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Toxicon 60 (2012) 324–328

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Toxicon

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Short communication

Poisoning by Indigofera lespedezioides in horses

Everton F. Lima a,b, Franklin Riet-Correa c,*, Dale R. Gardner d, Severo S. Barros e,Rosane M.T. Medeiros c, Mauro P. Soares e, Gabriela Riet-Correa f

aUniversidade do Estado do Amazonas, Escola Superior de Saúde, Av. Carvalho Leal, 1777, Cachoeirinha, Manaus, AM 69065-001, Brazilb Escola Superior Batista do Amazonas, R. Leonor Teles, 153, Adrianópolis, Manaus, AM 69057-510, BrazilcVeterinary Hospital, CSTR, Federal University of Campina Grande, Patos 58700-310, Paraíba, BrazildUSDA, ARS, Poisonous Plant Research Laboratory, 1150 E. 1400 N., Logan, UT 84341, USAeRegional Diagnostic Laboratory, Federal University of Pelotas, 96100-000 Pelotas, RS, BrazilfUniversidade Federal do Pará, Campus de Castanhal, Central de Diagnóstico Veterinário, Maximino Porpino da Silva, 1000, Pirapora, Castanhal 68740-080, Brazil

a r t i c l e i n f o

Article history:Received 20 December 2011Received in revised form 28 March 2012Accepted 17 April 2012Available online 25 April 2012

Keywords:HorsesIndigoferaIndospicineLipofuscinosisNeuronal degenerationToxic plants

* Corresponding author. Tel.: þ55 83 34239734; faE-mail address: franklin.riet@pq.cnpq.br (F. Riet-

0041-0101/$ – see front matter � 2012 Elsevier Ltddoi:10.1016/j.toxicon.2012.04.341

a b s t r a c t

Poisoning by Indigofera lespedezioides is reported in horses in the state of Roraima, northernBrazil. The main clinical signs are anorexia, sleepiness, unsteady gait, severe ataxia,weakness, stumbling, and progressive weight loss. To induce the disease experimentally,a 7-year-old horse was introduced in a small paddock invaded by the plant. The firstnervous signs were observed 44 days from the start of grazing. The animal was euthanizedon day 59. No significant gross lesions were observed upon necropsies of the experimentalhorse as well as one spontaneously affected horse. Upon histologic examination neuronallipofuscinosis was observed in the brain, cerebellum, and spinal cord. Wallerian-typedegeneration was observed on some mesencephalic tracts. Neuronal and axonal degener-ation and lipofuscinosis were observed on electron microscopy examination. Indospicinewas detected in four samples of I. lespedezioides with concentrations ranging from 63 to1178 mg/g whereas nitro toxins could be detected in only one of the samples at a concen-tration of 2.5 mg/g. In conclusion, poisoning by I. lespedezioides is very similar to thosepoisonings by Indigofera linnaei and Indigofera hendecaphylla. Based on the preponderanceof indospince and lack of nitro toxins in the samples it is proposed that indospicine is thetoxic compound responsible for the poisoning.

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The Indigofera genus from the Fabaceae family containsapproximately 700 different species (Aylward et al., 1987).Indigofera linnaei (¼ Indigofera dominii, Indigofera ennea-phylla) in Australia (Bell and Hall, 1952; Hooper et al., 1971;Carroll and Swain, 1983) and Indigofera spicata in Florida(Morton, 1989) have been reported as a cause of nervoussigns in horses. In Florida, I. spicata is now regarded as anincorrect identification of the plant, which is now recog-nized as Indigofera hendecaphylla (Wilson and Rowe, 2008).I. hendecaphylla contains indospicine (Hegarty and Pound,1968, 1970). There are no references on the indospicine

x: þ55 83 34239537.Correa).

. All rights reserved.

content in I. linnaei, but Hooper et al. (1971) cite a personalcommunication from Hegarty and Bolton that they detec-ted indospicine in this plant, Hegarty et al. (1988) showedthat horses fed I. linnaei accumulated indospicine in theirmuscle. It has not been fully demonstrated that indospicineis responsible for the clinical signs in horses; it is suspectedthat a nitro toxin maybe the cause of the disease (Majaket al., 1992). Indospicine is a liver toxin for dogs and hascaused secondary poisoning in dogs ingesting meat fromhorses (Hegarty et al., 1988; Kelly et al., 1992) and camels(FitzGerald et al., 2011) poisoned by I. linnaei.

