Duration: 10/01/2020 to 09/30/2025

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

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. There was a project SERA008 Fescue Endophyte Research and Extension but that project was terminated some years back. The International Symposium on Poisonous Plants is an international group that meets every four years. The W1193 program members have met together for some time with a narrow focus on locoweeds. Here we have expanded our previous locoweed focus to include the much broader community of poisonous plants. We hope that this will better unite experts on toxic plants with those that specialize on the veterinary aspects of toxicoses to better develop management tools.

Objectives

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

Methods

1. Assemble a group which will include 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 in the western US.

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 the new swainsonine producing endophytes will facilitates the understanding of plant-fungal relationship. Rationale: Locoweeds are the most widespread poisonous plant problem in the world and have been reported in the western United States since the 1800s, causing tremendous losses in livestock. The name locoweed is used for Astragalus, Swainsona and Oxytropis species known to contain swainsonine, the toxic principle produced by the plant endophytic fungus 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 eleven 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 will be extracted from plant materials and/or from seeds using aseptic techniques. Pieces of stem (length of 1 cm), and leaflets (3 or 4) will be removed from dried plant material using sterile forceps. Stem pieces and leaflets will be soaked in 70% ethanol for 30 s, 20% bleach for 3 min and sterilized water for 30 s. After drying, the pieces will be transferred onto water agar (WA) plates and observed daily. Seed isolation will be done following methods of Oldrup et al. (2010) with seed coats being placed on WA for endophyte isolation. Fungi that grow out of the plant material will be transferred to potato dextrose agar (PDA) and examined weekly for color of culture and presence of spores. For assessment of sporulation, fungal slides will be viewed with a Nikon Optiphot microscope (Nikon, Tokyo, Japan) for confirmation, then examined under Scanning Electron Microscopy SEM following fungal vapor-fixation procedure to record the length and width of the spores, and number of septa necessary for characterization. Fungal DNA then will be extracted and analyzed using Illumina sequencing technology. Finally, phylogenetic trees will be generated using Geneious Prime Version 2020.1.2 to aid plant-fungal relationship.  

 

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. They are currently investigating the biosynthesis of slaframine and developing methods to identify and quantify the toxin. Work to be led by Rebecca Creamer. Hypothesis: RNA-mediated down regulation of swnT gene 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 secretes the toxin out is poorly understood. This project is to study the role of the toxin transporter of this black patch fungus. This research will lead to a better understanding of the interaction of plant and fungus on slaframine and swainsonine secretion and subsequent effects on grazing animals. Through increased knowledge of this interaction, we may be able to develop strategies to eradicate swainsonine or slaframine poisoning in these poisonous plants. This work can benefit society by helping to more completely understand the plant-pathogen interaction and how these toxic alkaloids are transported out by the fungus, which will eventually safeguard the food supply by aiding ranchers whose livestock suffer from slobbers and locoism. Experimental Design:  We will generate a silencing construct pSilent-swnT. For construction of pSilent-swnT, silencing vector, pSilent-1 vector developed Nakayashiki et al., will be used, which will include inverted repeat transgenes (IRT) of the transmembrane transporter gene swnT. We will transform S. leguminicola protoplasts. Mycelia will be grown on potato dextrose agar (PDA) for 24 h at 28°C. Following incubation, the cell wall of mycelia will be digested with 5% lysing enzymes (Sigma, L1412) for 4-5 h at 30°C. Protoplasts (concentration ~10⁷/ml) will be transformed with ∼10 μg pSilent swnT -2 using 40% polyethylene glycol (PEG) 3350 (Sigma-Aldrich) and plated onto regeneration media with 0.6 M sucrose, 0.3% yeast extract, 0.3% casamino acid and 1.6% agar. Following 48 h of incubation at room temperature, plates will be overlaid with top agar containing hygromycin B (70g). We will also transform protoplast with only pSilent-1 plasmid (empty vector) as a control. We will transfer colonies that appear 7-14 days post transformation on hyg B resistance plates to Potato Dextrose Agar (PDA) plates. After 5-10 days there should be two different phenotypes: pSilent-1 transformants should retain wild type phenotype, and the swnT transformants will probably have colonies of irregular shape/color/texture. To check the stability of the transformant, the mutant colonies will be transferred three times onto selective PDA plates containing hyg B. If they can grow at least 2-5 cm on the PDA plates in ten days, they will be considered as stable transformants. On day 3 post transformation, we will check the differential expression pattern of swnT using qRT-PCR. Five selected swnT mutants, wild type and wild type transformed with empty vector pSilent-1 will be grown on PDA plates for eight days at 28 °C. The mycelial mass of each culture will be dried at 80°C for 3h. 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.

