Locoweed Research: Updates and Highlights 1999 9

TOXICOLOGYAstragalus and Oxytropis poison livestock

with different toxins ..................................... 10

Defining the chemistry and biochemistryof swainsonine .............................................. 12

How locoweed poisoning developsand progresses ............................................. 14

Locoweeds reduce reproductiveperformance in livestock ............................... 17

Grazing locoweed can increase incidenceof congestive right-heart disease .................. 19

Locoweed alters ewe and lamb behavior .......... 21

Low-level locoweed consumption altersblood chemistry in sheep.............................. 23

Looking for ruminant bacteria that candetoxify swainsonine .................................... 26

10 TOXICOLOGY

Astragalus and OxytropisPoison Livestock with Different ToxinsLynn F. James, Kip E. Panter, Bryan L. Stegelmeier,Michael H. Ralphs, James A. Pfister, and Dale R. Gardner

Locoweed species cancontain swainsonine orother toxins that poisonlivestock.

Poisonous plants rank high among the major causes of economic lossesto the livestock industry. Of all the poisonous plants, the Astragalus andthe closely related Oxytropis genera are probably the most destructive tolivestock. The taxonomy of these genera is complex and difficult evenfor trained plant taxonomists. These groups of plants are fascinating notonly because of their complexity, but also because of their many andvaried effects on animals that consume them.

Astragalus species are mostly perennial, stemmed or stemless herbs.The leaves are alternate and pinnately compound (fig. 1). Flowers areleguminous. The fruits are legume pods of various sizes and shapes. Oneof the most remarkable characteristics of the genus Astragalus is thatthere are hardly two species—even those closely related—that do notdiffer in fruit form or structure. The seeds are kidney-shaped. The seedsof some Astragalus species may remain viable in the soil for 40 years oreven longer.

Not all Astragalus species are toxic. Some that are nontoxic are valu-able as livestock forage. For example, livestock relish Nuttall’smilkvetch (Astragalus nuttallianus). The introduced species of cicermilkvetch (Astragalus cicer) has been used as livestock forage and alsoas a soil builder.

Oxytropis species are similar to Astragalus species except that theyhave erect stems and the keel of the corolla (of petals) is tipped with asharp, erect point.

The Astragalus and Oxytropis species can be divided into three gen-eral syndromes according to the toxin they contain. Astragalus andOxytropis species that contain swainsonine, the nitro-containingAstragalus species that have 3-nitropropanol or 3-nitropropionic acid,and the Astragalus species that accumulate selenium in toxic amounts.

LocoweedsThe term locoweed properly applies only to those species of theAstragalus and Oxytropis genera containing the toxic indolizidine alka-loid swainsonine in amounts that will poison animals. The term “loco”can be used as a noun to describe the plant, as a verb to describe poison-ing with locoweed, or as an adjective to describe abnormal behavior(crazy). Locoweeds are destructive to livestock not only because of theirwide distribution but also because of the broad variety of pathologicaleffects and the nature of the intoxication process.

The locoweeds must be grazed over a period of weeks before the in-toxication becomes apparent. By the time intoxication is recognized, theanimal is in trouble and correction is difficult if not impossible. Loco-weed affects animals in a number of ways. These include neurologicaldamage, which causes the animal to behave in an abnormal fashion;emaciation or wasting; habituation (there is some question regarding thiseffect); abortions; skeletal birth defects; and congestive right-heart fail-

Figure 1. Locoweed leaves, whichare arranged alternately on thestem and are pinnately compound.

Locoweed Research: Updates and Highlights 1999 11

ure associated with grazing at high elevations. Animals may appear tothrive for a short time after they start to graze locoweed. But with con-tinued grazing, they will lose weight and show other signs of poisoning.

Nitro-containing AstragalusSpecies of this group can be found growing from western Canadathroughout the western United States and into northern Mexico. Ex-amples include red-stemmed peavine (Astragalus emoryanus) in NewMexico, Texas, and Mexico; A. tetrapterus in Utah, Nevada, Arizona,and southern Oregon; and timber milkvetch (A. miser) and A. canadensisin the western United States and Canada. There are more than 250 spe-cies in this group.

These toxic plants contain the glycosides of 3-nitropropanol and 3-nitropropionic acid. Animals poisoned by these plants develop degen-erative lesions of the spinal cord and emphysema. Sheep, cattle, andhorses are all sensitive to poisoning by these plants. Signs of poisoninginclude weakness, knuckling at hocks, goose stepping (interference ofhind limbs), respiratory distress, cyanosis, sudden collapse, and death.Temporary blindness may occur. Wasting occurs with prolonged grazing.Death occurs from both acute (one large dose) and chronic (multipledoses over time) intoxications.

Selenium-accumulating AstragalusSelenium in certain soils may be taken up by plants in amounts that ren-der them toxic to grazing animals. Certain species (about 24) ofAstragalus accumulate relatively large amounts of selenium. Theseinclude plants such as two-grooved milkvetch (A. bisulcatus) andPatterson’s milkvetch (A. pattersonii). They are often called indicatorplants and can be used to identify areas where selenium poisoning maybe a problem. Certain forage grasses and desirable shrubs that grow inthese areas may accumulate sufficient selenium to make them toxic. Poi-soning of livestock is usually associated with the selenium-accumulatinggrasses. Some of the selenium-accumulating Astragalus also contain lowswainsonine levels. Selenium poisoning is a problem primarily west ofthe Mississippi River.

