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Poisoning by Indigofera lespedezioides in horses
Toxicon 60 (2012) 324–328
Contents lists available at SciVerse ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
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 a
Universidade do Estado do Amazonas, Escola Superior de Saúde, Av. Carvalho Leal, 1777, Cachoeirinha, Manaus, AM 69065-001, Brazil Escola Superior Batista do Amazonas, R. Leonor Teles, 153, Adrianópolis, Manaus, AM 69057-510, Brazil c Veterinary Hospital, CSTR, Federal University of Campina Grande, Patos 58700-310, Paraíba, Brazil d USDA, ARS, Poisonous Plant Research Laboratory, 1150 E. 1400 N., Logan, UT 84341, USA e Regional Diagnostic Laboratory, Federal University of Pelotas, 96100-000 Pelotas, RS, Brazil f Universidade Federal do Pará, Campus de Castanhal, Central de Diagnóstico Veterinário, Maximino Porpino da Silva, 1000, Pirapora, Castanhal 68740-080, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 December 2011 Received in revised form 28 March 2012 Accepted 17 April 2012 Available online 25 April 2012
Poisoning by Indigofera lespedezioides is reported in horses in the state of Roraima, northern Brazil. 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 first nervous signs were observed 44 days from the start of grazing. The animal was euthanized on day 59. No significant gross lesions were observed upon necropsies of the experimental horse as well as one spontaneously affected horse. Upon histologic examination neuronal lipofuscinosis was observed in the brain, cerebellum, and spinal cord. Wallerian-type degeneration was observed on some mesencephalic tracts. Neuronal and axonal degeneration and lipofuscinosis were observed on electron microscopy examination. Indospicine was detected in four samples of I. lespedezioides with concentrations ranging from 63 to 1178 mg/g whereas nitro toxins could be detected in only one of the samples at a concentration of 2.5 mg/g. In conclusion, poisoning by I. lespedezioides is very similar to those poisonings by Indigofera linnaei and Indigofera hendecaphylla. Based on the preponderance of indospince and lack of nitro toxins in the samples it is proposed that indospicine is the toxic compound responsible for the poisoning. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Horses Indigofera Indospicine Lipofuscinosis Neuronal degeneration Toxic plants
The Indigofera genus from the Fabaceae family contains approximately 700 different species (Aylward et al., 1987). Indigofera linnaei (¼ Indigofera dominii, Indigofera enneaphylla) 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 nervous signs in horses. In Florida, I. spicata is now regarded as an incorrect identification of the plant, which is now recognized as Indigofera hendecaphylla (Wilson and Rowe, 2008). I. hendecaphylla contains indospicine (Hegarty and Pound, 1968, 1970). There are no references on the indospicine * Corresponding author. Tel.: þ55 83 34239734; fax: þ55 83 34239537. E-mail address: [email protected] (F. Riet-Correa). 0041-0101/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2012.04.341
content in I. linnaei, but Hooper et al. (1971) cite a personal communication from Hegarty and Bolton that they detected indospicine in this plant, Hegarty et al. (1988) showed that horses fed I. linnaei accumulated indospicine in their muscle. It has not been fully demonstrated that indospicine is responsible for the clinical signs in horses; it is suspected that a nitro toxin maybe the cause of the disease (Majak et al., 1992). Indospicine is a liver toxin for dogs and has caused secondary poisoning in dogs ingesting meat from horses (Hegarty et al., 1988; Kelly et al., 1992) and camels (FitzGerald et al., 2011) poisoned by I. linnaei. Indigofera lespedezioides has been associated with a neurologic disease in horses in Roraima (Braga, 1998). The plant is also found in wet-lands in Mato Grosso where it is
E.F. Lima et al. / Toxicon 60 (2012) 324–328
suspected of being toxic for cattle (Pott and Pott, 1994) and fish (Braga, 1998). The objective of this paper is to report the poisoning by I. lespedezioides (¼ Indigofera pascuori) (Fig. 1A and B) in horses in the state of Roraima, northern Brazil, and report on the analyses of indospicine and nitro toxins in the plant. Data on the occurrence of the disease were collected during February 2010 during visits to farms in the affected region and in interviews with veterinary practitioners and farmers in the city of Boa Vista. The disease occurs in the northern region of the state of Roraima in at least five counties (Amajarí, Alto Alegre, Normandia, Cantá, and Bom Fim) and has been recognized by the farmers for more than 20 years. The plant is mostly found in the native vegetation (savanna) known as lavrado, mainly in the borders of the forest. The amount of I. lespedezioides was significantly reduced after pastures were planted primarily with Brachiaria spp. and the disease has ceased to occur in those pastures. In this region of the state of Roraima the climate is tropical with yearly rainfalls of 1100 to 1400 mm. The rainy season with monthly rainfalls of 150–300 mm is from April/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 dry season when I. lespedezioides is nearly the only green vegetation available. Typically, up to 10% of the horses can be affected, but in one case a farmer reported 100%
325
mortality in a herd of 30 horses. Cattle and sheep fed the plant were not affected. The main clinical signs are anorexia, sleepiness, unsteady gait, severe ataxia (Fig. 1C and D), weakness, stumbling, and progressive weight loss. Gait alterations are more marked in the hind limbs with the hind hooves dragging and causing excessive wear of the toes. Eye discharge and blindness are also observed. Some farmers have reported corneal opacity in affected horses. Horses of all ages are affected. If the animals are disturbed or forced to move, nervous signs increase and the animals can fall. Abortion is commonly observed in mares. Death occurs 2–4 months after the observation of first clinical signs. If the plant consumption is interrupted, some animals may recover. To induce the disease experimentally, a 7-year-old horse of the Lavradeiro breed was introduced into a small paddock invaded by the plant. First clinical signs were observed 44 days from the start of grazing. The animal was euthanized on day 59. Clinical signs were weight loss, general weakness, ataxia, hind limb dragging, and sleepiness. One spontaneously affected 10-years-old horse and the experimental animal were necropsied. No significant gross lesions were observed. Fragments of liver, kidney, spleen, heart, mesenteric lymph nodes, lung, thyroid, and large and small intestine and the whole brain and spinal cord were collected and fixed in 10% buffered formalin. After fixation, 1 cm thick serial sections were made from the brain and
Fig. 1. A and B) Indigofera lespedezioides. C and D) Horse spontaneously poisoned by I. lespedezioides showing severe ataxia.