 

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, as well as the mechanism by which the toxin causes disease.

Slafractonia leguminicola produces two toxins, swainsonine and slaframine, however, the effects of slaframine on animals has not been well characterized. NMSU and USDA PPRL are interested in determining the effects of slaframine, and/or swainsonine, on animals such as horses. Work to be led by Jason Turner and Rebecca Creamer. Hypothesis: The effects of slaframine independent of swainsonine will be milder on horses than the combination of toxins. Rationale: Slaframine is thought to cause slobbers syndrome that is characterized by excessive salivation, lacrimation, feed refusal, bloating, diarrhea, abortion, stiffness, and weight loss. Swainsonine consumption also induces feed refusal, abortion, stiffness and weight loss, so those symptoms may be caused by swainsonine instead of slaframine. Slaframine at concentrations of 50-100 ppm have been detected in red clover hay. The role of swainsonine in slobbers is not known and the relative contribution of slaframine to slobbers has not been determined. Some authors have suggested that an interaction between swainsonine and slaframine may be necessary for slobbers symptoms. Others suggested that injecting purified swainsonine or slaframine did not reproduce the symptoms associated with slobbers. Almost no research has been done looking at the effects of slaframine on horses. Experimental design: Wild type Slafractonia cultures will be assessed to determine that they are producing swainsonine and slaframine and the levels of both toxins determined using GC-MS. Mutant Slafractonia will be produced using pSilent vector containing the KS-SW. The vector will be introduced into Slafractonia protoplasts and the resulting transformed fungal colonies allowed to grow. Each colony will be screened for swainsonine and slaframine presence and levels to select the most stable mutants. All swainsonine and slaframine levels will be screened at the Poisonous Plant Laboratory, Logan, UT. Wild type and mutant Slafractonia colonies will be for 2 weeks, harvested, dried and ground. Ground mycelia will be pooled and tested for swainsonine and slaframine levels prior to use. Since purified slaframine was shown to induce elevated salivation at 12 or 24 ug/kg body weight in steers, this study will use a dose of 24 ug/kg body weight. Ground fungal mycelia will be mixed with the feed as listed below.

Horses will be fed a basal diet (2x/d) comprised of hay and a commercial concentrate at 2% of their body weight in order to meet or exceed NRC (2007) nutrient requirement recommendations. Horses will be offered water and trace mineralized salt ad libitum throughout the experiment. A total of 9 mature horses will be used for this study to determine the effect of fungal culture isolates on the initiation of symptoms of slaframine poisoning in horses. The three treatments (3 horses assigned per treatment) consist of: (A) the basal diet (untreated negative control); (B) the “wild” type fungal isolate (swainsonine and slaframine); or (C) the modified fungal isolate (slaframine only). On day 0, at the 0700 feeding, horses will be fed the morning ration of their basal diet with the appropriate treatment top-dressed onto the diet. At 0700, and every 4 hours for the next 24 hours, vital signs (temperature, pulse, respiration) and physical appearance and behavior will be recorded to determine outward signs of slaframine poisoning. Along with the outward measurements, a blood sample will be taken every four hours to evaluate blood chemistry marker changes, as well as serum cortisol levels to characterize the physiological stress, that may accompany slaframine poisoning. After the first 24 hours, these same measurements will be taken at 12-hour intervals through 96 hours post-treatment to characterize the return to normal following the single dose of the respective treatment. Data will be analyzed using ANOVA-procedures for a completely randomized design with repeated measures.

