Poisonous Plants

C H A P T E R

37 Poisonous Plants: Biomarkers for Diagnosis Kip E. Panter, Kevin D. Welch, Dale R. Gardner United States Department of Agriculture, Agricultural Research Service, Poisonous Plant Research Laboratory, Logan, UT, United States

INTRODUCTION Poisonous plants and the toxins they produce result in major economic losses to the livestock industries throughout the world. In the 17 western United States, it was estimated that more than $340 million in annual losses were attributed to poisonous plants (Nielsen et al., 1988; Nielsen and James, 1992). This cost estimate used 1989 figures and only considered death losses and measureable reproductive losses in cattle and sheep. A revised estimate of over $500 million annually using more current animal prices was recently reported (Holechek, 2002). Less obvious costs such as lost grazing opportunities, additional feed costs, increased healthcare costs, management adjustments, increased culling costs, lost weight gains, delayed or failed reproduction, and the emotional stress accompanying many poisonous plant cases were not included in the Nielsen and James or Holechek analyses. When one considers these other costs, inflation, and current animal values, and when all pastures and ranges in the United States are factored in, the economic cost of poisonous plants to the livestock industry is significant. In addition, an often ignored cost is the environmental impact on plant biodiversity from invasive species, many of which are poisonous. These invasive and poisonous species are often aggressive invaders and reduce optimum utilization of private, federal, and state-managed forest, range, and pasture lands. This aspect alone has far-reaching implications, not only for livestock producers but also for many other segments of society. Animals graze poisonous plants for a number of reasons. In some cases, it is a matter of survival. For example, in the arid and semiarid livestock-producing regions of the world, such as the western United States, regions of South Africa, Australia, China, South America, and others, browsing or grazing animals may have

Biomarkers in Toxicology, Second Edition https://doi.org/10.1016/B978-0-12-814655-2.00037-2

limited access to high-quality forage at certain times of the year and are forced to survive by grazing some poisonous species. In other instances, hay or harvested forages from areas where poisonous plants are abundant may be contaminated with a high percentage of poisonous plants, and when animals are fed contaminated hay, they may be poisoned. On many ranges, poisonous plants are a normal but small part of the animal’s diet as they are highly nutritious, i.e., larkspurs, locoweeds, and lupines, to name a few. Poisonous plant problems are often exacerbated during periods of below normal rainfall when the abundance of grasses is reduced. Frequently, the animal’s diet selection changes during the season when grasses and palatable forbs mature and senesce; for example, the consumption of some poisonous plants such as lupines, locoweeds, or larkspurs, which stay green longer into the season, may increase as the season progresses. Pine needles and junipers are often consumed in the winter. In other instances, poisoning occurs early in the season when poisonous plants such as lupine or death camas have emerged ahead of grasses. To restate the obvious, poisoning by plants only occurs when animals eat too much too fast or graze it over extended periods. Therefore, management strategies utilizing multiple factors are required to minimize losses from poisonous plants. Over the last 20 years, research at the USDA-ARS Poisonous Plant Research Laboratory in Logan, Utah, has provided information utilized by livestock producers and land managers to significantly reduce losses from poisonous plants and improve economic sustainability in rural communities. Part of this research has emphasized identifying biomarkers of poisoning for diagnostics and research. Although much more research is required, new information presented here will assist clinicians and diagnostic laboratories in diagnosing cases

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of poisonous plant toxicoses. This chapter is not intended to be all inclusive but focuses on some of the most economically important and geographically widespread poisonous plants to livestock producers in the United States.

ASTRAGALUS AND OXYTROPIS SPECIES (LOCOWEEDS, NITROTOXIN SPP., AND SELENIUM ACCUMULATORS) Of all the poisonous plants found on rangelands and pastures in the United States, the Astragalus and Oxytropis genera cause the greatest economic losses to the livestock industry in the western states (Graham et al., 2009; Cook et al., 2009c). There are three toxic syndromes associated with these species: (1) locoweed poisoning caused by species containing the indolizidine alkaloid swainsonine (w24 species); (2) species containing nitrotoxins (w356 species and varieties); and (3) selenium accumulators (w22 species).

animals. In the final stages of locoism, central nervous system tissue shows swelling of axonal hillocks (meganeurites) and growth of new dendrites and synapses. This altered synaptic formation in nervous tissue in severely affected animals is permanent and may be the cause of some irreversible neurological signs. Because of neurological dysfunction and apparent permanence of some lesions in the nervous system, horses are believed to be unpredictable and therefore unsafe to use for riding or draft, but they may remain reproductively sound once they have recovered from the poisoning. Toxin The indolizidine alkaloid swainsonine (1) was first isolated from the Australian plant Swainsona canescens (Colegate et al., 1979) and then soon thereafter isolated with its N-oxide from locoweeds (Molyneux and James, 1982). OH H

Locoweeds

OH

OH

Locoweed poisoning was one of the first poisonous plant problems recognized by stockmen and was reported as early as 1873. There are about 24 known species of Astragalus and Oxytropis that contain swainsonine and have been implicated in livestock poisonings. The term “loco” is Spanish, meaning crazy, and colloquially describes the aberrant behavior of locoweed-poisoned animals. All species of Astragalus and Oxytropis containing swainsonine are collectively referred to as locoweeds. Toxicology There are numerous effects of locoweed on animals, but the classic syndrome from which the term “locoism” derived is one of the central neurological dysfunctions, resulting in aberrant and often aggressive and unpredictable behavior. The disease is chronic, developing after weeks of ingesting locoweeds, and begins with depression, dull-appearing eyes, and incoordination, progressing to uncharacteristic behavior, including aggression, staggering, solitary behavior, wasting, and eventually death if continued consumption is allowed. Other problems associated with locoweed ingestion include reproductive failure, abortion, birth defects, weight loss, and enhanced susceptibility to brisket disease at high elevations (Panter et al., 1999b). Locoweed poisoning affects all animals but because of the transient nature of the poisoning, animals removed from the locoweed early in the toxicosis will recover most of their function and may be productive

N Swainsonine (1)

The locoweed poisoning syndrome is a lysosomal storage disease in which a-mannosidase is inhibited, resulting in prevention of hydrolysis of mannose-rich oligosaccharides in cells and accumulation of these oligosaccharides resulting in cellular dysfunction. Pryor et al. (2009) discovered that swainsonine in Astragalus and Oxytropis species is produced by a fungal endophyte, Undifilum oxytropis (formerly called Embellesia oxytropis). A positive correlation was shown to exist between swainsonine concentrations found in the plant and concentrations of swainsonine produced by the endophytic fungus cultured from the same plant (Ralphs et al., 2008; Cook et al., 2009a). This same correlation was demonstrated for Oxytropis glabra, an important poisonous plant in the Inner Mongolia steppe (Ping et al., 2009). Research results have shown that inhibition of a-mannosidase is relatively transient and quickly reversible once animals stop eating locoweed (Stegelmeier et al., 1994). Blood serum clearance of swainsonine is rapid (half-life of w20 h); thus, the effects of locoweed should be reversible if tissue damage has not become extensive and permanent. This suggests that intermittent grazing of locoweed could be an effective means of reducing locoweed poisoning. There is also an apparent threshold dosage where severity of cell damage is more timedependent than dosage-dependent. Once the threshold

IV. BIOTOXINS BIOMARKERS

ASTRAGALUS AND OXYTROPIS SPECIES (LOCOWEEDS, NITROTOXIN SPP., AND SELENIUM ACCUMULATORS)

dosage is reached, which appears to be relatively low (0.35 mg/kg BW in the rat), eating more locoweed does not accelerate the toxicosis. Therefore, increasing animal numbers on loco pastures and reducing time of grazing is also a logical method to reduce economic impact yet utilize infested pastures. Many locoweeds are biennials or perennials that flourish periodically under optimum environmental conditions. Historically, losses are regional and sporadic, with large regional economic impact. Individual cases of significant losses are frequent, and some historical cases are reported in James and Nielsen (1988). Conditions of Grazing In cows, preference to graze locoweed is relative to the amount and condition of other available forage. Many locoweeds are cool-season species that green up and start growth early in the spring, flower, set seed, and go dormant in summer, and then resume growth in fall. Livestock generally prefer the green, growing locoweeds to dormant grass. Sheep preferred the regrowth foliage of Green River milkvetch to dormant grasses during late fall and early winter on the desert range in eastern Utah (Ralphs et al., 2001). Horses selected green spotted locoweed instead of dormant grasses in the spring in Arizona (Pfister et al., 2003). Cattle readily grazed Wahweap milkvetch in proportion to its availability on desert winter range in southeastern Utah. In a series of grazing studies in northeast New Mexico, cattle readily grazed white locoweed in Marche May but stopped grazing it in June as warm-season grasses became abundant and white locoweed matured and became coarse and rank. Stocker cattle grazed white-point locoweed on shortgrass prairies in May and early June, but weight losses continued throughout the summer, even though they had stopped eating locoweed. On mixed-grass prairies on the eastern foothills of the Rocky Mountains in northern Colorado, cattle ceased grazing white-point locoweed when it matured following flowering in mid-June and became rank and less palatable. However, they continued to graze it throughout the subsequent summer when abundant summer precipitation caused the locoweed to remain green and succulent (Ralphs et al., 2001). Biomarkers of Poisoning (AST, GGTP, White Cell Count, Cellular Vacuolation, Serum Swainsonine, a-Mannosidase) Clinically, locoweed poisoning in all classes of livestock and wildlife include depression, loss of appetite, weight loss followed by intention tremors, dull hair coat, proprioceptive deficits, aberrant behavior, abortion in pregnant animals, and eventually death with prolonged exposure. Evaluation of serum swainsonine versus serum alpha mannosidase will indicate recent

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exposure to locoweed; however, elimination kinetics of swainsonine is short (t½ elimination of 20 h) and recovery rate of a-mannosidase is relative to disappearance of swainsonine. Therefore, serum evaluation of swainsonine or a-mannosidase is of limited value in diagnosis. Currently, serum biochemistry provides some support for a diagnosis as aspartate aminotransferase (AST), Lactic Acid Dehydrogenase (LDH), Gamma-Glutamyl Transferase (GGTP), and white blood cell counts are all elevated with locoweed poisoning. However, these are not pathognomonic to locoweed poisoning and without a history of locoweed ingestion would be of limited value as biomarkers. Currently, the best diagnosis for locoweed poisoning can only be obtained after necropsy and follow-up histopathology. Although there are few gross lesions seen in locoweed poisoned animals, there are many characteristic microscopic lesions. Most organ systems are affected; however, the nervous and endocrine systems are extremely sensitive and diseased cells of these organs are swollen and filled with dilated vacuoles described as cellular constipation. This cellular foamy vacuolation is readily observed in thyroid, pancreas, kidneys, testes, ovaries, and macrophages in nearly all tissues. Research at the Poisonous Plant Research Laboratory is focused on identifying selected abnormal proteins with a slow elimination kinetics as a biomarker to diagnose locoweed exposure and also as measure of prognosis for recovery. Prevention of Poisoning and Management Recommendations Prevention of poisoning remains a matter of management strategy adapted to individual grazing programs to minimize grazing of locoweed plants (Graham et al., 2009). Livestock should be denied access to locoweeds during critical periods when they are relatively more palatable than associated forages. On shortgrass prairies of northeastern New Mexico, stocker cattle should not be turned onto locoweed-infested rangelands until warm-season grasses start growth in late May or early June. Cattle on rangeland year-round should be removed from locoweed-infested areas in the spring when it is green and growing and warm-season grasses remain dormant. They can be returned to locoweedinfested pastures in summer when warm-season grasses are abundant. Most locoweed species are endemic, growing only in certain habitats or on specific soils. Fences can be used to provide seasonal control of grazing. Reserving locoweed-free pastures for grazing during critical periods in spring and fall can prevent locoweed poisoning. Locoweed-free areas can be created by strategic herbicide use. Currently, no broad management schemes or methods of treatment are known to generally prevent