Lynn F. James is the research leader,Kip E. Panter is a research animal sci-entist, Bryan L. Stegelmeier is aveterinary pathologist, Michael H.Ralphs and James A. Pfister are range-land scientists and Dale R. Gardner is achemist, all at USDA’s Agricultural Re-search Service, Poisonous PlantResearch Laboratory in Logan, Utah.

12 TOXICOLOGY

Defining the Chemistryand Biochemistry of SwainsonineRussell J. Molyneux

Locoweed’s toxic component is a natural constituent of the plant namedswainsonine. The compound was first discovered in the “poison peas”(Swainsona species) of Australia but has now been found in the loco-weeds (Astragalus and Oxytropis species) of North America and otherparts of the world, including South America and regions of China. It alsois present in some members of the morning-glory family (Ipomoea spe-cies), which are poisonous to sheep and goats in southern Africa andAustralia. The signs of poisoning are quite similar in animals that con-sume these plants.

Not all species of Astragalus and Oxytropis contain swainsonine,but it occurs in all of those that typically have been known as locoweeds(spotted loco, woolly locoweed, and white locoweed). Although thetoxin content of locoweeds is not very high—generally less than 0.2%of the plant’s dry weight—it appears to be highest in the flowers andseeds. However, because of its exceptional potency, it has been calcu-lated that levels greater than 0.001% can cause poisoning if the plant isconsumed over a sufficient time period. None of the above-ground partsof the plant are free of the toxin and even plants dead as long as twoyears retain enough of the toxin to cause locoism. An estimate of the po-tential hazard to livestock can be made by chemical analysis of a plantsample for swainsonine’s presence.

The chemical structure of swainsonine is not complex and is quitesimilar to simple sugars, such as mannose and glucose, which it appearsto mimic (fig. 1). As a result of this imitation, it stops the action of theenzyme α-mannosidase, which is essential for the proper functioning ofall animal cells. This enzyme trims sugar molecules from complex mol-ecules known as glycoproteins (sugar-proteins) within the cell. Once thecorrect number of sugars have been trimmed off, the smaller moleculescan be released from the cell to serve the functions (e.g. digestion) forwhich they have been prepared. Failure of the trimming process resultsin an increase in the number of complex molecules being retained withinthe cell, until the cell can no longer contain them and bursts open. This

Figure 1. The chemical structure of swainsonine.

Understandinglocoweed’s major toxiccomponentand its effects onlivestock

Locoweed Research: Updates and Highlights 1999 13

has been described as a “vapor-lock” of the cell’s production lines. Aftera sufficient number of cells have been damaged, the signs of poisoningappear in the animal. Because all cells depend on proper functioning ofα-mannosidase, many different organs can be damaged, including thebrain, heart, and reproductive and digestive systems. The particularorgans affected and signs of poisoning depend on the amount ofswainsonine consumed and the exposure period, as well as externalfactors such as nutritional status, grazing altitude, and pregnancy.

Swainsonine is very water-soluble and therefore distributes rapidly tomany parts of the body. It also is rapidly excreted, primarily in the urine.But in lactating animals, a portion of it is transferred to the milk, so thatnursing calves or lambs can become “locoed.” This fast excretion ratesuggests that occasional locoweed consumption for short periods is un-likely to have serious adverse effects. But continuous consumption, evenat relatively low levels, generally results in typical signs of locoweedpoisoning. Therefore, it is important to remove animals from locoweed-infested land as soon as they are observed grazing the plant.

Russell J. Molyneux is a research chem-ist at USDA’s Agricultural ResearchService, Western Regional ResearchCenter in Albany, Calif.

14 TOXICOLOGY

How Locoweed PoisoningDevelops and ProgressesBryan L. Stegelmeier, Lynn F. James, Kip E. Panter,Michael H. Ralphs, Dale R. Gardner, and James A. Pfister

Locoweed poisoning or “locoism” results when animals ingest locoweedfor several weeks. North American species that commonly poison live-stock include spotted locoweed, woolly locoweed, Wooton locoweed,white locoweed, and Lambert’s locoweed (Astragalus lentiginosus, A.mollissimus, A. wootoni, O. sericea, and O. lambertii) (fig. 1). Theselocoweeds are legume, pealike plants that are found throughout the west-ern United States and in many parts of the world. They often grow in theearly spring and late fall when other green feeds are not available. Mostlocoweed species are palatable and readily eaten by livestock and wild-life. Generally, the amount eaten is in proportion to locoweed availabil-ity compared with other green forages. Locoweeds remain toxicthroughout the season and even the dry stalks of dying plants arepoisonous.

Locoweed poisoning has an insidious onset with signs of poisoningnot becoming apparent until the animal has grazed the plant for severalweeks. Clinically, locoweed intoxication is characterized by depression,neurologic deficits, loss of muscular control, nervousness (especiallywhen stressed), dull hair coat, emaciation, decreased libido, infertility,abortion, water belly (hydrops amnii), cardiovascular disease, and death.