 

 

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 NMSU, Montana State University and the USDA PPRL is investigating the relative role of the locoweed endophyte in contributing to plant fitness. For example, at Montanta State, investigators are determining the relative role of endophyte in plant defense specifically the production of volatile compounds. Investigators at all three insititutions are collaborating to determine if the endophyte may influence other microbiota associated with the plant. Work to be led by Rebecca Creamer, Tracy Sterling, and Daniel Cook. Hypothesis: Endophyte-containing locoweed plants deter the growth of bacteria that would otherwise be associated with the plants. Rationale: Locoweed plants have long been associated with Alternaria section Undifilum fungi. The fungi don’t hurt the plants, but it has not been clear if the fungi are beneficial to the plants. Preliminary tests from Oxytropis sericea plants from Montana and Utah suggested that bacteria much less likely to be isolated from E+ plants that E- plants, while other fungi are found in equal abundance. The difference in bacterial abundance could be due to the presence of swainsonine or the presence of the endophyte or a combination of the two options. If it is due to the presence of the endophyte, it could work by competitive exclusion or niche presence. Experimental design: To determine if swainsonine versus fungus containing-swainsonine has an impact on bacterial growth, bacteria isolated from E- locoweed plants will be grown on agar media with wells containing swainsonine or swainsonin-producing fungi in the center of plates, compared to Alternaria alternata control cultures. Embryo-cultured locoweed plants with or without endophyte will be sprayed with the same bacteria and allowed to grow for 2 months, then tested for bacteria inside the plants.

Likewise investigators at NMSU are interested in researching the forage crop sorghum. Work to be led by Jason Turner. Hypothesis: Sorghum hay could be utilized for animal forage under highly regulated conditions. Rationale: Sorghum is a desirable summer forage crop and use of its hay would be useful, however it has the potential to release hydrogen cyanide by ruminants. Hay could be more toxic than the fresh forage, but producing silage should decrease the dhurrin content. Grain sorghum varieties have higher dhurrin content and can cause problems in horses, as well as cattle and sheep. Fertilization of the sorghum plants affects dhurrin content differentially depending on plant age and growth conditions. Drought conditions also increase dhurrin content, but decrease toxic high nitrate levels. More information is needed to decipher the conditions under which sorghum could be used as forage and for which animals it could best be utilized. Experimental design: Four different varieties of sorghum hay types will be grown under well watered and drought stress conditions in a greenhouse trial. At harvest, subsamples will be dried from each treatment and dhurrin content will be compared in dried and fresh samples. Subsamples will be used for silage and dhurrin levels will be tested. Nitrate levels will also be tested in all samples.

 

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: Toxins from poisonous plants are excreted in the earwax, hair, oral fluid, and nasal mucus of livestock that have consumed poisonous plants 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 tissue specimens of gut content for diagnosis of plant poisoning (Stegelmeier et al., 2009).  However, earwax, hair, oral fluid, and nasal mucus have been largely neglected as potential specimens in determining livestock consumption of poisonous plants. Earwax, hair, oral fluid (e.g., saliva), and nasal mucus are noninvasive specimens and may prove to be valuable tools in the assessment of livestock animals exposed to and poisoned by poisonous plants. Experimental Design: Cattle will be administered specific doses of poisonous plants. Initially, earwax and hair will be collected, and analytical methods developed to determine if the plant toxins can be detected.  Earwax, hair, oral fluid, and nasal mucus will then be collected at appropriate time points to determine the excretion and deposition time period of toxins in these specimens.  Samples of earwax, hair, oral fluid, and nasal mucus from livestock in herds not exposed to a poisonous plant along with herds suspected of being exposed to a poisonous plant on the range will be collected and analyzed for the toxin.  Since we expect earwax samples to be limited, prior to dosing plant material, earwax production will be monitored and the appropriate time intervals necessary for earwax accumulation and sampling will be determined.  Unexposed cattle (n=4) will be dosed at 1 g/kg BW dried ground lupine material, which is a level that can inhibit fetal movement but not cause severe clinical signs of intoxication (Green et al. 2015b). Earwax, hair, oral fluid, and nasal mucus will be collected from each animal prior to the start of dosing plant material and then every 2-3 days, post dosing, for 30 days when sufficient earwax hair, oral fluid, and nasal mucus is produced for collection.  All ear wax, hair, oral fluid, and nasal mucus samples will be analyzed for concentrations of several important lupine alkaloids including anagyrine by High Resolution Mass Spectrometry (HR-MS).   Appropriate doses and sampling intervals will be modified for other toxic plants such as larkspur and locoweed.

