping the inside of the ear canal with a tissue. Hair samples will be collected by shaving a vertical strip on the back of the animal near the front shoulder using electric clippers. The outer layer of hair will be discarded, and the remaining hair (1.25 cm) will be clipped to the skin and recovered for analysis. The only the 1.25 cm of hair closest to the skin will be analyzed. Initially, earwax and hair will be collected, and analytical methods developed to detect the plant toxins in these matrices. Earwax and hair will be collected at appropriate time points to determine the excretion and deposition time period of the plant toxins in these specimens. Samples of earwax and hair from livestock in herds not exposed to these poisonous plants along with herds suspected of being exposed to these poisonous plants on the range will be collected and analyzed for the toxins. Appropriate doses and sampling intervals will be modified for specific toxic plants. Experiment 1) Death camas causes an acute toxicity in livestock. Therefore, sheep (n=4) and cattle (n=4) will receive a single dose of 0.5 g/kg BW dried ground death camas plant material. Earwax and hair will be collected from each animal prior to dosing plant material and then every three days for 30 days post dosing. Earwax and hair will be analyzed by previously established analytical techniques (Lee et al. 2020). The methods will be adjusted as needed to establish a viable method for the detection of the death camas toxins, or metabolites, in these matrices. Experiment 2) Because locoweed presents as a chronic toxicity in livestock, two different dosing regimens will be investigated. 1) cattle (n=4) will receive a single dose of dried ground Oxytropis sericea at 2 mg/kg swainsonine (the toxin in locoweed). The earwax and hair samples from this experiment will be used to determine if swainsonine can be detected in these specimens from cattle after a single dose. 2) cattle (n=4) will receive 10 doses of dried ground Oxytropis sericea at 2 mg/kg swainsonine once a day for 10 days. Earwax and hair will be collected from each animal prior to the start of dosing plant material and every three days for 30 days post dosing. The samples from the second dosing regimen will be used to determine if swainsonine can be detected in the earwax and hair of livestock exposed to locoweed over a more extended time, as would be the case in grazing animals who show signs of intoxication. Earwax and hair will be analyzed by previously established analytical techniques (Gardner and Cook 2011). The methods will be adjusted as needed to establish a viable method for the detection of swainsonine in these matrices. Experiment 3) It has been previously determined that the teratogenic lupine alkaloids can be detected in earwax for 30 days after a single dose of lupine plant material, and the same alkaloids can be detected in animals grazing lupine-infested rangelands (Lee et al., 2019). Therefore, earwax from pregnant cattle (n=50-60) that have grazed on lupine-infested rangelands will be collected and analyzed. The samples will be collected at the livestock owners ranch in the fall after the cattle are done grazing on the lupine-infested summer rangelands. After the calves are born, the concentrations of teratogenic alkaloids from individual animals will be correlated with malformations observed in the offspring of the pregnant cows. All earwax and hair samples will be analyzed for toxins and their metabolites by HPLC-MS (Lee et al. 2019).

    