Generally, there are no visible lesions of locoism on the animal. How-ever, intoxication results in characteristic microscopic lesions that aredescribed as vacuolar degeneration of neurons (fig. 2) and other paren-chymatous cells. These lesions develop within 4 or 5 days after animalsbegin consuming locoweed. Most changes resolve in several days ifconsumption is discontinued. However, many neuronal lesions are notreversible as certain neurons die easily. These permanent neurologic

Figure 1. Locoweed (Astragalus lentiginosus).

A summary of thedevelopment andchanges caused bylocoweed poisoning,current diagnostictechniques to identifypoisoned animals, andinformation concerninglocoweed toxinmetabolism andexcretion.

Locoweed Research: Updates and Highlights 1999 15

changes can result in behavioral and functional deficits that make thevalue of any previously poisoned animal questionable. Additionalresearch is needed to determine how long animals can be poisoned with-out developing permanent neurologic lesions and to better understandthe development of these permanent secondary effects.

The diagnosis of locoweed poisoning is currently made by document-ing exposure to the plant and identifying the characteristic clinical andpathologic changes of poisoning. Recently, diagnostic tests using serumfrom living poisoned animals have been developed.

The locoweed toxin has been identified as swainsonine, a poly-hydroxy-alkaloid (with some sugar-like properties) that is a potent in-hibitor of several enzymes called mannosidases. One inhibited enzymeis lysosomal α-mannosidase that degrades unneeded or damaged oli-gosaccharides and glycoproteins. Mannosidase inhibition results in accu-mulations of incompletely processed compounds in cellular vacuoles(vacuolar degeneration). This process resembles cellular constipation asthe cells cannot metabolize or excrete waste material. Similar vacuola-tion caused by genetically abnormal lysosomal α-mannosidase is seen ingenetic mannosidosis of humans, Angus cattle, and cats. Swainsoninealso inhibits mannosidase II, an enzyme of glycoprotein metabolism re-sulting in abnormal hormones, membrane receptors, and enzymes. Con-sequently, locoweed poisoning results in abnormal endocrine,reproductive, immune, and gastrointestinal function. All of thesechanges reduce animal efficiency and production and may make poi-soned animals more susceptible to disease. Low swainsonine doses ofshort duration do not seem to be harmful, and swainsonine may be a use-

Figure 2. Photomicrograph of a vacuolated neuron from a cowpoisoned on locoweed. Bar is 25 microns.

16 TOXICOLOGY

ful pharmaceutical. Swainsonine has been shown to inhibit the spread ofsome cancers. It also protects certain bone marrow cells from the effectsof other cancer treatments.

The locoweed toxin, swainsonine, is very water soluble. It is absorbedrapidly from the gastrointestinal tract and is quickly excreted in theurine, milk, and feces. The swainsonine clearance rate (T1/2) from achronically poisoned animal is a little less than 20 hours from the serumand 60 hours from the liver. This suggests that after a withdrawal time of28 days (10 half lives), practically no toxin will remain in a poisonedanimal. Reversal of the locoweed toxin’s effects is slower. As normalproteins must be synthesized, packaged, and integrated into the correctlocation, it may take weeks or months for cell function to recover. Somecells never completely recover.

It also has been shown that after a certain dose, higher doses donot cause more severe disease. This suggests that once all the cellularmannosidase is inhibited, higher swainsonine doses are not directly toxicand do not cause more damage. This “threshold-like” response may beuseful in formulating management plans to graze locoweed-infestedranges. Animals could be managed so that they ingest low doses that donot inhibit all cellular mannosidase. Alternatively, management could bechanged so that all the animals get an extremely high dose of short dura-tion with periods of withdrawal, so that the permanent changes of poi-soning are avoided. More work is needed to define both safe doses andexposure duration.

Swainsonine also alters specific endocrine and cellular functionsincluding the immune system. At low doses of short duration,swainsonine promotes proliferation and antibody production of somelymphocytes. Higher locoweed doses of longer duration inhibit this re-sponse and the final result of locoweed poisoning appears to be a sup-pressed immune system. Current research is underway to define thelong-term, immunologic effects of locoweed poisoning.

In summary, locoweed poisoning is a chronic disease that developsafter livestock graze certain Astragalus and Oxytropis spp. for severalweeks. A diagnosis of poisoning can be made by documenting exposureto the plant, identifying the neurologic signs of poisoning, and analyzingserum for α-mannosidase activity and swainsonine. Many of the histo-logic lesions and loss of function seen in poisoned animals are regainedshortly after locoweed consumption is discontinued. However, some ofthe neurologic changes are irreversible and permanent. This makes theusefulness of many working animals questionable, if they are poisoned.

Bryan L. Stegelmeier is a research vet-erinary pathologist, Lynn F. James isthe research leader, Kip E. Panter is areproductive toxicologist, Michael H.Ralphs and James A. Pfister are range-land scientists, and Dale R. Gardner isa research chemist. All are at USDA’sAgricultural Research Service, Poison-ous Plant Research Laboratory inLogan, Utah.