Indigofera lespedezioides has been associated witha neurologic disease in horses in Roraima (Braga, 1998). Theplant is also found in wet-lands in Mato Grosso where it is

E.F. Lima et al. / Toxicon 60 (2012) 324–328 325

suspected of being toxic for cattle (Pott and Pott, 1994) andfish (Braga, 1998).

The objective of this paper is to report the poisoning byI. lespedezioides (¼ Indigofera pascuori) (Fig. 1A and B) inhorses in the state of Roraima, northern Brazil, and reporton the analyses of indospicine and nitro toxins in the plant.

Data on the occurrence of the disease were collectedduring February 2010 during visits to farms in the affectedregion and in interviews with veterinary practitioners andfarmers in the city of Boa Vista. The disease occurs in thenorthern region of the state of Roraima in at least fivecounties (Amajarí, Alto Alegre, Normandia, Cantá, and BomFim) and has been recognized by the farmers for more than20 years. The plant is mostly found in the native vegetation(savanna) known as lavrado, mainly in the borders ofthe forest. The amount of I. lespedezioides was significantlyreduced after pastures were planted primarily withBrachiaria spp. and the disease has ceased to occur in thosepastures. In this region of the state of Roraima the climate istropical with yearly rainfalls of 1100 to 1400 mm. The rainyseason with monthly rainfalls of 150–300 mm is fromApril/May to August/September. During the dry season,monthly rainfalls are of approximately 50 mm (Barbosa,1997). Most cases of poisoning occur at the end of the dryseason when I. lespedezioides is nearly the only greenvegetation available. Typically, up to 10% of the horses canbe affected, but in one case a farmer reported 100%

Fig. 1. A and B) Indigofera lespedezioides. C and D) Horse spontan

mortality in a herd of 30 horses. Cattle and sheep fed theplant were not affected.

Themain clinical signs are anorexia, sleepiness, unsteadygait, severe ataxia (Fig. 1C and D), weakness, stumbling, andprogressive weight loss. Gait alterations are more marked inthe hind limbs with the hind hooves dragging and causingexcessive wear of the toes. Eye discharge and blindness arealso observed. Some farmers have reported corneal opacityin affected horses. Horses of all ages are affected. If theanimals are disturbed or forced to move, nervous signsincrease and the animals can fall. Abortion is commonlyobserved in mares. Death occurs 2–4 months after theobservation of first clinical signs. If the plant consumption isinterrupted, some animals may recover.

To induce the disease experimentally, a 7-year-old horseof the Lavradeiro breed was introduced into a small paddockinvaded by the plant. First clinical signs were observed 44days from the start of grazing. The animal was euthanizedon day 59. Clinical signs were weight loss, general weak-ness, ataxia, hind limb dragging, and sleepiness.

One spontaneously affected 10-years-old horse and theexperimental animal were necropsied. No significant grosslesions were observed. Fragments of liver, kidney, spleen,heart, mesenteric lymph nodes, lung, thyroid, and large andsmall intestine and the whole brain and spinal cord werecollected and fixed in 10% buffered formalin. After fixation,1 cm thick serial sections were made from the brain and

eously poisoned by I. lespedezioides showing severe ataxia.

E.F. Lima et al. / Toxicon 60 (2012) 324–328326

kept in formalin, for observation of gross lesions. Trans-verse sections taken from the cervical, thoracic and lumbarspinal cord, medulla oblongata, pons, rostral colliculi,thalamus, internal capsule, cortex, cerebellar pedunclesand cerebellumwere examined histologically. Longitudinalsections of the spinal cord were also studied. All tissueswere embedded in paraffin, sectioned at 4–6 mm, andstained with hematoxylin and eosin and PAS for ceroid-lipofuscins. Selected sections of the CNS were also stainedwith Luxol fast blue for myelin.

Within 5–10 min after euthanasia, small fragments ofthe cerebrum, brain stem, cerebellum, and spinal cord ofthe experimental horse were fixed in 2% glutaraldehydewith 2% paraformaldehyde in 0.4 M cacodylate buffer(pH 7.4). Blocks were post fixed in 1% osmium tetroxidebuffered in 0.4 M sodium cacodylate (pH 7.4), andembedded in Epon 812. Semithin sections were stainedwith methylene blue. Ultrathin sections were stained withlead citrate and uranyl acetate and examinedwith an EM 10Zeiss electron microscope at 60 kV.