   Investigators at the USDA PPRL are collaborating with other USDA groups to idenitfy the potentail for betonite clay to sequester larkspur toxins in the rumen, thus preventing intoxication of cattle. Work to be led by Ben Green, Clint Stonecipher and Kevin Welch.  Research Hypothesis: Bentonite clay can sequester larkspur toxins in the rumen, which will prevent intoxication of cattle.  Rationale: Published research from the PPRL has shown that mineral supplementation of cattle can affect/decrease larkspur poisoning (Stonecipher et al., 2022a). Recently, we have examined selected components of mineral mixes for their ability to adsorb/bind larkspur toxins in vitro. Results from these experiments suggest that bentonite avidly binds methyllycaconitine (MLA) the principal toxin in larkspur. Based upon results from our in vitro research, we are extending this research into cattle. Experimental Design: Experiment 1) Dose optimization of larkspur and bentonite in cattle. A dose-response study will be conducted to determine the amount of bentonite that is optimal for binding larkspur toxins in cattle. Cattle (n=16) will be assigned to one of four treatment groups (n=4; control-no bentonite, 31, 125, or 250 mg bentonite/ gram of larkspur plant material) in a 4 x 4 Latin square experimental design. Cattle will be dosed orally with larkspur at 4 mg/kg MSAL-type alkaloids and then orally gavaged their respective bentonite treatment. Blood will be collected over a 48-hour period after dosing to monitor larkspur alkaloid toxicokinetics (Stonecipher et al., 2022a). It is our hypothesis that bentonite will bind to and sequester the larkspur alkaloids in the rumen thus preventing them from being absorbed and appearing in the serum. Serum alkaloid data will be analyzed as a replicated Latin square design. Treatment (bentonite dose), square, and period will be fixed effects. Animal within square will be a random effect. Experiment 2) Larkspur-bentonite challenge study. With the optimum dose of bentonite identified in experiment 1, a study will be conducted wherein cattle will be dosed with a highly toxic dose of larkspur (10 mg/kg larkspur alkaloids) followed with bentonite. The study will be a crossover design (two groups of six animals, AB/BA). Group A will be supplemented with bentonite following larkspur dosing and group B will not be supplemented with bentonite. After a three-week period to allow larkspur alkaloids to be eliminated the groups will be switched and the study repeated. Blood will be collected over a 48-hour period after dosing to monitor larkspur alkaloid toxicokinetics. Serum data will be analyzed using a two-treatment (bentonite vs no bentonite), 2-period crossover design with repeated measures ANOVA in Prism (Stonecipher et al. 2022a). If the bentonite inhibits the absorption of the larkspur alkaloids, the cattle will not become poisoned. The animals will be closely observed for 48 hours after dosing for the development of clinical signs of poisoning. Experiment 3) Larkspur-mineral mix study. A commercial mineral mix containing bentonite as an ingredient will be tested to determine if it can prevent cattle from becoming poisoned after a larkspur challenge. Cattle will be dosed with larkspur at 4 mg/kg MSAL-type alkaloids. The commercial mix will be compared to determine if it is equivalent to bentonite. Study design will be a 4 x 4 Latin square design (n=16) with four animals per treatment group. Treatments will be control (no mineral), a treatment of mineral based on the amount of bentonite discovered to be optimal in Experiment 1 and two doses below the optimal bentonite. Blood will be collected over a 48-hour period after dosing to monitor larkspur alkaloid toxicokinetics. Serum alkaloid data will be analyzed as a replicated Latin square design. Treatment (mineral dose), square, and period will be fixed effects. Animal within square will be a random effect. Experiment 4) Grazing study. A grazing study with cattle will be conducted in a larkspur-infested pasture. There will be two groups of cattle. One group will receive a bentonite-containing mineral supplement for 30 days prior to the study and the other group will be a control group. Cattle will graze for a two-week period when larkspur has reached the flower stage of phenological development. Bite counts will be recorded by forage class (grass, forbs, and larkspur) on each animal to determine diet. The cattle will also be closely observed to monitor for the development of clinical signs of larkspur poisoning. Forage will be clipped prior to grazing and every week during the study to determine forage availability and forage quality. Larkspur plants will be collected on a weekly basis to quantitate the larkspur alkaloid concentration during the grazing study (Gardner et al. 2021). Blood will be collected from animals on a weekly basis to determine alkaloid concentrations in the serum. The study will be repeated a second year with a new set of animals for two years of data. Bite count data will be analyzed using a linear mixed model (PROC MIXED) in SAS. Daily means will be calculated for each animal’s diet selection. The model will include treatment (mineral vs. no mineral), date, the treatment x date interaction, pasture nested within treatment, animals nested within treatment and pasture, and date x pasture within treatment. Pasture, animal, and date will be random factors in the model. Means and SE will be calculated for forage availability, nutritional analysis of forage, and the alkaloid concentration in larkspur.