Locoweed Research: Updates and Highlights 1999 17

Locoweeds Reduce ReproductivePerformance in LivestockKip E. Panter, Lynn F. James, Michael H. Ralphs, Bryan L. Stegelmeier,James A. Pfister, Dale R. Gardner, and Russell J. Molyneux

Rams and bulls shouldnot graze locoweed-infested pastures within60 to 90 days ofbreeding.

Locoweeds include those plants of the Oxytropis or Astragalus generathat contain the toxic alkaloid swainsonine. Ingesting locoweeds inter-feres with all aspects of reproductive function in livestock. The severityof these effects is determined by the length of time and the amount ofplant material animals graze. Certain reproductive parameters such asmale and female breeding behavior may be affected within a few weeks.Other effects (ovarian and testicular dysfunction) may go unnoticed untilweight loss and certain nervous disorders are evident. Abortion is themost obvious result of locoweed poisoning in pregnant animals. How-ever, altered sexual behaviors, increased embryonic loss, decreased fetaldevelopment, increased neonatal loss, and abnormal mothering instinctor nursing behavior in the newborn may be just as costly, although not asobvious.

There is no 100% safe grazing period for livestock on locoweed as faras reproduction is concerned. Once animals begin to graze locoweedthere are measurable changes such as an immediate increase in swain-sonine in the animal’s blood and an accompanying decrease inα-mannosidase activity, an enzyme that is critical in glycoproteinmetabolism described in Defining the Chemistry and Biochemistry ofSwainsonine (p. 12). Most reproductive functions are controlled by hor-mones that are glycoproteins. Many hormone receptors also are glyco-proteins. Abnormal hormones and receptors result in both behavioral andreproductive dysfunction. Pathological changes in some reproductive or-gans have been reported within a few days of locoweed ingestion.

Research at USDA’s Agricultural Research Service, Poisonous PlantResearch Lab has demonstrated that locoweed ingested at 10 to 15% ofthe diet for as little as two to three weeks in cows and ewes altered thelength of the estrous cycle, changed estrus behavior, reduced embryonumbers and viability, and reduced conception rates. Most reproductiveeffects are reversible when locoweed ingestion stops. The recoveryperiod for some aspects like the development of sperm may be relativelylong. Some neurological signs may not be reversible, resulting in pro-gressive wasting and eventual death. By the time outward signs are obvi-ous (20 to 30 days of ingestion), reproductive processes may already beseverely compromised. Abortion in pregnant animals may occur afteringestion of locoweed for 20 to 30 days. Locoweed is toxic to fetuses,causing almost immediate changes in the fetal heart function. Livestockingesting locoweed early in pregnancy may resorb or abort the fetuswithout obvious signs. Increased numbers of open cows or ewes maybe all that ranchers observe.

Ranchers have reported significant reproductive losses in cow-calfoperations from cows grazing locoweed. Therefore, we fed four maturecows white locoweed at 20% of their diet for 30 days during the estrouscycle to determine the effects of locoweed on cycling cows. The estrouscycle length was increased (fig. 1) in all cows and conception was de-

Figure 1. Progesterone profiles intwo cows fed white locoweedshowing extended estrous cycles.Extended follicular phase (41 days;low progesterone) in cow 153 andextended luteal phase (35 days;high progesterone) in cow 156.Both cows showed relativelynormal estrous cycles soon afterlocoweed feeding stopped, butcow 156 bred three times beforeconceiving.

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Cow 153Pre-treatmentEstrousCycle Locoweed

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18 TOXICOLOGY

layed one to three estrous cycles. Serum progesterone is a good indicatorof normal estrous cycle activity. Once cows conceived, pregnancy pro-gressed normally until seven months gestation when two of the fourcows were fed locoweed again for 30 days. One cow aborted one weekafter locoweed feeding was stopped. Cycling heifers were fed whitelocoweed at low, medium, and high doses representing approximately 3,10, and 30% of the diet for 42 days. Those heifers fed the high doses oflocoweed had severe changes in ovarian function by day 20, which per-sisted 15 to 20 days after we stopped feeding them locoweed. By day 42after locoweed feeding stopped, ovarian function had returned to normal.

Experiments on young rams fed locoweed 60 to 70 days showed thatbreeding behavior and libido were altered by 20 to 30 days. Semen qual-ity was not changed until 50 to 60 days, when numbers of abnormalsperm increased and motility decreased. While semen parameters re-turned to normal by 60 to 70 days after locoweed feeding stopped, neu-rological dysfunction persisted and eventually progressed to wasting,emaciation, and the need to euthanize six of nine rams. Therefore, werecommend that breeding rams and bulls be maintained on locoweed-free pastures and not be allowed access to locoweed within 60 to 90 daysof breeding.

Other conditions caused by locoweed that may indirectly contribute toreduced reproductive performance include emaciation, abnormal breed-ing behavior, lack of coordination, delayed puberty, abnormal motheringinstinct soon after birth, reduced nursing instinct in newborns, reducedlactation, lengthened calving intervals from year to year, and reducedgrowth rates in offspring.

Kip E. Panter is a research animal sci-entist, Lynn F. James is the researchleader, Michael H. Ralphs is a range-land scientist, Bryan L. Stegelmeier is aveterinary pathologist, James A. Pfisteris a rangeland scientist, and Dale R.Gardner is a research chemist, all atUSDA’s Agricultural Research Service,Poisonous Plant Research Laboratory,in Logan, Utah. Russell J. Molyneux is aresearch chemist at USDA’s AgriculturalResearch Service, Western Regional Re-search Center in Albany, Calif.