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locoweed poisoning. Management strategies for individual operations have been developed once the grazing practices and options are identified, allowing utilization of the particular range and yet minimizing losses. It was determined that cattle generally rejected woolly loco even under extreme grazing conditions, but once they were forced to start eating it, they continued to graze it and became intoxicated. Ranchers should watch for these “loco eaters” and move them to locoweed-free pastures. Shortage of feed with high grazing pressure, social facilitation (loco eaters teaching nonloco eaters to accept loco), or supplementing with alfalfa hay or cubes may compel cattle to start grazing woolly locoweed. Whitepoint locoweed is more palatable than woolly locoweed and is green before spring grasses begin to grow in northeastern New Mexico. Therefore, cattle readily graze white-point locoweed in early spring while grasses are dormant, but once green grass starts to grow, cattle switch off of locoweed. Recommendations include creating locoweed-free pastures through herbicide use, fencing, or selection of low locoweed-infested pastures for early spring grazing and also to provide a place to move the identified locoweed eaters. This practice appears to reduce the impact of locoweed on these ranges. Grazing pressure can also force cattle to begin grazing locoweed when they run short of desirable forage (Ralphs et al., 1994). Ranchers should not overstock locoweed-infested ranges but, rather, should ensure adequate forage is always available. Improper use of some grazing systems may encourage livestock to graze locoweed. In a grazing study comparing breeds, Brangus steers consumed more locoweed than did Hereford or Charolais steers (Ralphs et al., 1994). It was suggested that the gregarious nature of Brangus steers facilitated their acceptance of locoweed first among the other breeds. Observations of breed differences were acknowledged as early as 1909. C.D. Marsh suggested that black cattle and black-faced sheep were more likely to graze locoweed and become poisoned. Although this has not been experimentally substantiated, we do believe breed difference may play a role in an animal’s propensity to graze locoweeds and become poisoned.

plants are very diverse and concentrated on the deserts, foothills, and mountains of the west. Toxicology The nitro-containing Astragalus species cause acute and chronic poisoning in sheep, cattle, and horses. The acute form results in weakness, increased heart rate, respiratory distress, coma, and death. Although blood methemoglobin is high (induced from nitrotoxin metabolism to nitrites) and a contributing factor to the respiratory difficulties, administration of methylene blue in cattle does not prevent death. Therefore, the methemoglobinemia is apparently not the primary cause of death. The chronic form is the most frequent form of poisoning observed and follows a course of general weakness, incoordination, central nervous system involvement resulting in knuckling of the fetlocks, goose stepping, clicking of the hooves, and “cracker heels” progressing to paralysis and death. A respiratory syndrome is also present in the chronic and acute forms, with emphysema-like signs causing the animals to force respiration: “roaring disease.” Sheep manifest the respiratory syndrome more than the central nervous syndrome and are more resistant to poisoning compared with cattle. The toxic principles are b-d-glycosides of 3-nitro-1propanol (NPOH) or 3-nitropropionic acid (NPA). The glycoside conversion occurs more readily in the ruminant because of the microflora and is apparently the reason for increased toxicity. The glycoside (miserotoxin 2) is metabolized to the highly toxic NPOH in the gastrointestinal (GI) tract of ruminants (Williams et al., 1970). Thus, NPOH is absorbed in the gut and apparently converted to NPA by the liver. Further metabolism yields inorganic nitrite and an unidentified metabolite that may be involved in toxicity. It appears that NPOH is more rapidly absorbed from the gut than is NPA; therefore, forage containing the alcohol form is the most toxic. O O

HO

Nitro-Containing Astragalus (Milkvetches) There are more than 260 species and varieties (356 taxa) of nitro-containing Astragalus in North America (Barneby, 1964; Welsh et al., 2007). They are frequently referred to as milkvetches, as are some of the other Astragalus species. Nitrotoxins are therefore the most common toxin in the Astragalus, followed by swainsonine (locoweeds), then selenium. Major livestock losses occur in many regions of the western United States. These

O

HO

N+

O-

OH OH Miserotoxin (2)

Biomarkers of Poisoning (Clinical, Pathology, History of Ingestion, Methemoglobin) The diagnosis of nitrotoxin-poisoned animals can be made by documenting exposure to the plant, identifying

IV. BIOTOXINS BIOMARKERS

ASTRAGALUS AND OXYTROPIS SPECIES (LOCOWEEDS, NITROTOXIN SPP., AND SELENIUM ACCUMULATORS)

the characteristic clinical signs of poisoning, measuring serum 3-nitropropionic acid concentrations, and histological examination. Studies have shown that 3-nitropropionic acid is rapidly absorbed, distributed, and eliminated. Consequently serum 3-nitropropionic acid concentrations may be below detectable limits within days of removal from the plant. This limits the diagnostic usefulness of serum 3-nitropropionic acid detection, as none may be detected in the serum even though the animals are demonstrating severe clinical signs. There are no hematological or serum chemistry changes of diagnostic value. However, cattle and sheep have been shown to have elevated methemoglobin concentrations, in excess of 20%. The gross pathological changes that occur are not distinctive but include hepatic congestion, pulmonary emphysema and edema, excess pericardial fluid, and abomasal ulceration. Microscopic lesions are also nonspecific, with the exception of changes in the brains of cattle with chronic exposure. These changes include the presence of foci of necrosis in the thalamus, moderate necrosis of the Purkinje cells in cerebellar folia, spongiosis in the white matter of the globus pallidus, distension of the lateral ventricles, and wallerian degeneration of the ventral and lateral columns of the spinal cord and the sciatic nerve. Clinical signs of respiratory distress from the pulmonary emphysema “roaring disease” and rear leg paresis “cracker heels” would suggest 3-nitropropionic acid (milkvetch or miserotoxin) poisoning and should be considered in the differential diagnosis. Prevention and Treatment There is no preferred treatment for milkvetch poisoning, although treatment with methylene blue appears to reverse the methemoglobinemia but may not prevent death in cattle. Oxidation of NPOH to NPA is prevented if alcohol dehydrogenase is saturated with ethanol or inhibited with 4-methylpyrazole before NPOH is given. This suggests that NPOH is a good substrate for the enzyme, alcohol dehydrogenase. This information could be useful in acute cases; however, its value in treatment of poisoning in the field is limited. Livestock losses can be reduced by decreasing the density of the Astragalus species with herbicides or avoiding grazing livestock on infested areas when the plant is most poisonous. Wasatch milkvetch contains the highest concentration of miserotoxin from bloom to immature seed pod stage of growth. Nitro compounds are found in all parts of the plant, but the leaves contain the highest concentration. Once the leaves begin to dry and lose their green color, the nitro levels drop very rapidly, and the plant is relatively nontoxic. However,

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the toxins in plants pressed green and preserved in herbaria appear to remain stable for years (Williams and Barneby, 1977). Herbicide treatment decreases the density of plants and also decreases the toxicity of the plants once they start to dry; therefore, spraying milkvetch appears to be the best method to reduce losses and still utilize infested ranges.

Seleniferous Astragalus Approximately 22e24 species of Astragalus known to accumulate selenium (Se) have been identified (Rosenfeld and Beath, 1964; Welsh et al., 2007). These are less numerous and more geographically restricted than the nitro-containing species. Many of these species are referred to as Se-indicator plants because they only grow on soils high in bioavailable selenium; therefore, they are helpful in locating and identifying areas or soils high in selenium. The Astragalus species are deep-rooted plants and are thought to bring selenium (pump) from deeper soil profiles to the surface where shallow-rooted forbs and grasses will take up toxic levels. It is these facultative accumulators that create most of the subacute or chronic toxicity problems for livestock. Toxicity With selenium poisonings, one may observe acute, subacute, or chronic selenosis depending on the daily dose, Se type (organic vs. inorganic), and duration of exposure. Acute cases of selenium poisoning are rare and usually involve animals that have been exposed by one of four methods. (1) Forages that have superaccumulated selenium from seleniferous soils. (2) Environmental contamination from agricultural drain water, reclaimed soils from phosphate or ore mining, or from fly ash. (3) Accidental overdosing with pharmaceutical grade selenium such as Bo-Se in the treatment of white muscle disease. (4) Misformulated feed mixes. The signs of acute selenium poisoning include diarrhea, unusual postures, increased temperature and heart rate, dyspnea, tachypnea, respiratory distress, prostration, and death (Tiwary et al., 2006). Gross pathological findings are usually limited to pulmonary congestion, hemorrhage, and edema. Histologically, multifocal myocardial necrosis and pulmonary alveolar vasculitis are common (Tiwary et al., 2006). Chronic selenium poisoning is most common and referred to as alkali disease because most areas with high concentrations of available selenium are alkaline in nature. Chronic selenosis occurs from prolonged ingestion of seleniferous forages containing 5e40 ppm

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Se. Clinical signs include rough coat, hair or wool loss, poor growth, emaciation, abnormal hoof growth, lameness, dermatitis, and depressed reproduction (Rosenfeld and Beath, 1964; Raisbeck, 2000). In swine, a condition of paralysis (poliomyelomalacia or polioencephalomalacia) often occurs with cervical or lumbar involvement (Panter et al., 1996b). The description of a second chronic syndrome in cattle called “blind staggers” has been redefined and is now believed to be polioencephalomalacia induced by high sulfate water or high sulfate forage sources. Selenium is found in soil and plants in both organic (3) and inorganic (4) forms. The organic forms are more bioavailable than the inorganic forms, resulting in higher animal tissue concentrations when administered at equivalent selenium doses (Tiwary et al., 2006; Davis et al. 2012). Although a dramatic difference in tissue selenium uptake between organic (selenomethionine) and plant (Astragalus bisulcatus) forms and inorganic (sodium selenate) forms occurs, the clinical and pathological syndromes are similardi.e., poliomyelomalacia in pigs (Panter et al., 1996b) and pulmonary edema and hemorrhage in sheep and cattle (Tiwary et al., 2006; Davis et al. 2012).