On histologic examination of the central nervous systemof both horses, neurons of the cerebrum, brain stem, spinalcord and cerebellum showed a PAS positive pigment withthe characteristics of lipofuscins. Myelin ellipsoids, occa-sionally with presence of axonal residues andmacrophages,suggesting Wallerian-like degeneration were observedin some mesencephalic tracts (Fig. 2). No lesions wereobserved in other organs examined.

In the ultrastructure of the cerebrum, brain stem,cerebellum, and spinal cord different degrees of axonaldegeneration characterized by swollen axons, degenerationand disappearance of organelles, neurotubules, and neu-rofilaments were observed; in some cases the axoplasmwas occupied by a flocculent material (Fig. 3A). Concomi-tantly the number of myelin lamellae decreased and anextraordinary disproportion among the diameter of theaxon and the number of lamellae of the myelin sheathwas seen (Fig. 3A). In the perikarion of numerous neurons,the mitochondria were swollen with disorganization,disruption, and disappearance of cristae; degranulation of

Fig. 2. Brain stem. Telencephalon. Horse poisoned by I. lespedezioides.Numerous myelin ellipsoids, some with axonal remnants (black arrow) ormacrophages (white arrow) are observed in the medial longitudinal fascic-ulus. HE �200.

the rough endoplasmic reticulum (Fig. 3B); lipofuscingranules ranging from lipoid, membranous and granularappearances (Fig. 3B and C) were also observed. Lipofuscinswere also observed in swollen astrocytes, pericytes, andendothelial cells (Fig. 3D).

Clinical signs of the neurologic disease observed inhorses in Roraima are very similar than those reported inBirdsville disease caused by I. linnaei in Australia (Carrolland Swain, 1983) and in I. hendecaphylla poisoning in US(Morton, 1989). This similarity and the reproduction of thediseases in a horse introduced to a paddock severely invadedby I. lespedezioides after 44 days of grazing confirmed thatthis is most likely responsible for the poisoning.

Gross, histologic, and ultrastructural lesions have notbeen previously reported in horses poisoned by I. linnaei andI. hendecaphylla. In the poisoning by I. lespedezioides electronmicroscopy showed neuronal and axonal degeneration. TheWallerian-type degeneration observed in light microscopy(Fig. 2) represents the axonal degeneration observed onelectron microscopy. Lipofuscins in different regions of thecentral nervous system were observed in light microscopyand electron microscopy. Ceroid-lipofuscinosis has beenreported as a hereditary lysosomal storage disease ofdifferent animal species (Myers et al., 2012). Lipofuscinsaccumulates in a time-dependent manner in lysosomes ofneurons and other cells and are normally observed in oldhealthy animals (Myers et al., 2012). Lipofuscinosis has beenreported in the Purkinje cells in horses with Gomen disease(Hartley et al., 1982). In the poisoning by I. lespedezioides theaccumulation of lipofuscins in the central nervous systemprobably occurs as a consequence of chronic cell injury.Presence of lipofuscins in neurons, astrocytes, and pericytes,and axonal degeneration, are also observed in sheepintoxicatedwith the plantHalimium brasiliensis (Riet-Correaet al., 2009).

One sample of I. lespedezioides collected in the munici-pality of BomFim in 2008, two samples collected in BomFimand Amajarí (state of Roaraima) in 2010, and one samplecollected in Manaus (state of Amazonas) in 2010 wereanalyzed for indospicine and nitro toxins (typically glyco-sides of 3-nitropropanol and 3-nitropropionic acid). Thesample from Manaus was from plants collected in Roraimathat were then introduced one year before in a place wherethe neurologic disease has not occurred. Samples wereanalyzed for indospicine by liquid chromatography-tandemmass spectrometry (LC-MS/MS) as reported by Gardnerand Riet-Correa (2012). Nitro toxins were analyzed byboth Fourier transform infrared spectroscopy spectroscopy(FT-IR) (Schoch et al., 1998) and spectrophotometricmethods (Matsumoto et al., 1961; Williams, 1981; Majaket al., 1992).