Locoweed Research: Updates and Highlights 1999 19

Grazing LocoweedCan Increase Incidenceof Congestive Right-Heart FailureLynn F. James, Kip E. Panter, Bryan L. Stegelmeier,Michael H. Ralphs, James A. Pfister, and Dale R. Gardner

Consumption oflocoweed at elevationsabove 7,000 feetincreases incidence ofcongestive right-heartfailure.

In cattle, congestive right-heart failure (CRHF) associated with livingat high elevations (above 7,000 feet) is commonly referred to as “highmountain disease.” The cause of this condition is an hypoxic-inducedpulmonary hypertension associated with the reduced oxygen found athigh elevations. The incidence of the disease in Wyoming and Coloradovaries from 0.5 to 2%, annually. The higher the elevation, the higher theincidence. As elevations increase, the time required to develop hypoxic-induced pulmonary hypertension and, subsequently, the time to produceheart failure and signs of the disease decreases. Chronic congestive heartfailure can result from a number of causes, which include traumatic peri-carditis, parasitism, chronic anemia, valvular endocarditis, pneumonia,and any condition that causes a work overload on the heart’s right ven-tricle. Cattle and especially calves grazing locoweeds at high elevationshave an increased incidence of high mountain disease. The incidence ap-proaches 100% if cattle are left to graze on locoweed more than 45 days.

CRHF has been produced by feeding the locoweed toxin, swain-sonine, to cattle living at high elevations (10,000 ft). Swainsonine ishighly soluble in water and is excreted in the cow’s milk. Calves nursingcows that are grazing locoweed can develop CRHF. Calves can get anincreased dose of the toxin by nursing an infected cow and grazing theplant. After eating locoweed for only a few days, calves become sensi-tive to exercise, showing weakness and reluctance to walk. Cold andfluctuating temperatures seem to enhance the development of CRHF incattle grazing locoweed-infested ranges at high elevations. Pneumonialikewise will predispose calves at high elevations to CRHF.

Locoweed poisoning results when cattle graze white locoweed over aperiod of several weeks. Clinical signs of poisoning include depression;rough, dry hair coat; dull, lusterless eyes; and excitement when placedunder stress. No outstanding large lesions are seen, but there are micro-scopic lesions of neurovisceral cytoplasmic vacuolation. Emaciation,abortion, birth defects, and interference with other reproductive pro-cesses also have been associated with locoweed poisoning. No associa-tion between CRHF and locoweed intoxication has been found at lowerelevations.

The clinical signs and lesions associated with high mountain diseaseinclude dry hair coat, weakness, visible jugular pulse, right-ventricularhypertrophy and dilation, subcutaneous edema ascites, hydrothorax,chronic passive congestion of the liver, and hypervolemia with hemodi-lution. In advanced cases, death follows slight exertion. Medial thicken-ing with luminal restriction of the small pulmonary arteries andarterioles develops and becomes a prominent histological feature of thecondition.

20 TOXICOLOGY

Clinical signs in calves developing CRHF when grazing locoweed athigh elevations are not typical of locoweed poisoning observed underusual field conditions at low altitude or as recorded in the literature. Thesigns of CRHF enhanced by the grazing of locoweed are similar to thoseseen in the typical right-heart failure condition. The clinical signs in-clude dry nose; rough, dry hair coat; depression; dark fluid diarrhea;edema (accumulation of fluid) under the jaw, brisket, and underline;labored respiration; visible jugular pulse; weakness; and death. How-ever, some calves appear to dehydrate rather than become edematous,but after death the gross lesions are the same. A few calves first developthe edema and then appear to become dehydrated. In some calves, theskin along the sides of the jaw becomes tight and the hairs stand erect.These calves become ill and die in a short time. Gross and microscopiclesions are the same as those observed in calves showing the more typi-cal signs of CRHF.

At high elevations, pathologic changes of CRHF in calves fed loco-weed are similar in right-heart failure to those not fed locoweed. Thegross changes include right ventricular dilation and hypertrophy, subcu-taneous edema, ascites, hydrothorax, profuse fluid diarrhea, and chronicpassive congestion of the liver. The heart has a rounded contour due tothe dilation and hypertrophy. In advanced cases, the lungs have varyingamounts of atelectasis or consolidation. Microscopic lesions reflect con-gestive heart failure and the neurovisceral vacuolation characteristic oflocoweed poisoning.

Fetal lambs from ewes fed spotted locoweed (Astragalus lentiginosus)during days 60 to 90 of gestation have an enlarged right ventricle of theheart and edema about the neck area. Fetal lambs from ewes fed loco-weed also have lesions of locoweed poisoning. The changes observed inthese fetal lambs are reminiscent of calves consuming locoweed at highelevations. The changes may be related to the CRHF observed in calvesfed locoweed at high elevations. Locoweed also can cause hydrops am-nii (an accumulation of fluid in the placenta) in cattle grazing locoweed.Locoweed poisoning lesions are found in the fetal calves of these cows.These calves have large fluid-filled abdominal cavities. This could bethe result of heart or other vascular degeneration related to consumingthe locoweed toxin.