Selenomethionine (3)

O Se NaO

ONa

Sodium selenate (4)

Biomarkers of Poisoning (Liver Biopsy Se, Whole Blood Se, Enzyme, Hair, Mane, Tail, Hoof Samples) A complete health history of the animals with excess selenium in the forages or feed supplementation providing the basis for suspecting selenium poisoning (acute or chronic) should be evaluated. Selenium is slowly excreted, requiring relatively long withdrawal times (weeks). Therefore, acute Se poisoning can be diagnosed by sampling numerous biological tissues, including whole blood, serum, liver biopsies, hair, tail, and hooves. Acute selenium poisoning can be easily diagnosed by sampling hair 3e4 weeks postexposure. Selenium appears to be

excreted slightly slower from the liver; however, liver biopsies are a much more invasive procedure for diagnosis. Interestingly, selenium is excreted relatively slowly from the red blood cell, thus making whole blood a good tissue for analysis (Davis et al., 2012). The primary lesion in acute selenosis in cattle has been found to be myocardial necrosis with subsequent heart failure seen as passive congestion and centrilobular hepatic congestion with hepatocellular degeneration and necrosis. Consequently increases in serum creatine kinase and troponin can be used as biomarkers of acute selenosis. Chronic selenium poisoning results in bilateral hair loss over the withers of cattle and horses, tail and mane hair loss, overgrown coronary bands or separation and sloughing of the outer shell of the hooves, and an overall unkept appearance. In cattle excess selenium, or selenium deficiency, may reduce overall reproductive performance. In horses, sampling of the tail hair then cutting into 1 inch increments and performing selenium analysis on each of the 1 inch increments will provide a good history of selenium ingestion for up to a 3-year period depending on how long the tail hair is (Davis et al., 2014). In cattle, a hair sample should be clipped from the shoulder area close to the skin and divided into 2 or 3 samples based on hair length. This will provide a good evaluation of selenium status and an approximate time when selenium ingested. The 0.5 cm sample closest to the skin would indicate recent selenium exposure (0e7 days), whereas the center and outer samples would indicate more long-term exposure (Davis et al., 2012). In pigs, selenium poisoning (subacute and acute) manifests with cervical or lumbar paresis and lesions of polioencephalomalacia or poliomyelomalacia and chronic poisoning by hair loss, overgrown hooves, reduced growth rate, and overall poor or unkept appearance. In sheep, acute selenium poisoning is manifest by respiratory distress, depression, lethargy, and death within hours to a day or two after ingestion. There are few clinical signs associated with chronic selenium ingestion in sheep; however, reproductive performance may be impaired. Prevention of Poisoning There is no treatment for selenium poisoning except removal from the source, allowing spontaneous recovery in chronic cases. Treat overgrown hooves, i.e., trim horse’s hooves frequently until normal hoof growth resumes (6e12 months). Monitoring soils in a particular area and understanding the plant communities can provide the management information to avoid poisoning. In areas where selenium is a problem, many ranchers have switched to grazing steers to avoid decreased

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LARKSPURS (DELPHINIUM SPP.)

reproductive efficiency in cows. Sheep appear to be more resistant to chronic selenosis compared with cattle and are better adapted for some of these ranges. However, sheep are sensitive to acute selenium poisoning, as was observed when a large number of sheep died within days after grazing on mine reclamation sites that contained very high soil and plant selenium concentrations (Panter, personal communications, 2004). Monitoring for selenium concentrations and forms in soil, vegetation, as well as animal tissues and hair, can help avoid poisoning incidents. Likewise, deficiency problems can be rapidly resolved with frequent monitoring and trace mineral supplementation.

LARKSPURS (DELPHINIUM SPP.) Methyllycaconitine (5)

There are more than 80 wild species of larkspurs in North America, classified into two general categories based primarily on mature plant height and distribution: low larkspurs and tall larkspurs. The larkspurs are a major cause of cattle losses on western ranges. As early as 1913, C.D. Marsh reported that more cattle deaths on western ranges are caused by larkspurs than by any other poisonous plant except locoweed. Large sporadic losses continue to occur on ranges with both low and tall larkspur species.

Toxicology Larkspurs (Delphinium spp.) are a serious economic problem for cattle producers utilizing valley, foothill, and mountain rangelands in western North America. The toxicity of larkspur plants is due to norditerpenoid alkaloids, which occur as one of two chemical structural typesdthe N-(methylsuccinimido) anthranoyllycoctonine (MSAL) type (5) and the 7,8methylenedioxylycoctonine (non-MSLA) type (6). Although the MSAL-type alkaloids are much more toxic (typically more than 20 times) (Panter et al., 2002), the non-MSAL-type alkaloids are generally more abundant in Delphinium barbeyi and Delphinium occidentale populations and will exacerbate the toxicoses (Gardner et al., 2002, Welch et al., 2010, 2012). The effects of 7, 8-methylenedioxylycoctonine-type diterpenoid alkaloids may enhance the overall toxicity of tall larkspur (Delphinium spp.) in cattle. Consequently, for a larkspur plant to be toxic to livestock, a sufficient quantity of MSAL-type alkaloids is required. However, MDL-type alkaloids appear to potentiate the overall toxicity of the MSAL-type alkaloids and should be considered when predicting potential toxicity of larkspur populations (Welch et al., 2010, 2012).

Deltaline (6)

Toxicity declines rapidly in tall larkspurs once pods begin to shatter. Measuring plant toxicity early in the growing season may allow prediction of grazing risk throughout the season (Ralphs et al., 2002). Because of the fact that the MSAL-type alkaloids are much more toxic than the MDL-type alkaloids, management recommendations for grazing cattle on larkspur-containing ranges are based primarily on the concentration of MSAL-type alkaloids in larkspur (Pfister et al., 2002; Ralphs et al., 2002). Clinical signs of intoxication include muscular weakness and trembling, straddled stance, periodic collapse into sternal recumbency, respiratory difficulty, and death while in lateral recumbency. The primary result of larkspur toxicosis is neuromuscular paralysis from blockage at the postsynaptic neuromuscular junction (Benn and Jacyno, 1983). Cattle generally begin consuming tall larkspur after flowering racemes are elongated, and consumption increases as larkspur matures. Consumption usually peaks during the pod stage of growth in late summer, when cattle may eat large

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quantities (25%e30% of diet as herd average; >60% on some days by individual animals). Because larkspur toxicity generally declines throughout the growing season, and cattle tend to eat more larkspur after flowering, this risk period of greatest danger has been termed the “toxic window” (Pfister et al., 2002). This toxic window extends from the flower stage into the pod stage, or approximately 5 weeks, depending on temperature and elevation. Many ranchers typically defer grazing on tall larkspur-infested ranges until the flower stage to avoid death losses. This approach is dangerous as it places cattle in larkspur-infested pastures when risk of losses is high. This approach also wastes valuable, high-quality forage early in the season when risk of grazing is much lower. An additional 4e6 weeks of grazing may be obtained by grazing these ranges early, before larkspur elongates flowering racemes. The risk of losing cattle is low when grazing before flowering, even though larkspur is very toxic, because cattle prefer the highly palatable lush grasses. Once pods mature and begin to shatter, larkspur ranges can usually be grazed with impunity because pod toxicity declines rapidly, and leaf toxicity is low. Managing cattle on low larkspur pastures is quite different from that on tall larkspur pastures. Not all low larkspurs are high risk, and alkaloid analyses should be done before grazing. On pastures where toxic species of low larkspur grow, delayed grazing until after pods shatter and plants begin to dry up is the only safe approach. Based on limited studies, cattle increase consumption of low larkspur after flowering, and increases in grazing pressure increase amounts of low larkspur eaten by cattle (Pfister et al., 2002).

Biomarkers of Poisoning (Blood Alkaloid, Liver Alkaloid, Plant Fragment, or Rumen Sample) For diagnostic purposes, the toxic larkspur alkaloids can usually be detected in the whole blood, liver, kidney, sera, and rumen content of poisoned animals. Because of the acute nature of larkspur toxicosis, if the animals survive the initial intoxication they will recover and have no lasting effects. Consequently, in general, when a diagnosis of a larkspur-poisoned animal is needed, it is for a dead animal. The diagnosis of larkspur poisoning is currently made by documenting exposure to the plant, identifying the characteristic clinical signs, identifying larkspur in the rumen contents, and measuring serum alkaloid concentrations. Studies have shown that the larkspur alkaloids are rapidly absorbed, distributed, and eliminated. Serum alkaloid concentrations can be detected for approximately 7 days after intoxication. Because of the difficulty for most diagnostic laboratories to accurately identify plant parts in rumen contents and

measure serum for larkspur alkaloids, experiments are being conducted to use PCR technology to identify larkspur plants in rumen contents. The use of a common and robust technology such as PCR will allow for most any veterinary diagnostic laboratory to determine if the plant was present in the rumen of the animal.

Prevention and Management of Poisoning Grazing Management A simple and low-risk grazing management scheme can often be used based simply on tall larkspur growth and phenology: (1) graze during early summer when sufficient grass is available until larkspur elongates flowering racemes (4e6 weeks, depending on elevation and weather); (2) remove livestock, or contend with potentially high risk from flowering to early pod stages of growth (4 or 5 weeks); and (3) graze with low risk during the late season when larkspur pods begin to shatter (4e6 weeks). This scheme can be refined substantially if livestock producers periodically obtain an estimate of the toxicity of tall larkspur, and if ranchers spend time periodically observing and documenting larkspur consumption by grazing cattle. Management to reduce losses to low larkspur begins with recognition of the plant during spring. Vegetative low larkspur plants will typically begin growth before the major forage grasses. Low larkspur populations fluctuate with environmental conditions (Pfister, unpublished data). Risk of losing cattle is much higher during years with dense populations. During those years, recognizing the plant, and finding alternative pasture or waiting to graze infested pastures for 4e 6 weeks until the low larkspur has dried up, will reduce losses. In addition, it is recommended that animals not be watered or provided mineral supplementation in areas that have high densities of larkspurs. Drug Intervention A variety of remedies have been applied in the field when ranchers find intoxicated animals, but most are without a solid scientific rationale. Treatment for overt poisoning is usually symptomatic, and recovery is often spontaneous if animals are not stressed further. Once the animal is observed showing muscular tremors, it should be allowed to drop back and proceed at its own pace. Poisoned animals should never be forced to continue moving because this will exacerbate the clinical effects and can result in death. Drugs that increase acetylcholine effectiveness at the neuromuscular junction have potential for reversing larkspur toxicosis or reducing susceptibility. The cholinergic drug physostigmine (0.08 mg/kg intravenous (i.v.)) has been successfully used under field and pen conditions to reverse clinical

IV. BIOTOXINS BIOMARKERS

635

LUPINES (LUPINUS SPP.)

larkspur intoxication (Pfister et al., 1994). Similarly, i.v. administration of neostigmine (0.04 mg/kg) significantly reduced clinical signs in cattle (Green et al., 2009), and neostigmine administered intramuscularly at 0.02 mg/ kg can be used as a rescue treatment for cattle that are recumbent. This reversal lasts approximately 2 h, and repeated injections of neostigmine are sometimes required. Under field conditions, neostigmine temporarily abates clinical signs and animals quickly (w15 min) become ambulatory. Depending on the larkspur dose, the intoxication may recur. The use of physostigmine-based treatments may aggravate losses in the absence of further treatment if intoxicated, yet ambulatory animals later develop increased muscular fatigue, dyspnea, and death.

in changing levels of toxicity within and between species and populations. The chemical phenology has been studied in Lupinus caudatus and Lupinus leucophyllus (Lee et al., 2007). Total alkaloid concentration is high in the new early growth but diluted as the plant biomass increases. Pools of total alkaloids increase during the phenological growth stages and peak at the pod stage, concentrating in the pods. The teratogenic alkaloid anagyrine appears to be an end product in the biosynthetic pathway and accumulates in the floral parts and is stored in the seed. Following seed shatter, both concentration and pools of all alkaloids decline precipitously, leaving the senescent plant relatively nontoxic. H N

LUPINES (LUPINUS SPP.)

N H H

The Lupinus genus contains more than 150 species of annual, perennial, or soft woody shrub lupines. More than 95 species occur in California alone. The lupines are rich in alkaloids, responsible for most of the toxic and teratogenic properties. There are domestic lupines that, through plant breeding, are low in alkaloid content and have been selected for ornamental purposes or for animal and human food. Only the range lupines known to cause poisoning or birth defects are discussed here. Stockmen have long recognized the toxicity of lupines when livestock, particularly sheep, were poisoned in the late summer by the pods and seeds of lupine. Major losses in sheep were reported in the 1950s, and individual flock losses of hundreds and even thousands were reported. Lupines are also poisonous to other livestock, and field cases of poisoning in cattle, horses, and goats have been reported. However, the most recognized condition of lupine ingestion is the “crooked calf syndrome,” a congenital condition in calves resulting in skeletal contracture-type malformations and cleft palate after their mothers have grazed lupines during sensitive periods of pregnancy (Panter et al., 1999a). The condition was first reported in 1959 and experimentally confirmed after large outbreaks in Oregon and Montana in 1967.