Chemical analysis demonstrated the presence of indo-spicine in all samples of I. lespedezioides analyzed (Table 1).The concentration ranged from a low of 63 mg/g up to1178 mg/g. In a previous analysis of I. lespedezioides, Aylwardet al. (1987) reported an indospicine concentration of 0.02%(200 mg/g). Nitro toxins were detected only in the samplecollected from Amajari. The FT-IR spectrum showed a weaksignal at 1556 cm�1 indicative of 3-nitropropionic acid.The presence of nitro toxins was verified in the use ofa colorimetric assay (Williams, 1981) in which a slightly

Fig. 3. Electron microscopy. Horse poisoned experimentally by I. lespedezioides. A) Frontal cortex. An axon markedly swollen with flocullar axoplasm, absence oforganelles, cytoskelektal elements, and axolema is observed (Bar ¼ 1 mm). Inset: Magnification of myelin sheet showing only two lamellae and absence of theaxolema (Bar ¼ 100 nm). B) Cerebellum. Purkinje cell. Swollen mitochondria with disorganization, rupture and lysis of cristae (black arrow) are observed.Lipofuscin eletrodense granules are present in the perikarion (white arrows). Degranulation is observed in the rough endoplasmic reticulum (Bar ¼ 2 mm).C) Cervical spinal cord. A neuron showing swollen mitochondria and damage cristae (arrow) is observed. Numerous lipofuscin granules are observed in theperikarion. D)Thalamus. Lipofuscin granules are observed in the cytoplasm of endothelial cell of capillary and pericyte (black arrow in endothelial cell end whitearrow in pericyte) (Bar ¼ 2 mm).

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pink solution was observed but the concentrationwas below the level of quantitation. To confirm thepresence of nitro toxins the samples were analyzed usinga third method reported by Matsumoto et al. (1961); onlythe sample fromAmajairi was found to contain a detectablelevel of nitro toxin at a concentration of 2.5 mg/g as3-nitropropionic acid equivalents. Majak et al. (1992)reported a slightly lower concentration at 1.5 mg/g 3-NPAin a sample of I. linnaei.

I. linnaei and I. hendecaphylla also contain indospicinebut it has not been shown that this toxin is responsible forthe clinical syndrome. In Australia the disease in horses wastreated and prevented with arginine or arginine containingsubstances (Hooper et al., 1971), and it has been suggestedthat indospicine may competitively interfere with the

Table 1Content of indospicine and nitro toxins in samples of I. lespedezioides.

Sample Indospicine (mg/g) Nitro toxins (mg/g)a

Bom Fim (2010) 263 n.d.Manaus (2010) 63 n.d.Amajari (2010) 1178 2.5Bom Fim (2008) 488 n.d.

a Measured as 3-nitropropionic acid equivalents.

incorporation of arginine into proteins due to inhibition ofarginase activity and nitric oxide synthase (Madsen andHegarty, 1970; Pass et al., 1996). The presence of indospi-cine in the three Indigofera species causing nervous signs inhorses highly suggests that this amino acid is responsible forthe clinical signs of the disease as suggested previously(Hegarty and Pound, 1968; Hooper et al., 1971). However,the disease has not been reproduced dosing indospicine toexperimental animals. Anitro toxin has also been suspectedas a cause of the disease (Majak et al., 1992), and similarconditions have been observed in other livestock ingestingnitro toxin-containing plants (Shenk et al.,1976; James et al.,1981), in possums and rats dosed with 3-nitropropionicacid (Hamilton and Gould, 1987; Gregory et al., 2000), andin humans with moldy sugar cane poisoning which isconsidered a 3-nitropropionic acid toxicosis (Liu et al., 1970;Hu, 1992). However, we found the nitro toxins to be eithernon-detectable or low compared to knownnitro toxic plantssuch as some Astragalus species and would question ifthese levels would be toxic as Williams (1981) previouslysuggested and reported.

In conclusion, I. lespedezioides causes nervous signsin horses in the state of Roraima. The toxic amino acidindospicine was detected in all samples collected as well as

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a low concentration of nitro toxin in one sample and thusthe toxic compound responsible for the poisoning isproposed to be indospicine. However, the presence of nitrotoxins might exasperate the toxicological problemsencountered with animals grazing I. lespedezioides.

Acknowledgments

This work was supported by National Institute forScience and Technology for the Control of Plant Poisonings,CNPq, grant 573534/2008-0.

Conflict of interest

The authors declare that there are no conflicts ofinterest.

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