It has been found that the CRHF in calves whose mothers are grazingon locoweed-infested ranges at high elevations can largely be reversedby moving them off the locoweed and to lower elevations within 40 daysof starting to graze locoweed.

Lynn F. James is the research leader,Kip E. Panter is a research animal sci-entist, Bryan L. Stegelmeier is aveterinary pathologist, Michael H.Ralphs and James A. Pfister are range-land scientists, and Dale R. Gardner isa chemist, all at USDA’s AgriculturalResearch Service, Poisonous Plant Re-search Laboratory in Logan, Utah.

Locoweed Research: Updates and Highlights 1999 21

Locoweed AltersEwe and Lamb BehaviorJames A. Pfister, Bryan L. Stegelmeier, Kip E. Panter,Lynn F. James, Michael H. Ralphs, and Dale R. Gardner

Eating locoweed caninterfere with ewe-lambbonding.

Behavioral studies were conducted several years ago to examine some ofthe possible effects of locoweed on livestock behavior. The first studiesfocused on the effects of locoweed on ewe-lamb bonding at birth and onnewborn lamb behavior shortly after birth.

Thirty ewes were hand-mated to lamb within a certain time period.When the ewes were pregnant for 100 days, they were divided into treat-ment and control groups. The locoweed treatment group received whitelocoweed for 30 days as a 10% locoweed pellet (90% alfalfa hay). Twoewes aborted in 15 to 30 days; the other ewes lambed normally.

The locoweed-treated ewes did not bond normally with their lambs.The poisoned ewes were nervous and circled their lambs excessively,making nursing virtually impossible. The lambs from poisoned eweswere developmentally delayed at birth in every measurement comparedto normal lambs. They were slow to stand, slow to seek the udder, andwere unable to nurse without human assistance. In a behavioral test bat-tery, these lambs were less mobile than control lambs and had difficultyrecognizing their dam from another ewe. Poisoned ewes did not havedifficulty recognizing their own lamb from a strange lamb. The lambspoisoned during gestation recovered much of their ability to functionwithin 7 to 10 days. When retested a few months later, lambs from poi-soned ewes were indistinguishable from normal lambs.

We also studied if lambs from ewes eating locoweed would eat morelocoweed than lambs from ewes that would not eat locoweed. Threegroups of ewes were conditioned: loco-eaters, loco-avoiders, and thosewith no experience with locoweed. Lambs were then exposed to loco-weed with their mothers and later tested alone. A fourth treatment group

22 TOXICOLOGY

included orphan lambs with no maternal interaction. Lambs from all thegroups ate locoweed when tested, and mother’s experience had only aslight impact on the lamb’s eating behavior. Short-term maternal influ-ence apparently was not sufficient to teach lambs to eat locoweed com-pared to control lambs. Nonetheless, other studies we have done suggestthat even limited consumption of locoweed by young animals may setthe stage for increased locoweed consumption later in life.

Cyclic or “on-off” grazing programs have been suggested as a meansof limiting how poisoned livestock become when grazing locoweed-infested pastures combined with locoweed-free pastures. We examinedthe behavioral effects of intermittent locoweed consumption in sheep.Sheep were fed a 10% locoweed pellet for 3- to 5-week periods with 3-to 5-week recovery periods for 22 weeks.

Sheep showed evidence of poisoning after the initial, 4-week “on”locoweed period and never fully recovered behaviorally even though theinitial dose of locoweed was low. Some sheep were sacrificed at the endof locoweed feeding and showed brain lesions, whereas other sheepwere allowed to recover for 5 weeks then euthanized. After five weeksrecovery (“off” locoweed), sheep continued to show persistent behav-ioral signs of locoweed poisoning even though brain lesions had largelyresolved. These findings suggest that even a 4-week low dose oflocoweed may be excessive and could permanently jeopardize animalhealth.

James A. Pfister is a rangeland scien-tist, Bryan L. Stegelmeier is a veterinarypathologist. Kip E. Panter is a repro-ductive toxicologist, Lynn F. James isthe research leader, Michael H. Ralphsis a rangeland scientist, and Dale R.Gardner is a research chemist, all withUSDA’s Agricultural Research Center,Poisonous Plant Research Laboratory inLogan, Utah.

Locoweed Research: Updates and Highlights 1999 23

Low-Level Locoweed ConsumptionAlters Blood Chemistry in SheepJoshua B. Taylor and James R. Strickland

Finding a tolerablelocoweed consumptionlevel for sheep could helpranchers managelocoweed-infestedpastures.

Locoweeds are common legume plants found on rangelands in the west-ern United States. These plants, when consumed in adequate amounts,result in a toxicity syndrome known as “locoism,” which is characterizedby depression, emaciation, abortion, hyperexcitability, lack of coordina-tion, decreased feed intake, and rough hair coats. The toxin apparentlyresponsible for this syndrome is swainsonine.