Toxicology Most lupine species contain quinolizidine alkaloids, a few contain piperidine alkaloids, and some contain both. The specific alkaloids responsible for crooked calf syndrome are anagyrine (7), ammodendrine (8), Nacetylhystrine, and N-methyl ammodendrine. Hence, risk is based on chemical profile and the presence and concentration of these teratogenic alkaloids. It is known that chemical profile and concentration differ, resulting

O

Anagyrine (7)

Ammodendrine (8)

The toxicity of lupines was first recognized in the early 1900s. Lupines can be toxic to sheep at 0.25% e1.5% of their body weight depending on alkaloid composition. A few cases of poisoning have occurred on young plants. Losses of 80e100 sheep in multiple bands have been reported during the past 5 years in Idaho and Wyoming (Panter, personal communication, 2005). Poisoning by lupine plants should not be confused with lupinosis reported in Australia. Lupinosis is a condition that is entirely different, as it is a mycotoxicosis of livestock caused by toxins produced by the fungus Phomopsis leptostromiformis, which colonizes domestic lupine stubble. It affects livestock that graze lupine stubble and limits the use of this animal forage in Australia. Historically, lupines were responsible for more sheep deaths than any other single plant in Montana, Idaho, and Utah. Most losses occurred from hungry sheep grazing seed pods. Poisoning occurred following trucking or trailing bands in late summer or fall, or after getting caught in early snowstorms that covered herbaceous vegetation. Hungry sheep nonselectively grazed

IV. BIOTOXINS BIOMARKERS

636

37. DIAGNOSTIC BIOMARKERS FOR PLANT POISONINGS

lupine pods, which are high in alkaloids, and were poisoned. Large losses have also occurred when lupine hay harvested in the seed pod stage was fed in winter. Lupine-induced crooked calf syndrome was first reported in 1959 and 1960 and experimentally confirmed in 1967 (Panter et al., 1999a). Crooked calf syndrome includes various skeletal contracture-type birth defects and occasionally cleft palate. The skeletal defects are similar to an inherited genetic condition reported in Charolais cattle. Based on epidemiologic evidence and chemical comparison of teratogenic and nonteratogenic lupines, the quinolizidine alkaloid anagyrine was determined to be the teratogen (Keeler, 1973). A second teratogen, a piperidine alkaloid, ammodendrine, found in Lupinus formosus, was also demonstrated to cause the condition (Keeler and Panter, 1989). Further research determined that the anagryrine-containing lupine only caused birth defects in cattle and did not affect sheep or goats; however, the piperidine alkaloid-containing lupine, L. formosus, induced similar birth defects in cattle and goats (Keeler and Panter, 1989).

Cattle Grazing Different lupines produce varying toxic syndromes in livestock, apparently because the alkaloid profile varies remarkably among plant species and between populations. Season and environment influence alkaloid concentration in a given species of lupine. Generally, alkaloid content is highest in young plants and in mature seeds. Alkaloids are not lost on drying, so wild hay may be highly toxic if young lupine plants or seed pods are present. For many lupines, the time and degree of seeding vary from year to year. Most losses occur under conditions in which animals consume large amounts of plant or pods in a brief period, such as when they are being driven through an area of heavy lupine growth, unloaded into such an area, trailed through an area where the grass is covered by snow but the lupines are exposed, or when feeding lupine hay with pods. Most serious poisonings may occur in the late summer or early fall because lupine remains green after other forage has dried, and seed pods may be present. Once the poisonings were understood, the practice of harvesting lupine hay for winter sheep feed was discontinued. Lupine is not very palatable to cattle, although it has been considered fair to good quality feed on some ranges that are heavily utilized. Its palatability or acceptability depends on availability and maturity of other forage. In a grazing study of velvet lupine (L. leucophyllus) on annual cheatgrass ranges in eastern Washington (Ralphs et al., 2006), cows selected lupine in July and August after cheatgrass dried and other forbs were depleted or matured and became less palatable. The deep-rooted lupine remained green and

succulent longer into the summer than the other forage. Lupine was higher in crude protein and lower in fiber (NDF) than the other forages throughout the season (the crude protein level in foliage was 15%, and in seeds it was 36%). The abundance of lupine is another factor influencing the amount of lupine consumed. Lupine population cycles are influenced by weather patterns. Catastrophic losses from lupine-induced crooked calves occurred in the Channel Scabland region of eastern Washington in 1997. Annual precipitation from 1995 to 1997 was 33% above average, precipitating a population outbreak of lupine throughout the region. The density of velvet lupine plants has declined since then (Ralphs, unpublished data), and the incidence of crooked calves has returned to what has become an acceptable tolerance of 1%e5% incidence. Clinical signs of poisoning are of muscular weakness (neuromuscular blockade) beginning with nervousness, frequent urination and defecation, depression, frothing at the mouth, relaxation of the nictitating membrane, ataxia, muscular fasciculations, weakness, lethargy, collapse, sternal recumbency followed by lateral recumbency, respiratory failure, and death. Signs may appear within 15 min to 1 h after ingestion or as late as 24 h, depending on the amount and rate of ingestion. Death usually results from respiratory paralysis. The incidence of crooked calves is variable geographically and from year to year within a given herd. Up to 100% of a given calf crop may be affected, and individuals may be more severely affected than others. Affected calves are generally born alive at full term. Dystocia may occur when calves are severely deformed and assistance is required, often resulting in cesarean section. Arthrogryposis is the most common malformation observed and is often accompanied by one or more of the following: scoliosis, torticollis, kyphosis, or cleft palate. Elbow joints are often immobile because of malalignment of the ulna with the articular surfaces of the distal extremity of the humerus. The part of the limb distal to the elbow joint is often rotated laterally. In crooked calf syndrome, the osseous changes observed are permanent and generally become progressively worse as the calf grows and its limbs are subjected to greater load-bearing stress. Frequently, minor contractions such as “buck knees,” often attributed to lupine, will resolve on their own, and the calf will appear relatively normal. The sensitive gestational period in the pregnant cow for exposure is 40e70 days with periods extending to day 100 (Panter et al., 1997). The condition has been experimentally induced with dried ground lupine at 1 g plant/kg BW and with semipurified preparations of anagyrine (the apparent teratogen) at 30 mg anagyrine/kg BW fed daily from 30 to 70 days of gestation

IV. BIOTOXINS BIOMARKERS

POISON HEMLOCK (CONIUM MACULATUM)

(Keeler et al., 1976). The dose range of anagyrine to cause crooked calves is 6.5e11.9 mg/kg BW/day for 3 or 4 weeks during gestation days 40e70. Crooked calf disease has also been induced by feeding the piperidine alkaloid-containing lupine, L. formosus (Keeler and Panter, 1989). The teratogenic piperidinesd ammodendrine, N-acetylhystrine, and N-methyl ammodendrinedare absorbed quickly after ingestion and can be detected in blood plasma by 0.5 h, with peak levels maintained for more than 24 h (Gardner and Panter, 1993). The mechanism of action has been determined to be an alkaloid-induced reduction in fetal movement by a neuromuscular blocking effect during the critical stages of gestation (Panter et al., 1990a). This inhibition of fetal movement is due to stimulation followed by desensitization of skeletal muscle-type nAChR (Lee et al., 2006). This mechanism is a common factor for multiple alkaloids found in many species of lupines, poison hemlock (Conium), and wild tree tobacco (Nicotiana glauca), and research using TE-671 cells that express human fetal muscle-type nAChR and SH-SY5Y cells that express human autonomic-type nAChR supports this mechanism (Green et al., 2010).

Biomarkers of Poisoning (Clinical Effects, History of Ingestion, Serum Analysis, Liver, Urine, Crooked Calf Syndrome (CCS)) There are few biomarkers for diagnosing lupine poisoning. However, there are clinical signs that should be considered in a differential diagnosis such as incoordination, nictitating membrane partially covering the eye, muscular weakness especially after mild exertion and death after a history of potential lupine exposure. Alkaloid screening of the serum, liver, rumen content, or urine of poisoned animals with positive analysis for quinolizidine or piperidine alkaloids is diagnostic, especially if a history of lupine exposure exists. When newborn calves are presented with skeletal contractures especially arthrogryposis, scoliosis, torticollis, kyphosis, and/or cleft palate, lupine ingestion during pregnancy should be suspected. Because of the acute nature of lupine toxicosis, if mature animals survive the initial intoxication they will recover and have no lasting effects. However, if a pregnant cow is poisoned between the GD 40 and 100, the calf will likely be born with skeletal contractures. The diagnosis of lupine poisoning is currently made by documenting exposure to the plant, identifying the characteristic clinical signs, identifying lupine in the rumen contents, and measuring serum alkaloid concentrations. Studies have shown that the lupine alkaloids are rapidly absorbed, distributed, and eliminated. Serum alkaloid concentrations can be detected for approximately 3e4 days after intoxication.

637

Prevention, Management, and Treatment Keeler et al. (1977) proposed a simple management solution to prevent crooked calves: graze lupineinfested pastures so that the susceptible period of gestation (40e70 days) does not overlap the flower and pod stage of growth when anagyrine is highest. Ralphs et al. (2006) refined Keeler’s recommendations to restrict access during the susceptible period of gestation, when anagyrine concentration is still high in the flower and pod stage, only when cattle are likely to eat lupine and in years when it is abundant. Panter et al. (2013) suggested that intermittent grazing between lupineinfested pastures and lupine-free pastures would allow the fetus to regain normal movement for a few days during the sensitive stage of gestation. It has been hypothesized that inhibited fetal movement over a prolonged period is required for severe malformations to occur (Panter et al., 1999a). Lupines are easily controlled with 2,4-D-type broadleaf herbicides (Ralphs et al., 1991); however, herbicide treatment alone rarely provides long-term solutions to poisonous plant problems. Seed reserves in the soil will rapidly reestablish the stands if grazing management practices are not implemented. Death losses in sheep can be reduced by recognizing the variability in lupine toxicity with stage of growth and the conditions under which animals graze the plant. Providing a choice of other quality forages usually prevents excess lupine grazing. The dangerous period of plant growth for sheep exists mainly with plants in the pod stage. The hazard increases if sheep are hungry, as is often the case with crowding, hauling, driving, or overgrazed conditions. The hazard is reduced or eliminated after seed pods shatter. Treatment for overt poisoning is usually symptomatic, and recovery is often spontaneous if animals are not stressed further. Once the animal is observed showing muscular tremors, it should be allowed to drop back and proceed at its own pace. Poisoned animals should never be forced to continue moving because this will exacerbate the clinical effects and can result in death. The elimination of the toxic alkaloids in the urine is quite rapid (t½ w 6.3e6.9 h) and begins within hours of ingestion (Lopez-Ortiz et al., 2004). Therefore, allowing the animal to rest and move slowly will often result in full recovery within 24 h. There is no treatment for the malformations, and euthanasia is recommended for the serious skeletal defects and cleft palate. However, less severe contracture defects, particularly of the front legs (buck knees), will often resolve if the knee joint can be locked within 1 week after birth. If not, the defect generally becomes worse with growth and size, and although the animal will continue to grow, the front legs will break down until the animal is unable to survive.

IV. BIOTOXINS BIOMARKERS

638

37. DIAGNOSTIC BIOMARKERS FOR PLANT POISONINGS

POISON HEMLOCK (CONIUM MACULATUM) Poison hemlock (C. maculatum) was introduced into the United States as an ornamental plant from Europe and has become naturalized throughout the United States (Kingsbury, 1964). The plant generally grows in waste places or habitats where there is adequate moisture to sustain populations. It is a biennial, however, where temperatures and adequate moisture allow seed germination in the fall the 2-year cycle for seed production may occur in one season. Poison hemlock has an interesting history as it is believed to be the plant from which a decoction (hemlock tea) was used to execute Socrates and others from that era. An interesting review describes the historical perspective of poison hemlock (Daughtery, 1995).