    

   6. Develop and coordinate management of toxicoses.

   Collaborating and coordinating research will streamline both development of management tools and identification of areas of high need for management. There is also a need to develop specific management strategies for different types of animals. For example, horses and ruminants respond differently to some toxic plants. The multi-state approach will allow for review of management tools effective at both local and regional levels. A new assessment of losses suffered by the livestock industry in the western United States is also of high necessity to provide context and direction to ongoing research efforts. We anticipate collaborations with agricultural economists to conduct new county-level assessments of the economic impacts of poisonous plants.  The research resulting from this project will be used to create management plans for ranchers, veterinarians, livestock producers, land managers, extension agents, and government agencies to assist in managing livestock on ranges, pastures, and fields where poisonous plants grow.  Improved techniques and tools for diagnosis, prognosis, and treatment of poisoning will be developed to assist livestock producers, veterinarians, and diagnosticians to improve animal health and welfare.  Information bulletins, pamphlets, presentations, and peer-reviewed scientific articles will be developed to provide current information to stakeholders and the general public on poisonous plants and best methods to avoid or reduce losses.  These resources will be made available through our website and websites of our extension partners. 

    

Measurement of Progress and Results

Outputs

• The primary product of this project will be data and information, as well as management recommendations. The types of expected data include plant range information, nucleic acid sequence from plants and fungi, new species descriptions for novel plant and fungal species, phylogenetic trees, toxin biosynthtetic pathways, data on ecological interaction parameters, data on efficacy of management strategies, and management recommendations. A joint publication reviewing the group accomplishments is the anticipated output at the end of the 5 year cycle.

Outcomes or Projected Impacts

• The primary outcome for the project is increased comprehension of poisonous plants. This information will support progress in managing poisonous plants by developings tools to minimize the losses associated with toxic plants. Overall, this work can benefit society by improving understanding of poisonous plants, which in turn may ultimately protect the food supply by aiding ranchers whose livestock suffer from them.

Milestones

(2029):Most of the projected research is highly dependent on prior research. Development of better tools for assesing toxins is one early milestone. Assessing populations and plant fitness, with and without associated microbial organisms can be accomplished across several states in parallel and is an ongoing project. Some specifics can be obtained through sequence for genetic fingerprinting of plants and fungi. Development of management strategies for specific plants and animals is a key component of the work. Once identified, management strategies will be investigated for efficacy and application feasibility at local and regional levels.

Projected Participation

View Appendix E: Participation

Outreach Plan

The results of the project will be disseminated through refereed publications, extension bulletins, field days, and a jointly produced review publication. Daniel Cook, Rebecca Creamer and Christopher Schardl have already published joint refereed publications on aspects of poisonous plants. Jason Turner is an extension animal scientist that has published extension bulletins on the impact of poisonous plants on horses. The USDA-ARS Poisonous Plant Laboratory (PPL) regularly puts on field days in Utah on the effects of poisonous plants. As such, the PPL and Jason Turner have already have developed stakeholder communication pathways with ranchers in their states (Utah and New Mexico, respectively) and further pathways for direct communication with ranchers in Montana and Colorado will be strengthened through this project.

      Current knowledge of poisonous plant problems is inconsistent among the public, and is highly dependent on location and prior experience. Additionally, there are likely non-research based management practices used by ranchers that have not been communicated outside of local groups. Combining team members from several different states and establishing a route of communication directly to ranchers will help bridge these gaps in knowledge.

            Schardl is the PI and Creamer and Cook are participants on a NSF-funded collaborative project 2021-2026 that includes researchers in 5 states and China that involves collection of seed from poisonous plants and identification of the fungi growing in them. That group has had joint meetings with the W2193 group that have provided an outlet for relevant research information for interested scientists. In addition, that grant has and will provide education and research training on poisonous plants and microbiology for under-represented minority undergradute students in Kentucky. The collaborative grant has provided funds for graduate students from New Mexico and Kentucky to attend the meetings of this multistate research project and present their research results

Organization/Governance

The group will be governed by an executive committee composed of 3 representatives from participating institutions. The committee will be composed of the chair, vice chair, and secretary, as well as the appointed administrative leader. Initially that composition will include representatives from New Mexico State University, the USDA Poisonous Plant Laboratory, and Montana State University and the project administrator. Individual  participation on the executive committee is likely to change every two years with changes in elected leadership. The executive committee will help plan and organize annual meetings, produce annual reports, and establish subcommittees for specific tasks such as the review publication.

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Attachments

Land Grant Participating States/Institutions

NM

Non Land Grant Participating States/Institutions

Pacific West Area