Large amounts of data have been published concerning locoism.However, the vast majority of the data has been concerned with loco-weed consumption levels that are high enough to cause clinical (visual)locoism. This research has allowed the identification of visual symptomsand some subclinical indications (require laboratory tests to identify) oflocoism. These include alterations in blood chemistry values, such as in-creased alkaline phosphatase activity. In addition, most of the studies todate have resulted in blood swainsonine concentrations of 150 ng/mL orgreater. As such, the issue of low-level locoweed consumption associ-ated with swainsonine levels of <150 ng/mL has not been adequatelyaddressed.

Additionally, most of the research to date has focused on locoweedconsumption in sheep or cattle consuming diets adequate in nutritionalcontent. However, range forage availability during periods of locoweedprevalence may not be adequate to fully meet the animal’s nutritionalrequirements. Therefore, we have conducted experiments designed toaddress the issues of low-level locoweed consumption and nutrientrestriction on range animal health.

Our objective is to define a level of locoweed consumption in sheepeating a nutrient-restrictive diet—animals receive only 65% of theirenergy requirements—that does not cause clinical or subclinical loco-ism. Defining a tolerable locoweed consumption level will allow us tomake more definitive recommendations concerning the use of locoweed-infested rangelands.

To address our objective, we fed 28 sheep energy-restricted dietscontaining varying levels of swainsonine (supplied as locoweed) for 28days. Because swainsonine content varies greatly among different loco-weeds, we fed locoweed on the basis of its swainsonine content. There-fore, our findings are discussed in relation to the levels of swainsoninein the plant and in the animal’s blood. This is necessary if we wish to de-fine a locoweed consumption level that can be tolerated by a grazinganimal. Recovery of sheep from locoweed’s effects was monitored foran additional 21 days following removal from locoweed. We collectedblood (serum) samples 24 hours after feeding and performed immunefunction tests weekly to assess the dose-response effects of locoweed(swainsonine).

The consumption of locoweed by sheep in our trial did not appear toadversely affect the lymphocyte component (a type of white blood cellsincluding T-cells and B-cells) of the animal’s immune system. Lympho-cytes are only a portion of the immune system. Thus, these findings do

24 TOXICOLOGY

not mean that locoweed does not have any adverse effects on the im-mune system as a whole. In fact, in vitro (test-tube) studies previouslyconducted in our laboratory (not reported here) indicate the potentialfor decreased function of T-cells due to swainsonine consumption.

In addition, other researchers have reported effects of locoweed orswainsonine on the immune system of cattle and sheep. Inadequatenutrition can, at least potentially, adversely affect the immune system.Therefore, the lack of an effect in this set of sheep experiments may bethe result of an already compromised immune system. This may pre-clude the chances of detecting effects due to swainsonine on theimmune system. As such, further research is needed to fully exploreswainsonine’s effects on the immune system of range livestock.

Analysis of serum from sheep consuming locoweed confirmed thepresence of swainsonine in the blood (table 1). Taking into account thehalf-life of swainsonine in the blood (about 20 h), serum swainsoninelevels in our experiment only approached or exceeded the serum level ofswainsonine (150 ng/mL) that is known to cause clinical and subclinicallocoism in the two highest dosage levels fed. We succeeded in feedinglocoweed levels that induced serum levels below those previously re-ported. This allowed us to begin studying the effects of low-levelswainsonine consumption on animal health.

Blood was analyzed for the presence of several diagnostic enzymesand nutrients. Alkaline phosphatase (fig. 1), aspartate aminotransferase(fig. 2), and lactate dehydrogenase (table 1) enzyme levels were elevatedin sheep consuming locoweed. Elevations of these enzymes indicate tis-sue damage, including liver and kidney damage. In addition, blood ironand cholesterol levels were decreased. Reductions in serum levels ofthese nutrients may indicate damage to the kidney, liver, and/or gas-trointestinal tract.

Figure 1. Effect of white locoweedconsumption on ovine serumalkaline phosphate activity.Treatments (TRT) were 0, 0.2, 0.4,0.8, and 1.6 mg swainsonine/kgbody weight.

Figure 2. Effect of white locoweedconsumption on ovine serumaspartat amino transferase.Treatments (TRT) were 0, 0.2, 0.4,0.8, and 1.6 mg swainsonine/kgbody weight.

Locoweed Research: Updates and Highlights 1999 25

Although these blood chemistry values indicate that damage occurreddue to locoweed consumption, there is evidence that lower locoweedconsumption levels may be tolerated by the animals. For instance, alka-line phosphatase levels were only elevated in animals consuming loco-weed at doses of >0.4 mg of swainsonine/kg body weight/day. Likewise,cholesterol was only reduced at 1.6 mg of swainsonine/kg body weight/day. Thus, it may be possible to define a locoweed consumption levelbased on swainsonine content that animals tolerate. However, this levelwill most likely be below the levels fed in this study. Establishing alocoweed consumption level that is safe will be useful in developingmanagement systems for using rangelands infested with locoweed.

Joshua B. Taylor is a graduate researchassistant and James R. Strickland is anassistant professor of nutritional toxi-cology, both in the Department ofAnimal and Range Sciences.

Table 1. The effects of white locoweed consuption (28 days) onsheep serum constituents and serum swainsonine 24 hours afterfeeding.