Toxicology Eight piperidine alkaloids are known in poison hemlock, five of which are commonly discussed in the literature. Two alkaloids (coniine (9) and g-coniceine (10)) are prevalent and likely responsible for toxicity and teratogenicity of the plant. g-Coniceine is the predominant alkaloid in the early vegetative stage of plant growth and is a biochemical precursor to the other Conium alkaloids (Panter and Keeler, 1989). Coniine predominates in late growth and is found mainly in the seeds. g-Coniceine is seven or eight times more toxic than coniine in mice (Lee et al., 2013). This makes the early growth plant most dangerous in the early spring, and the seedlings and regrowth again in the fall. This is also the time when green feed is limited to livestock and may impact their propensity to graze this plant. Frequently, poison hemlock encroaches into hay fields where it can be problematic in green chop or silage. Ensiling usually reduces toxicity; however, hot spots in silage pits increase risk of poisoning. Seeds, which are very toxic, can contaminate poultry and swine cereal grains (Panter and Keeler, 1989). Plants often lose their toxicity on drying, but seeds remain toxic as long as the seed coat is intact.

N H Coniine (9)

The clinical signs of toxicity are the same in all species and include initial stimulation (nervousness), resulting in frequent urination and defecation (no diarrhea), rapid pulse, temporarily impaired vision from the nictitating membrane covering the eyes, muscular weakness, muscle fasciculations, ataxia, incoordination followed by depression, recumbency, collapse, and death from respiratory failure (Panter et al., 1988). Conium plant and seed are teratogenic, causing contracturetype skeletal defects and cleft palate like those of lupine. Field cases of teratogenesis have been reported in cattle and swine and experimentally induced in cattle, swine, sheep, and goats (Panter et al., 1999a). Birth defects include arthrogryposis (twisting of front legs), scoliosis (deviation of spine), torticollis (twisted neck), and cleft palate. Field cases of skeletal defects and cleft palate in swine and cattle have been confirmed experimentally. In cattle, the susceptible period for Conium-induced terata is the same as that described for lupine and is between days 40e70 of gestation. The defects, susceptible period of pregnancy, and probable mechanism of action are the same as those of crooked calf syndrome induced by lupines (Panter et al., 1999a). In brief, these alkaloids and their enantiomers in poison hemlock, lupines, and N. glauca were more effective in depolarizing the specialized cells TE-671, which express human fetal muscle-type nAChR, relative to SH-SY5Y, which predominantly express autonomic nAChRs, in a structureeactivity relationship (Panter et al., 1990a; Lee et al., 2006, 2008; Green et al., 2010). In swine, sheep, and goats, the susceptible period of gestation is 30e60 days. Cleft palate has been induced in goats when plant, or toxins, was fed from 35 to 41 days of gestation (Panter and Keeler, 1992). Field cases of poisoning have been reported in cattle, swine, horses, goats, elk, turkeys, quail, chickens, and Canada geese (Panter et al., 1999a). Poisoning in wild geese eating small seedlings in early spring was most recently reported (Panter, personal communication). Human cases of poisoning are occasionally reported in the literature, and a case of a child and his father mistakenly ingesting the plant has been reported. Field cases of teratogenesis have been reported in cattle and swine and experimentally induced in cattle, sheep, goats, and swine (Panter et al., 1990a). Pigs become habituated to poison hemlock, and if access to the plant is not limited, they will eat lethal amounts within a short time.

Biomarkers of Poisoning

N γ-Coniceine (10)

There are no diagnostic lesions or serum biomarkers in poisoned animals, and diagnosis is based on clinical history of exposure and/or alkaloid (coniine, N-methyl coniine, or g-coniceine) detection in the liver, urine, blood, or stomach contents. At necropsy, the presence IV. BIOTOXINS BIOMARKERS

639

WATER HEMLOCK (CICUTA SPP.)

of plant fragments in the rumen and a characteristic pungent odor in the contents with chemical confirmation of the alkaloids is diagnostic. Characteristic contracture-type skeletal birth defects or cleft palate in cattle, sheep, or goats similar to those produced by lupines should be included in the differential diagnosis.

Prevention and Treatment Prevention of poisoning is based on recognizing the plant and its toxicity and avoidance of livestock exposure when hungry. If a lethal dose has not been ingested, the clinical signs will pass spontaneously, and a full recovery can be expected. Avoidance of stressing animals poisoned on Conium is recommended. However, if lethal doses have been ingested, supporting respiration, gastric lavage, and activated charcoal are recommended. Control of plants is easily accomplished using broadleaf herbicides; however, persistent control measures are recommended because seed reserves in the soil will quickly reestablish a population. The mechanism of action of the Conium alkaloids is twofold. The most serious effect occurs at the neuromuscular junction, where they act as nondepolarizing blockers such as curare. Systemically, the toxins cause biphasic nicotinic effects, including salivation, mydriasis, and tachycardia, followed by bradycardia as a result of their action at the autonomic ganglia. The teratogenic effects are undoubtedly related to the neuromuscular effects on the fetus and have been shown to be related to reduction in fetal movement (Panter et al., 1990a). Likewise, cleft palate is caused by the tongue interfering in palate closure during reduced fetal movement and occurs during days 30e40 of gestation in swine, 32e41 days in goats, and 40e50 days in cattle (Panter and Keeler, 1992).

WATER HEMLOCK (CICUTA SPP.) Water hemlock (Cicuta spp.) is among the most violently poisonous plants known to humans. It is often confused with poison hemlock because of its name, growth patterns, and appearance. However, there are distinct differences in morphology and habitat.

Toxicology The primary toxic principle in water hemlock is a long-chain, highly unsaturated alcohol called cicutoxin (11). Water hemlock acts on the central nervous system inducing violent grand mal seizures and death from respiratory failure.

OH

HO

H Cicutoxin (11)

Tubers are the most toxic part of the plant, especially in early spring. The parsnip-like roots extending from the tuber are two to four times less toxic, and as the vegetative parts of the plant grow and mature, they become less toxic. Preliminary studies suggest that mature leaves and stems are much less toxic and are nontoxic after drying (Panter et al., 1988). Historically, water hemlock was believed to be most dangerous in early spring, and poisoning usually occurred when animals milled around in streambeds or sloughs and exposed tubers, which were then ingested. Although this is true, a recent case of poisoning and death in cattle after ingesting flower and green seed heads implicates this phenological stage as dangerous also (Panter et al., 2011). Chemical comparison of green seed and tubers combined with mouse bioassay studies showed that green seed is also toxic. Like tubers, the more mature vegetation, including leaves, flowers, and green seed heads, was palatable. The parsnip-like roots given free choice to hamsters showed that they were very palatable, and the hamsters actually preferred these roots to their normal laboratory chow. No signs of toxicoses were observed when hamsters had free choice to these roots. The roots are much less toxic compared with the tubers. Observations of cattle grazing early in spring suggest that the young shoots of water hemlock are very palatable because young plants growing in streambeds were frequently and extensively grazed without toxic sequelae (Panter, personal observation). Clinical signs of poisoning appear within 10e15 min after ingestion and progress from nervousness, frothy salivation, ataxia, dyspnea, muscular tremors, and weakness to involuntary, spastic head and neck movements accompanied by rapid eye blinking and partial occlusion of the eyes from the nictitating membranes. This is quickly followed by collapse and intermittent grand mal seizures lasting 1 or 2 min each followed by relaxation periods of 8e10 min. Depending on the dosage, recovery may occur or seizures continue until death from exhaustion or respiratory failure. There appears to be a very narrow safety threshold in which a very small increase in dosage is all that is required to induce grand mal seizures (Panter et al., 1996a). On necropsy, gross lesions are confined to pale areas in heart muscle and skeletal muscles, particularly the long digital extensor muscle groups (Panter et al., 1996a). Microscopic lesions include multifocal, subacute

IV. BIOTOXINS BIOMARKERS

640

37. DIAGNOSTIC BIOMARKERS FOR PLANT POISONINGS

to chronic myocardial degeneration characterized by granular degeneration of myofiber cytoplasm necrosis and replacement fibrosis in the heart. These areas correspond to the pale areas observed grossly. There is bilateral symmetrical, subacute to chronic myofiber degeneration, and necrosis of the long digital extensor muscle groups. Clinical serum chemistry changes of elevated lactic dehydrogenase, aspartate aminotransferase, and creatine kinase occur in relation to severity of seizures. The extent of gross and microscopic lesions in muscles and elevated clinical chemistries are a result of the muscle damage from the severe seizures. In an experimental setting, administration of barbiturates at the onset of clinical signs prevented the seizures, death, and lesions in sheep. Interestingly, the lethal effects of a 3 lethal dose of water hemlock could be prevented with pentobarbital and no elevated serum chemistries or death occurred (Panter et al., 1996a).

Biomarkers of Poisoning (Blood Enzymes, CPK, AST, GGTP, Histological Lesions in the Heart, Long Muscles, Clinical Signs, History of Ingestion, Rumen Analysis) Diagnosis can be made by documenting exposure to the plant, with positive identification of plant parts in the rumen, especially tubers or seeds. Additionally, cicutoxin may or may not be detected in stomach contents as the toxin is quite unstable and readily oxidized. Because of the acute nature of water hemlock poisoning, animals that survive the intoxication will not have any lasting effects, whereas severely poisoned animals are found dead, normally with indications of violent terminal grand mal seizures. Death normally occurs within 1e8 h after ingestion of the plant. In animals that do survive poisoning, elevations in serum LDH, AST, and CK from muscle damage induced by the seizures peaks 3 days after exposure then declines to normal levels within 8e10 days (Panter et al., 1996a). There are no consistent gross lesions, although bruises and lacerations may be evident on the skin, muscles, tongue, etc., resulting from the severe seizures. Small hemorrhages may be present in the long skeletal muscles, and white streaking may be evident on the surface of the heart. Microscopically, cardiac lesions, including multifocal pale areas in the left, right, and interventricular myocardium, may be evident with myocardial fibrosis. In the long digital extensor muscle groups there maybe acute to chronic, moderate, random myofiber necrosis with regeneration of skeletal myofibers. Because of the acute nature of water hemlock poisoning and narrow threshold between death and survival, successful treatment would require immediate response with appropriate drugs.

Prevention and Treatment Prevention of poisoning is accomplished by recognizing the plant and avoiding exposing animals to it early in the spring or when in the flower/seed stage. Water hemlock is easily controlled with herbicides (2,4-D per manufacturer’s specification); however, herbicide use is often restricted near natural water sources. If few plants are present, hand pulling (using appropriate gloves) may be accomplished using caution to discard tubers away from possible exposure to animals or humans. Successful treatment with barbiturates or perhaps tranquilizers prevents death, lesion formation, and serum chemistry changes; however, treatment must be prompt (Panter et al., 1996a). This treatment has been successful in humans, but in animals it has never been demonstrated in the field and would require a veterinarian to respond very rapidly with appropriate drugs soon after the ingestion of the plant occurred.

PONDEROSA PINE NEEDLES (PINUS SPP.) The needles of ponderosa pine have been known for years to induce abortion in pregnant cows when grazed during the last trimester of pregnancy (Gardner et al., 1999). Occasional toxicosis in pregnant cows occurs; however, cases of toxicosis in nonpregnant cows, steers, or bulls are not reported.

Toxicology The toxin in ponderosa pine that induces abortion in cattle is the labdane resin acid isocupressic acid (12) (ICA; Gardner et al., 1994). Two related derivatives (succinyl ICA and acetyl ICA) also contribute to the induction of abortion after hydrolytic conversion to ICA in the rumen (Gardner et al., 1996). Other related labdane acids (agathic acid, imbricatoloic acid, and dihydroagathic acid) that are found in ponderosa pine needles at low levels may also contain abortifacient properties based on their similar chemical structure to ICA. Other genera and species have also been implicated in abortions, such as Monterey cypress (Parton et al., 1996), Korean pine (Kim et al., 2003), common juniper, lodgepole pine (Gardner et al., 1998), and other juniper species (Gardner et al., 2010; Welch et al., 2011a). Current research indicates that the concentration of labdane acids in ponderosa pine needles and western juniper bark is not uniform throughout the same tree, the concentrations can vary from location to location, and there is evidence for seasonal fluctuations as well (Cook et al., 2010, Welch et al., 2013a 2015).