 

Investigators at the USDA PPRL are collaborating with other USDA groups to identify a genetic marker that may predict the relative susceptibility of cattle to larkspur. Better diagnostic and management tools will help livestock producers minimize their losses. Work to be led by Ben Green and Kevin Welch. Hypothesis: There are sex-dependent differences in susceptibility to larkspur intoxication in Angus cattle. Rationale: Larkspurs (Delphinium spp.) have been a long-term problem for cattle grazing on rangelands of the western United States (Marsh et al., 1916). The scope of this problem can be as low as 2 to 5% cattle death losses, or catastrophically large when losses are as great as 15%. A simple, practical solution is needed for western U.S. cattle ranching operations. With this in mind, during the past we have focused on identifying genetic markers that associate with susceptibility to larkspur poisoning in cattle. These markers could then be used to identify animals most negatively impacted by larkspur (i.e., susceptible) and those animals would only be grazed on non-larkspur containing pastures. Over the course of our work we have phenotyped yearling steers from multiple cattle breeds by having them exercise after administering a standardized dose of larkspur. We have chosen the Angus breed for more research due to the distribution of steer responses to a standardized dose of Delphinium barbeyi and have observed that there are approximately 10% each of susceptible and resistant steers and bulls in every Angus herd sampled to date (Green et al., 2014; unpublished observations). Preliminary novel data suggest that yearling Angus heifers appear to be more susceptible to larkspur poisoning. Information on individual animal variation and sex-dependent responses to poisonous plants in cattle is lacking as well as the identification of genetic markers of susceptibility. Experimental Design: The following approach will be used to evaluate 15 Angus bull calves and 15 Angus heifer calves (Green et al., 2014; Appendix, p. 95). The Angus calves will be obtained from USMARC at the same time, housed in the same feedlot, and fed and gentled using the same procedures.  If more calves are needed they will be obtained from USMARC.  For the treatment protocol, animals will be dosed a plant slurry of about 200 g of dried, ground larkspur in 6 L of water through an intragastric tube providing a dose of 8 mg MSAL alkaloid/kg BW.  Toxin concentrations in the plant material will be determined in advance by FT-IR.  After oral gavage, each animal will be challenged with forced exercise for up to 40 min to compare stamina followed by continuous monitoring for clinical effects. The dependent variables include serum larkspur alkaloid concentration at 24 hours after oral plant dosing and time to clinical signs during exercise and will be analyzed using an incomplete block design. DNA will be saved from all screened animals.

 

A project led by Clint Stonechipher. Hypothesis:Providing a supplement high in crude protein will provide a nutritional context which is redundant to locoweeds causing livestock to avoid consuming locoweeds. Rationale: Locoweed species (Astragalus and Oxytropis spp.) are a serious toxic plant problem for grazing livestock resulting in significant economic losses. It is possible that supplements (e.g., high in protein) which complement the nutritional profile of the grazed plant community (e.g., high in fiber and low in nitrogen) will entice livestock to consume plants other than locoweeds (e.g., high in protein).  High protein feeds can be used to alter food choices by livestock (Perez et al. 1996; Villalba and Provenza, 1999).  Supplements high in nitrogen may antagonize intake of poisonous plants like locoweeds since the forb is high in nitrogen and thus provide a nutrient profile which is redundant to the animal after nitrogen supplementation.  Stonecipher et al. (2016) reported a decrease in forb consumption in cattle supplemented with protein compared to non-supplemented cattle.

Experimental Design: Two groups of livestock will be individually fed an iso-caloric diet with one group (n=6) supplemented with protein (soybean meal) and the other group (n=6) not supplemented.  Both groups will be acclimated to their respective diets for a 21-day period. Pellets consisting of 80-90% grass hay and 10-20% locoweed will be formed (a pilot study will be conducted to determine the concentration of locoweed in a pellet that animals will consume). Pellets will be offered to both groups of animals starting on day 22 for a 30-day period. Locoweed pellets will be offered for a 120-minute period each morning and refusals weighed back to determine locoweed consumption.  After locoweed pellets have been offered the groups will be fed their respective diet. Blood samples will be collected at day 22, prior to locoweed exposure, and every 3 days thereafter to determine serum swainsonine concentrations.  Treatment differences in locoweed consumption and serum swainsonine concentrations will be determined using a repeated measures, mixed linear model in SAS software (PROC Mixed; (SAS 9.4, SAS Institute Inc., Cary, NC).

 

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. Comments: 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

(0):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. Some participants already have developed stakeholder communication pathways with ranchers in their states (New Mexico and Utah) and further pathways for direct communication with ranchers in other states will be established. 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.

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 Chris Davies, Utah State University, 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

KY, MT, NM, UT

Non Land Grant Participating States/Institutions

Pacific West Area