Treatment1

Serum constituents 0 mg 0.2 mg 0.4 mg 0.8 mg 1.6 mg SE

Lactate dehydrogenase (U/L) 390.9 408.3 414.2 454.1 499.2 31.4Iron (µg/dL) 146.8 113.5 94.5 101.9 103.8 9.6

DayCholesterol (mg/dL) 7 90.4 85.7 95.1 96.3 73.3 5.8

14 88.4 87.7 89.1 89.6 63.8 7.021 83.4 84.7 81.0 78.6 55.3 6.728 80.4 79.3 73.6 76.7 60.0 5.4

Swainsonine (ng/mL) 1 ND 15.7 11.8 46.3 96.5 10.07 ND 20.3 16.1 63.4 109.3 14.0

14 ND 16.3 26.0 58.9 62.3 14.021 ND 23.0 17.3 69.4 12.3 11.028 ND 18.0 23.0 66.9 78.0 14.0

1Treatments are 0, 0.2, 0.4, 0.8, and 1.6 mg swainsonine/kg body weight. ND = not detectable.

26 TOXICOLOGY

Looking for Ruminant Bacteriathat Can Detoxify SwainsonineMarilee Morgan and Tammy May

Swainsonine, thetoxicant in locoweeds,is an organic compoundsimilar to a simple sugarand may be used as afood source by micro-organisms found in therumen.

Legumes of the genera Astragalus and Oxytropis found in the southwest-ern United States are commonly referred to as locoweeds. These loco-weeds often are the only forage available to grazing cattle at certaintimes of the year and are as nutritious as alfalfa (figs. 1 and 2). Manyspecies of these genera contain swainsonine, the chemical believed to beresponsible for locoism in grazing cattle. This chemical inhibits impor-tant enzymes involved in glycoprotein (sugar-protein) processing andresults in the accumulation of these compounds in the brain, liver, andkidneys.

Animals that graze locoweed suffer from neurological effects, ema-ciation, reproductive disorders, and eventually, death. The rumen (multi-compartment stomach) of these animals is teeming with diversepopulations of microorganisms with all major bacteria groups repre-sented. Furthermore, swainsonine is similar structurally to mannose, asimple sugar like glucose. It is not unreasonable to assume that a poten-tial for swainsonine metabolism exists in the rumen and may only needto be stimulated.

The goal of our research is to investigate this potential in order toeliminate or decrease the concentration of swainsonine in the rumen and,therefore, reduce its effect on the animal. Improving tolerance of loco-weed would eliminate revenue losses for ranchers from locoism, as wellas provide sufficient forage at no extra cost.

Bovine rumen samples were collected to obtain ruminal microorgan-isms that could grow on the sugar mannose as a preliminary step togrowing them on swainsonine. Mannose and swainsonine share similari-ties in their chemical structures. Swainsonine was not used directly dueto its cost and lack of availability.

Several bacterial types isolated from rumen were obtained for furtherexperiments. Two of the isolates were chosen, based on their differingcell morphologies. A cell growth experiment was performed usingswainsonine by itself (100 g/ml), mannose alone (4 mg/ml) as a viabilitycontrol, and swainsonine + mannose (100 g/ml + 2.4 mg/ml, respec-tively) to determine if an additional carbon (food) source in the form ofmannose was needed for swainsonine to degrade. A control set contain-ing no carbon source also was included.

Swainsonine concentrations were determined indirectly at the begin-ning and end of growth, using an enzyme assay (the α-mannosidase in-hibition assay). The two bacterial isolates grew well when mannose wasadded, but growth on swainsonine alone was insignificant (figs. 3 and4). Additionally, no loss of swainsonine was detected in the cultureswhere it was added (table 1). This experiment was repeated after severaltransfers using media with swainsonine as the sole carbon source toallow the microorganisms to adapt. The same results were observed(data not shown).

A previous study in our lab investigated swainsonine use by Kleb-siella and Pseudomonus species, with swainsonine concentrations at 5.0,

Figure 1. Cattle grazing innorthern New Mexico in earlyspring.

Locoweed Research: Updates and Highlights 1999 27

1.0, 0.5, and 0.25 g/ml. Once more, growth was not observed for any ofthe swainsonine cultures (unpublished data). It is possible that adaptingto swainsonine use may take longer than was allowed in these studies.Additionally, the swainsonine concentrations used may either be toxic tothe organisms or unavailable because it was in such low concentration,the latter being the more probable explanation. Furthermore, the cowused as a source of inoculum for this study was not exposed to locoweedand it would be more appropriate to use ruminal fluid from an animal in-gesting swainsonine. Swainsonine disappearance was not detected withthe analytical method used. The α-mannosidase inhibition assay ishighly variable and hard to reproduce. An alternative or parallel methodof determining swainsonine content is desperately needed to continuethis type of research. The present effort is to develop a reliable methodof swainsonine analysis.

Marilee Morgan is a laboratory techni-cian/research associate and TammyMay is an assistant professor of animalscience, both in the Department of Ani-mal and Range Sciences.

Figure 2. A white locoweed plantsurrounded by dormant, dryfoliage.

Figure 3. Absorbance measured for bacterial type 1 to determine ifswainsonine will support growth.

Figure 4. Absorbance measured for bacterial type 2 to determine ifswainsonine will support growth.