IV. BIOTOXINS BIOMARKERS

PONDEROSA PINE NEEDLES (PINUS SPP.)

641

Biomarkers of Poisoning (Premature Parturition; Live-Premature Calf; Retained Fetal Membrane; Agathic or Tetrahydroagathic Acid in Serum, Rumen Content, or Calf Tissues; History of Ingestion)

CH2OH

CO2H Isocupressic acid (12)

The primary toxicological effect of ponderosa pine needles in cattle is premature parturition and associated complications, such as retained fetal membranes, metritis, and occasional overt toxicosis and death (Gardner et al., 1999). Pine needleeinduced abortion appears to mimic normal parturition except that it is premature. The abortions generally occur in the last trimester of pregnancy in the late fall, winter, or early spring. Abortions have been induced as early as 3 months of gestation and have been reported by ranchers to occur any time; however, the closer to the time of normal parturition that ingestion of pine needles occurs, the higher the risk of abortion. Abortions may occur following a single exposure to the needles, but results from controlled experiments indicate the highest incidence of abortion is in cows eating the needles over a period of 2e3 days. Abortions have been associated with grazing of green needles from trees, needles from slash piles following lumber activity, and dead, dry needles from the ground. Abortions are generally characterized by weak uterine contractions, incomplete cervical dilation, dystocia, birth of weak but viable calves, agalactia, and retained fetal membranes (Gardner et al., 1999). Calves born after 255 days of gestation will often survive with extra care but need to be supplemented with colostrum and milk from other sources until the dam begins to lactate. Cows with retained fetal membranes may need antibiotic therapy to avoid uterine infections. Pine needles will induce abortion in buffalo, but sheep, elk, and goats do not abort. Pine needles, pine bark, and new growth tips of branches are all abortifacient, and new growth tips are also toxic (Panter et al., 1990b). A separate toxic syndrome has been described in addition to abortion in which the abietane-type diterpene resin acids cause depression, feed refusal, weakness, neurological problems, and death. Specific compounds include abietic acid, dehydroabietic acid, and other related compounds (Stegelmeier et al., 1996). At 15%e30% of the diet, pine needles have been shown to alter rumen microflora and affect rumen fermentation (Pfister et al., 1992). Rumen stasis is part of the toxic syndrome (Stegelmeier et al., 1996).

Diagnosis of pine needleeinduced abortions is currently made by documenting exposure to needles, birth of the calf at least 2 weeks early, retained fetal membranes (RFM) in the cow, and agalactia. Recent studies have demonstrated that metabolites of ICA, including tetrahydroagathic acid, can be detected in the serum of the cow and calf for several days after parturition. Additional experiments are being conducted to determine what tissues/fluids in the calf, including dead calves, are optimum for a positive diagnosis. Isocupressic acid and related labdane diterpene acids found in pine needles are metabolized in both the rumen and the liver. The three major metabolites include agathic acid, dihydroagathic acid, and tetrahydoagathic acid (Gardner et al., 1999). Of these metabolites, tetrahydroagathic acid remains in the cow serum for the longest period and has been found to be the most relevant diagnostic component from abortion case samples submitted (Gardner, personal communications). There is research evidence that tetrahydoagathic acid is also found in fetal fluids (stomach and thoracic fluids; Snider et al., 2015). For diagnostic purposes, sera samples should be taken from the cow as soon as possible to optimize detection of the ICA metabolites. Additional samples from the aborted fetus should include blood (sera), stomach, and thoracic fluids.

Prevention and Treatment The only recommendation to prevent pine needle abortion is to limit access of late term pregnant cows to pine trees. There is no known treatment for cattle once ingestion of pine needles has occurred. Open cows, steers, or bulls are apparently unaffected by pine needles; likewise, sheep, goats (pregnant or not), and horses can graze pine needles with impunity and experience no adverse effects. Supportive therapy (antibiotic treatment or uterine infusion for retained fetal membranes) is recommended for cows that have aborted, and intensive care of the calf may save its life. Grazing of pine needles intensifies during cold, inclement weather and if other forage is in short supply. In spring, before green grass is available, cows will leave bedding grounds in search of green forage and frequently graze green needles from low-hanging branches or old, dry needles from surrounding trees where the snow has

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37. DIAGNOSTIC BIOMARKERS FOR PLANT POISONINGS

melted. These cows are at risk and should be kept away from the pines. Research has also determined that cattle with low body condition are more likely to eat pine needles than cattle in adequate body condition (Pfister et al., 2008b). Consequently, it is recommended that pregnant cattle grazing in ponderosa pine areas be maintained in good body condition (Pfister et al., 2008). Anecdotal information suggests that pregnant llamas may be at risk from ingesting pine needles, but no research has been done to substantiate this (Panter, personal communications).

RAYLESS GOLDENROD (ISOCOMA PLURIFLORA) White Snakeroot (Eupatorium rugosum) Rayless goldenrod (I. pluriflora) and white snakeroot (E. rugosum) are toxic range plants of the southwestern and midwestern United States, respectively. The disease associated with toxicity has been referred to as “alkali disease” because originally it was associated with drinking alkali water. Currently, it is referred to as “milk sickness” or “trembles” (the same as white snakeroot in the midwest) because the toxin tremetone (13) is excreted in the milk and subsequently results in poisoning of humans and nursing offspring.

Toxicology The toxic constituents of rayless goldenrod and white snakeroot are similar and the first reported toxin, tremetol, is actually a mixture of ketones and alcohols. Tremetone (13) (5-acetyl-2,3-dihydro-2-isopropenylbenzofuran) was thought to be the principle toxic factor; however, 11 different compounds have now been isolated and identified (Lee et al., 2010). The elucidation of different chemotypes of white snakeroot partially explains the sporadic and unpredictable toxicoses reported in livestock throughout the midwestern United States.

constipation, nausea, vomition, rapid labored respiration, progressive muscular weakness, stiff gait, standing in a humped-up position, dribbling urine, inability to stand, coma, and death. Signs are similar in cattle, sheep, and goats. The disease is often more acute and severe in horses than in cattle, and horses may die of heart failure after subacute ingestion of white snakeroot and presumably rayless goldenrod. Cattle have also been poisoned on a related plant (Isocoma acradenia) in southern California (Galey et al., 1991). In this case, 21 of 60 cattle died and 15 of 60 were affected but recovered.

Biomarkers of Poisoning (Blood Enzymes, CPK, Troponin, Histology, Lesions in Heart, Long Muscles, Clinical Signs, History of Ingestion, Myonecrosis) Diagnosis of rayless goldenrod poisoning is made by documenting exposure to the plant and identifying the characteristic clinical and pathological signs. The primary lesion and cause of the clinical trembles in rayless goldenrod intoxication in goats is skeletal muscle degeneration and necrosis. As these subtle lesions are most consistently seen in the quadriceps femoris and diaphragm at low dose, these may be the best tissues to examine. However, if there are clinical signs of poisoning and lesions are severe, the large appendicular muscles, such as the triceps brachii, biceps femoris, quadriceps femoris, and adductor, are likely to show significant histologic change. Higher doses produced myocardial lesions. Serum enzyme activities, including CK, LDH, AST, and alanine aminotransferase (ALT), have been shown to be significantly changed in poisoned animals. Although the mean cardiac troponin-I concentrations are not consistently elevated in different poisoned animals, individual animals do have elevated amounts. Creatine phosphokinase and ketones were elevated, and severe myonecrosis was described in the dead animals.

Prevention and Treatment O O Tremetone (13)

Clinical signs of poisoning may occur after ingestion of 1%e15% BW during a 1- to 3-week period. Signs begin with depression or inactivity, followed by noticeable trembling of the fine muscles of the nose and legs. Most cases of poisoning reported

Rayless goldenrod is not readily palatable, and toxicity results from animals being forced to graze the plant because of lack of good quality forage. Avoiding overgrazing will usually minimize poisoning in livestock. White snakeroot is relatively palatable and may be ingested as part of the diet in cattle and horses. Treatment is generally symptomatic and supportive, providing dry bedding, good shelter, and fresh feed and water. Activated charcoal and saline cathartic may be beneficial. Treatment may include fluids, B vitamins, ketosis therapy, and tube feeding. Hay and water

IV. BIOTOXINS BIOMARKERS

643

HALOGETON (HALOGETON GLOMERATUS)

should be placed within reach if the animal is recumbent. In lactating cows, frequent milking may facilitate a more rapid and complete recovery.

O Na

O Na

O O

HALOGETON (HALOGETON GLOMERATUS) Halogeton is an invasive, noxious, and poisonous weed introduced into the western United States from central Asia in the early 20th century. It was first collected along a railroad spur near Wells, Nevada, in 1934 and rapidly invaded 11.2 million acres of the cold deserts of the western United States (Young, 1999). There has been no appreciable spread since the 1980s because halogeton has filled all the suitable niches within its tolerance limits. It currently infests disturbed areas within the salt-desert shrub and sagebrush plant communities in the Great Basin, Colorado Plateau, and Wyoming’s Red Desert physiographic provinces, which have 3e15 in of annual precipitation. Halogeton’s infamy began in the 1940s and 50s by causing large, catastrophic sheep losses. There were many instances of large dramatic losses; sometimes entire bands of sheep died overnight from halogeton poisoning. Life magazine ran a cover story titled “Stock Killing Weed” that focused national attention on halogeton (Young, 1999). Congress passed the Halogeton Act in 1952 with the intent to (1) detect the presence of halogeton; (2) determine its effects on livestock; and (3) control, suppress, and eradicate this stock-killing weed. Federal research was reallocated from the Forest Service Experiment Stations to the Bureau of Plant Industries, creating a Range Research unit devoted specifically to “solving” the halogeton problem. It was realized that halogeton was not the problem but a symptom of a larger problemdthat being degradation of desert rangelands (Young, 1999). It invaded disturbed sites where sheep congregated around railroad loading sites, trail heads, stock trails, road sides, and water holes. When hungry sheep were turned loose to graze, halogeton was the only feed available, and they consumed too much, too rapidly, and were poisoned.

Toxicology The toxins are sodium and potassium oxalates (14), and Halogeton plants are frequently high (17%e29% total oxalates) in these oxalates in the fall and early winter resulting in death losses in sheep and cattle (Rood et al., 2014). Poisoning occurs when sheep consume more oxalates than the body can detoxify (James, 1999). Rumen microbes can adapt if animals are introduced slowly and prevented from eating too much halogeton.

Sodium oxalate (14)

Treatment of Poisoned Animals Animals can be given excess water through a stomach tube to flush oxalates out in the urine, or supplemented with dicalcium phosphate in a drench to provide Ca that will combine with oxalates in the rumen and facilitate oxalate excretion. Intravenous injection of calcium gluconate can maintain blood Ca levels, but Ca oxalate crystals will continue to damage kidneys (James, 1999). Prevention is the key to avoid poisoning. Only hungry sheep are poisoned. Research has demonstrated that as little as 1 oz of soluble oxalates can be lethal to hungry sheep. Well-fed sheep grazing nutritious forage throughout the day can tolerate more than 4 oz of soluble oxalate. Sheep grazed in a desert plant community infested with halogeton consumed it in 5%e25% of their diets without ill effect. If other forage is available, they will likely not get a lethal dose. Historically, sheep were most often affected; however, multiple cases of cattle poisoning have recently been reported (J. Hall, personal communication, 2013).

Biomarkers of Poisoning (Blood or Ocular Calcium, Oxalate Analysis of Blood, History of Ingestion Biomarkers of halogeton poisoning include serum hypocalcemia and oxalate crystals in the rumen and kidney. Diagnosis may be made by documenting exposure to the plant, identifying characteristic clinical and pathological signs of poisoning and histological examination. Blood calcium declines in poisoned animals, with concurrent increases in magnesium and phosphorus. There may also be an increase in serum LDH and Blood Urea Nitrogen (BUN), although these are not pathognomonic. Gross pathological lesions include the presence of fluid in the abdominal and chest cavities, as well as the pericardial sac. The lungs maybe filled with blood-tinged foam, and splotchy hemorrhages may be found on the surfaces of the heart, rumen, and other organs. The kidneys will be pale and swollen and contain oxalate crystals resembling glass shards and easily detected when the kidney tissue is cut with a knife. Microscopically the renal tubules will be filled with proteinaceous casts and calcium oxalate crystals, and the epithelium flattened and necrotic. There may also be edema and

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37. DIAGNOSTIC BIOMARKERS FOR PLANT POISONINGS

hemorrhage of the rumen wall and oxalate crystals present in the submucosa stomach compartment and in the walls of vessels.

Compositae (Senecio spp.), Leguminosae (Crotalaria spp.), and Boraginaceae (Heliotropium, Cynoglossum, Amsinckia, Echium, and Symphytum spp.). Not all of these occur in the western United States.

Management to Prevent Poisoning Never turn hungry sheep or cattle onto dense halogeton-infested sites. Provide supplemental feed and plenty of fresh water following trucking or trailing. Introduce sheep and cattle gradually to halogeton to allow rumen microbes to adjust. Some sheep producers graze their sheep on shadscale ranges (which contain low oxalate levels) before going into halogeton areas. Do not overgraze; maintain desert range in good condition. This prevents halogeton invasion and provides an alternative food source. Herbicide control of halogeton is not recommended because the waxy surface of the leaves hinders absorption of most herbicides. More importantly, however, desirable desert shrubs are killed, leaving the site open for further invasion and degradation by halogeton and other invasive weeds.

PYRROLIZIDINE ALKALOID-CONTAINING PLANTS Pyrrolizidine alkaloid (PA)econtaining plants are numerous and worldwide in distribution and in toxic significance (Cheeke and Shull, 1985). Three plant families predominate in PA-producing genera and species:

Toxicology More than 150 DHPAs (Dihydro Pyrrolizidine Alkaloids) have been identified, and their structural characteristics elucidated. The PAs contain the pyrrolizidine nucleus and can be represented by the basic structures of senecionine (15) and heliotrine (16). The toxic effects of all PAs are somewhat similar, although their potency varies because of their bioactivation in the liver to toxic metabolites called pyrroles (Fig. 37.1). These pyrroles are powerful alkylating agents that react with cellular proteins and cross-link DNA, resulting in cellular dysfunction, abnormal mitosis, and tissue necrosis. The primary effect is hepatic damage; however, many alkaloid and species-specific extrahepatic lesions have been described. Small amounts of pyrrole may enter the blood and be transported to other tissues, but there is debate on this issue because most pyrroles are highly reactive and not likely to make it into the circulation (Stegelmeier et al., 1999). When PA metabolites circulate, they probably do so as protein adducts that may be recycled. Some alkaloids (monocrotaline) may dissociate from their carrier proteins and damage other tissues such as the lungs. Pigs seem more prone to develop extrahepatic lesions. O

O RO

CH2

O

C

RO

R

CH2

O

C

R

liver enzymes N

N Pyrrolizidine alkaloid

Pyrrole derivative

conjugation

liver tissue

CH2

glutathione

OCOR

CH2

OCOR

N

N Pyrrole bound to liver tissue (toxic reaction)

Urinary excretion

FIGURE 37.1 Simplified schematic of the hepatic metabolism of pyrrolizidine alkaloids to the highly reactive pyrroles that result in liver damage.

IV. BIOTOXINS BIOMARKERS

PYRROLIZIDINE ALKALOID-CONTAINING PLANTS

645

(Brown et al., 2015). In addition, in vivo studies demonstrated that a higher pyrrole production rate occurred in cattle compared with sheep (Cheeke and Shull, 1985). Simple induction of liver microsomal enzymes by phenobarbitone increased pyrrole production and increased PA toxicity (LD50 in guinea pigs from z800 to 216 mg/kg). PA toxicity may disrupt other hepatic functions. Abnormal copper metabolism, clotting factors, NH3 metabolism, protein metabolism, etc., may be affected in PA poisoning.

Senecionine (15)

Biomarkers of Poisoning (Liver Enzymes, AST, LDH, GGTP, Cirrhosis, Histology, Pyrrole Analysis of Liver)

Heliotrine (16)

There are marked differences in susceptibility of livestock and laboratory animals to PA toxicosis. Cows are most sensitive, followed by horses, goats, and sheep, respectively. In small laboratory animals, rats are most sensitive, followed by rabbits, hamsters, guinea pigs, gerbils, and mice, respectively. Among avian species, chickens and turkeys are highly susceptible, whereas Japanese quail are resistant (Cheeke and Shull, 1985). Humans, especially fetuses, neonates, and children are particularly susceptible to DHPAs in herbal products, and many countries limit what a pregnant women should consume (Edgar et al., 2015). Detoxification mechanisms of PAs generally involve the liver and GI tract. Evidence of ruminal detoxification in sheep suggests that this contributes to the reduced toxicity in that species. There are also substantial species-specific differences in the rate of PA metabolism. Both probably contribute to species susceptibility. For example, Echium and Heliotropium PAs are easily degraded by certain rumen microflora, but there is little evidence of ruminal degradation of Senecio PAs. The PAs in Senecio are macrocyclic esters of retronecine as opposed to the open esters found in heliotridine. Therefore, the reason for the difference in Senecio toxicity between sheep and cattle is unlikely to be the rumen detoxification but more likely differences in speciesspecific enzymatic activation of Senecio PAs. For example, in in vitro studies, retrorsine metabolism has been shown to be high in those species that are most susceptible and lowest in animals of least susceptibility

Toxicity of Senecio, Heliotropium, and Echium is largely confined to the liver, whereas Crotalaria will also cause significant lung damage. Typical histologic lesions are swelling of hepatocytes, hepatocyte necrosis, periportal necrosis, megalocytosis (enlarged parenchymal cells), karyomegaly (enlarged nuclei) fibrosis, bile duct proliferation, and vascular fibrosis and occlusion. Hepatic cells may be 10e30 times normal size, and DNA content may be 200 times normal (Brown et al., 2015). In most species affected by PA poisoning, the liver becomes hard, fibrotic, and smaller. Because of decreased bile secretion, bilirubin levels in the blood rise, causing jaundice. Common clinical signs include ill thrift, depression, diarrhea, prolapsed rectum, ascites, edema in the GI tract, photosensitization, and aberrant behavior. In horses, “head pressing” or walking in straight lines regardless of obstacles in the path may occur. These neurological signs in horses are due to elevated blood ammonia from reduced liver function. PA poisoning may cause elevated blood ammonia, resulting in spongy degeneration of the central nervous system. Elevated levels of serum enzymes such as ALT, AST, g-glutamyl transferase (GGT), and alkaline phosphatase (ALP) are reported (Stegelmeier et al., 1999). Use of these tests for diagnosis is supportive but should not be relied on exclusively because they vary with animal species and other conditions. They may also be in the normal range even though liver damage has occurred, and they tend to be transient. Liver function tests such as bilirubin, bile acids, or sulfobromophthalein (BSP) clearance may be useful estimates of the extent of liver damage. The sulfur-bound pyrrolic metabolites of PAs have been detected in tissues of poisoned animals after hydrolysis with an alcoholic silver nitrate solution and detection either by colorimetric methods or by GC-MS (Mattocks and Jukes, 1990; Seawright et al., 1991; Winter et al., 1993; Schoch et al., 2000). Mostly recently a modification of these methods has been used for detection of

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646

37. DIAGNOSTIC BIOMARKERS FOR PLANT POISONINGS

the sulfur bond metabolites using LC-MS (Lin et al., 2011; Brown et al., 2015). The pyrrolic metabolites also bind to DNA, which can be isolated, enzymatically hydrolyzed and then specific DNA baseepyrollic adducts detected by LC-MS (Fu et al., 2010).

Prevention and Treatment There are no effective methods of prevention or treatment except avoidance of the plant and controlling plant populations with herbicides or biological control. Resistance to PA toxicosis in some species suggests that the possibility may exist to increase resistance to PAs. Dietary factors such as increased protein, particularly those high in sulfur amino acids, had minor protective effects in some species. Antioxidants such as Butylated Hydroxytoluene (BHT) and ethoxyquin induced increased detoxifying enzymes such as glutathione Stransferase and epoxide hydrolase (Mattocks and Jukes, 1990; Seawright et al., 1991; Winter et al., 1993). Zinc salts have been shown to provide some protection against hepatotoxicosis from sporidesmin or lupinosis in New Zealand and Australia, and zinc supplementation reduced toxicity in rats from Senecio alkaloids (Burrows and Tyrl, 2013; Knight and Walter, 2001). Many of these plants were introduced either inadvertently or intentionally. Without natural predators to keep populations in check, they experienced explosive growth and distribution followed by epidemic proportions of toxicity. Introduction of biological controls and natural population controls has reduced many of the plant populations and thus toxicoses have declined. Sheep, a resistant species, have been used to graze plants, particularly Senecio jacobaea.

PHOTOSENSITIZING PLANTS Numerous plants cause photosensitization resulting in lost production to livestock producers. Photosensitization is the development of abnormally high reactivity to ultraviolet radiation or natural sunlight in the skin or mucous membranes. Primarily induced in livestock by various poisonous plants, the syndrome in livestock has been defined as primary or secondary photosensitization.

Toxicology Primary In primary photosensitization, the photoreactive agent is absorbed directly from the plant and reaches the peripheral circulation and skin, where it reacts with the ultraviolet rays of the sun and results in sunburn, particularly of unprotected areas of the body. Hypericin (17) and fagopyrin are polyphenolic

derivatives from St. John’s wort and buckwheat, respectively, and are primary photodynamic agents (Cheeke and Shull, 1985). By definition, primary photosensitization does not induce hepatic damage. Most agents are ingested, but some may induce lesions through skin contact. Several of these plants are weedy in nature and can contaminate pastures and feed. Exposure to some plants is increasing as they are becoming widely used as herbal remedies and naturopathic treatments. In most cases, the photodynamic agent is absorbed from the digestive tract unchanged and reaches the skin in its “native” form (Stegelmeier, 2002). OH

O

OH

HO

CH3

HO

CH3

OH

O

OH

Hypericin (17)

Secondary In secondary or hepatogenous photosensitization, the photoreactive agent is phylloerythrin, a degradation product of chlorophyll. Phylloerythrin is produced in the stomach of animals, especially ruminants, and absorbed into the bloodstream. In normal animals, the hepatocytes conjugate phylloerythrin and excrete it in the bile. However, if the liver is damaged or bile secretion is impaired, phylloerythrin accumulates in the liver, the blood, and subsequently the skin, causing photosensitivity. This is the most common cause of photosensitization in livestock (Knight and Walter, 2001). Because chlorophyll is almost always present in the diet of livestock, the etiologic agent of secondary photosensitization is the hepatotoxic agent. The dermatologic signs of photosensitization in livestock are similar regardless of the plant or toxicant involved. Degree or severity varies, depending on the amount of toxin or reactive phylloerythrin in the skin, degree of exposure to sunlight, and amount of normal physical photoprotection (hair, pigmentation, or shelter). First signs in most animals are restlessness or discomfort from irritated skin, followed by photophobia, squinting, tearing, erythema, itching, and sloughing of skin in exposed areas (i.e., lips, ears, eyelids, udder, external genitalia, or white pigmented areas) (Burrows and Tyrl, 2013). Swelling in the head and ears (edema) of sheep after ingestion of Tetradymia has been

IV. BIOTOXINS BIOMARKERS

647

DEATH CAMAS

referred to as big head. It was determined that sheep grazing black sagebrush (Artemesia nova) before Tetradymia were three times more likely to develop this photosensitization (Johnson, 1974). Tissue sloughing and serum leakage may occur where tissue damage is extensive. Primary photosensitization rarely results in death. However, in secondary or hepatogenic photosensitization, the severity of liver damage and secondary metabolic and neurologic changes of hepatic failure may ultimately result in death. Recovery may leave sunburned animals debilitated from scar tissue formation and wool or hair loss.

climate, soils, and geographical locations. Poisoning in sheep, cattle, horses, pigs, fowl, and humans has been reported. Because of their grazing habits, the largest losses generally occur in sheep. Death camas is generally not palatable to livestock but is one of the earliest species to emerge in the spring. Poisoning most frequently occurs in spring when other more palatable forage is not available, or on overgrazed ranges where there is a lack of more desirable forage. Poisonings have resulted due to management errors in which hungry animals were placed in death camaseinfested areas (Panter et al., 1987).

Biomarkers of Poisoning (Bilirubin, Liver Enzymes, Skin Biopsies, History of Ingestion)

Toxicity of Death Camas to Livestock

Identifying chronic hepatic disease is complicated because many of the serum markers for hepatic disease have returned to normal. As normal hepatocytes become replaced with fibrous connective tissue, there are fewer damaged cells to elevate serum enzymes. Percutaneous liver biopsies are invaluable in identifying and diagnosing these cases (Stegelmeier et al., 1999). Plant-induced hepatopathy generally results in characteristic histologic lesions. For example, PAs generally cause bridging portal fibrosis with hepatocellular necrosis, biliary proliferation, and megalocytosis. Panicum and Tribulus species generally produce a crystalline cholangiohepatitis. Liver biopsy also provides prognostic information. The degree of damage is correlated directly with the animal’s ability to compensate, recover, and provide useful production. Note that the liver reacts to insult in a limited number of ways, and most histologic changes are not pathognomonic. Hepatic cirrhosis (necrosis, fibrosis, and biliary proliferation) involves nonspecific changes that can be initiated by a variety of toxic and infectious agents (Stegelmeier et al., 1999).

The toxins in death camas are of the cevanine steroidal alkaloid typedi.e., zygacine (18). Zygacine is a very potent compound with an i.v. LD50 of 2 mg/kg and an oral LD50 of 130 mg/kg in mice (Welch et al., 2011b). Clinical signs of toxicosis are similar in all livestock poisoned by Zigadenus, irrespective of the species of plant involved. Excessive salivation is noted first, with foamy froth around the nose and muzzle that persists, followed by nausea and occasionally vomition in ruminants (Panter et al., 1987). Intestinal peristalsis is dramatically increased, accompanied by frequent defecation and urination. Muscular weakness with accompanying ataxia, muscular fasciculations, prostration, and eventual death may follow. The pulse becomes rapid and weak, and the respiration rate increases but the amplitude is reduced. Some animals become cyanotic, and the spasmodic struggling for breath may be confused with convulsions. H

H N H

OH

Prevention and Treatment Prevention of poisoning lies in controlling plants with photosensitizing potential and providing adequate quality forage to animals. Treatment after poisoning involves removing animals from sun exposure, treating areas of necrosis and sunburn, antibiotic therapy, and supplementing young animals when access to sunburned udders is prevented because of nursing discomfort to dams.

DEATH CAMAS All death camas (Zigadenus) species are assumed to be toxic; however, variation in toxicity exists between species and even within species depending on season,

H

H

H OH

OH OH CH3C

O H

O

OH Zygacine (18)

Similarity in clinical signs of toxicosis between certain species of these plants suggests that the same alkaloids are present; however, differences in concentrations can explain the differences in relative toxicity of different species.

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37. DIAGNOSTIC BIOMARKERS FOR PLANT POISONINGS

Biomarkers of Poisoning (Alkaloid Analysis of Serum, Liver, Pathology, History of Ingestion) The heart fails before respiration, and at necropsy the heart is usually found in diastole. A comatose period may range from a few hours to a few days before death. Pathological lesions are those of pulmonary congestion. Gross lesions of sheep include severe pulmonary congestion, hemorrhage, edema, and subcutaneous hemorrhage in the thoracic regions. Microscopic lesions include severe pulmonary congestion with infiltration of red blood cells in the alveolar spaces and edema. Diagnosis of poisoning may be established by clinical signs of toxicosis, evidence of death camas being grazed, histopathological analysis of tissues from necropsied animals, and identification of death camas in the rumen or stomach contents (Panter et al., 1987). Zygacine has been detected in rumen content from field cases and in serum from sheep and mice experimentally dosed (Welch et al., 2011b, 2013b, 2016).

Management and Prevention Conditions conducive to poisoning by death camas include driving animals through death camaseinfested ranges; not allowing animals to graze selectively; unloading hungry animals in infested areas; lambing, bedding, watering, or salting livestock in death camase infested areas; or placing animals on range where little forage is available. Poisoning generally occurs in the early spring when death camas is the first green forage available, and the young immature foliage is the most toxic. Single flock losses of 300e500 sheep have been reported (Panter et al., 1987). Three key factors contribute to sheep losses: (1) driving sheep through heavily infested areas of death camas when the sheep are hungry; (2) bedding sheep for the night near death camase infested areas, providing immediate access to death camas the following morning; and (3) forcing sick sheep to travel will contribute to the stress, exacerbating the toxic effects and increasing the losses.

KNAPWEEDS: CENTAUREA SPP. The knapweeds are a large group of plants with primarily noxious, invasive characteristics. Although this genus is not a great risk for livestock producers, a serious disease of horses called nigropallidal encephalomalacia warrants its inclusion in this chapter. There are 450e500 species of Centaurea, and 29 species have been described in North America (Burrows and Tyrl, 2013). Most of these have been introduced and have had a

huge negative impact on rangelands in the western United States. Although most species are opportunists and will aggressively invade rangelands, especially those that have been overgrazed, burned, or disturbed, only two species are of any toxicologic significanced Centaurea repens (Russian knapweed) and Centaurea solstitialis (yellow star thistle).

Toxicology The compounds isolated from knapweeds include a large class called sesquiterpene lactones. Although the putative toxin causing the neurological disease in horses has not been specifically identified, six of these compounds have been screened for cytotoxicity in an in vitro neuronal cell bioassay. The rank order of activity is repin (19) > subluteolide > janerin > cynaropicrin > acroptilin > solstitialin (Riopelle and Stevens, 1993). Toxicity of solstitialin A-13 acetate and cynaropicrin to primary cultures of fetal rat substantia nigra cells has been demonstrated. These sesquiterpene lactones are quite unstable, and it has been hypothesized that they are precursors to the ultimate neurotoxin. Also, there are aspartic and glutamic acids present in these plants, and they possess neuroexcitatory properties. H O HO

O

O

H O

O O Repin (19)

Clinical Signs Thus far, only yellow star thistle and Russian knapweed have been implicated in toxicoses in the United States, and only in horses (Panter, 1991). Apparently, ruminants are not affected, and the Centaurea spp. may be useful forage for sheep and goats. However, in other countries, toxicoses in ruminants have been reported. For example, in South Africa, C. repens fed to sheep at 600 g doses for 2 days caused an acute digestive upset and pulmonary edema and ascites. In Azerbaijan, C. repens is reported to cause a neurological disease in buffalo similar to that which has been described in horses. However, no neuropathology similar to that seen in

IV. BIOTOXINS BIOMARKERS

CONCLUDING REMARKS AND FUTURE DIRECTIONS

horses was observed in the buffalo. Toxicity generally occurs in summer and fall when forage is depleted and horses are forced to graze less palatable species. Ingestion often occurs for several months or more before an abrupt onset of neurological dysfunction is observed. Often, the disease progresses to dehydration, starvation, and bizarre behavior, including submergence of the head in water to allow water to flow into the esophagus or lapping water like a dog. C. repens appears to be more toxic than C. solstitialis, but prolonged ingestion is required by both before disease appears. The amount of plant ingested to induce the clinical effects is reported to be 60% or more of body weight for C. repens and 100% or more of body weight for C. solstitialis. Intermittent grazing can prevent disease, indicating that there is not a cumulative effect but, rather, a threshold must be exceeded before neurological signs are observed (Cordy, 1978). Once neurological signs are observed in horses, prognosis for recovery is poor and euthanasia should be considered.

Biomarkers of Poisoning (History of Ingestion, Pathology Brain Lesions, Horses Only) Impaired eating and drinking are often the first observable signs. Depression and hypertonicity of the lips and tongue follow, and a constant chewing may be observed, hence the name “chewing disease.” Abnormal tongue and lip postures may be observed, and other neurological signs include locomotor difficulties such as aimless walking, drowsy appearance, and inactivity with the head held low. The neurological disease is considered permanent, and although some improvement may be seen, difficulty eating and drinking may preclude long-term recovery. The lesions are very specific and limited to the globus pallidus and the substantia nigra (nigropallidal encephalomalacia), where distinct pale yellowish to buffcolored foci or softening and cavitation are seen (Cordy, 1978). The lesions are typically bilateral and symmetrical. This specificity of the lesions for the basal ganglia has prompted more investigations into unraveling the mysteries of human diseases associated with dopaminergic pathways, such as Parkinson’s or Huntington’s disease, and tardive dyskinesia. This disease in horses is often called equine Parkinsonism. This unusual disease is manifest by an almost immediate onset after prolonged ingestion, suggesting an all-or-none type of acute neurological crisis. The lesions develop quickly and completely, and progressive stages of degeneration rarely occur except for some changes in the adjoining neurons adjacent to the necrotic foci in the globus pallidus and the pars reticularis of the substantia nigra (Cordy, 1978). Microscopically, there is extensive

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necrosis of neurons, glia, and capillaries within sharply defined margins of the involved brain centers. Occasionally, lesions may be observed in the gray and white matter of the brain.

Prevention and Treatment Good veterinary care and supportive therapy, including good feed, easy access to water, supplemental vitamins, and good nursing care, is essential for survival. Treatment of the disease once it is manifest is not generally successful. However, in Argentina, affected horses have been treated with glutamine synthetase and a bovine brain ganglioside extract given daily intramuscularly for 1 month with some success (Selfero et al., 1989). When animals are first observed grazing Centaurea spp., they should be immediately removed to better pastures. Prevention of the disease is easily accomplished by knowing the plants that exist in one’s pastures, by providing good quality and adequate amounts of forages and feed, and by frequent observation of one’s animal’s grazing patterns and behavior. Control of plant invasion by good range/pasture management to prevent overgrazing and loss of other competitive grasses and forbs is important. Herbicide control is quite easily accomplished with broadleaf products, including 2,4-D, dicamba, and picloram. These plants are prolific seed producers, and followup treatment is required to eliminate the populations. Seeds are often distributed through contaminated hay or other feed sources, and initial populations often start near feed bunks and spread from there. Because of their morphology, size, and parachute-like structures, seeds are easily spread by wind and water. Understanding one’s weeds and close monitoring of populations will help in the control of these highly invasive species.

CONCLUDING REMARKS AND FUTURE DIRECTIONS Even with our ever-increasing knowledge about poisonous plants and their toxins, poisonings continue to occur, some catastrophic, on livestock operations. Poisoning in humans and companion animals from toxic plants also continues to be a significant risk, especially to pets and children. Identification of specific biomarkers of poisoning from plants is relatively new and is often limited to pathology after the fact or chemical analysis of biological fluids or tissues. Research at the Poisonous Plant Research Laboratory continues to emphasize methods and biomarkers that will improve early diagnosis and ultimately reduce losses from poisonous plants.

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