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kept in formalin, for observation of gross lesions. Transverse sections taken from the cervical, thoracic and lumbar spinal cord, medulla oblongata, pons, rostral colliculi, thalamus, internal capsule, cortex, cerebellar peduncles and cerebellum were examined histologically. Longitudinal sections of the spinal cord were also studied. All tissues were embedded in paraffin, sectioned at 4–6 mm, and stained with hematoxylin and eosin and PAS for ceroidlipofuscins. Selected sections of the CNS were also stained with Luxol fast blue for myelin. Within 5–10 min after euthanasia, small fragments of the cerebrum, brain stem, cerebellum, and spinal cord of the experimental horse were fixed in 2% glutaraldehyde with 2% paraformaldehyde in 0.4 M cacodylate buffer (pH 7.4). Blocks were post fixed in 1% osmium tetroxide buffered in 0.4 M sodium cacodylate (pH 7.4), and embedded in Epon 812. Semithin sections were stained with methylene blue. Ultrathin sections were stained with lead citrate and uranyl acetate and examined with an EM 10 Zeiss electron microscope at 60 kV. On histologic examination of the central nervous system of both horses, neurons of the cerebrum, brain stem, spinal cord and cerebellum showed a PAS positive pigment with the characteristics of lipofuscins. Myelin ellipsoids, occasionally with presence of axonal residues and macrophages, suggesting Wallerian-like degeneration were observed in some mesencephalic tracts (Fig. 2). No lesions were observed in other organs examined. In the ultrastructure of the cerebrum, brain stem, cerebellum, and spinal cord different degrees of axonal degeneration characterized by swollen axons, degeneration and disappearance of organelles, neurotubules, and neurofilaments were observed; in some cases the axoplasm was occupied by a flocculent material (Fig. 3A). Concomitantly the number of myelin lamellae decreased and an extraordinary disproportion among the diameter of the axon and the number of lamellae of the myelin sheath was 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) or macrophages (white arrow) are observed in the medial longitudinal fasciculus. HE 200.
the rough endoplasmic reticulum (Fig. 3B); lipofuscin granules ranging from lipoid, membranous and granular appearances (Fig. 3B and C) were also observed. Lipofuscins were also observed in swollen astrocytes, pericytes, and endothelial cells (Fig. 3D). Clinical signs of the neurologic disease observed in horses in Roraima are very similar than those reported in Birdsville disease caused by I. linnaei in Australia (Carroll and Swain, 1983) and in I. hendecaphylla poisoning in US (Morton, 1989). This similarity and the reproduction of the diseases in a horse introduced to a paddock severely invaded by I. lespedezioides after 44 days of grazing confirmed that this is most likely responsible for the poisoning. Gross, histologic, and ultrastructural lesions have not been previously reported in horses poisoned by I. linnaei and I. hendecaphylla. In the poisoning by I. lespedezioides electron microscopy showed neuronal and axonal degeneration. The Wallerian-type degeneration observed in light microscopy (Fig. 2) represents the axonal degeneration observed on electron microscopy. Lipofuscins in different regions of the central nervous system were observed in light microscopy and electron microscopy. Ceroid-lipofuscinosis has been reported as a hereditary lysosomal storage disease of different animal species (Myers et al., 2012). Lipofuscins accumulates in a time-dependent manner in lysosomes of neurons and other cells and are normally observed in old healthy animals (Myers et al., 2012). Lipofuscinosis has been reported in the Purkinje cells in horses with Gomen disease (Hartley et al., 1982). In the poisoning by I. lespedezioides the accumulation of lipofuscins in the central nervous system probably occurs as a consequence of chronic cell injury. Presence of lipofuscins in neurons, astrocytes, and pericytes, and axonal degeneration, are also observed in sheep intoxicated with the plant Halimium brasiliensis (Riet-Correa et al., 2009). One sample of I. lespedezioides collected in the municipality of Bom Fim in 2008, two samples collected in Bom Fim and Amajarí (state of Roaraima) in 2010, and one sample collected in Manaus (state of Amazonas) in 2010 were analyzed for indospicine and nitro toxins (typically glycosides of 3-nitropropanol and 3-nitropropionic acid). The sample from Manaus was from plants collected in Roraima that were then introduced one year before in a place where the neurologic disease has not occurred. Samples were analyzed for indospicine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as reported by Gardner and Riet-Correa (2012). Nitro toxins were analyzed by both Fourier transform infrared spectroscopy spectroscopy (FT-IR) (Schoch et al., 1998) and spectrophotometric methods (Matsumoto et al., 1961; Williams, 1981; Majak et al., 1992). Chemical analysis demonstrated the presence of indospicine in all samples of I. lespedezioides analyzed (Table 1). The concentration ranged from a low of 63 mg/g up to 1178 mg/g. In a previous analysis of I. lespedezioides, Aylward et al. (1987) reported an indospicine concentration of 0.02% (200 mg/g). Nitro toxins were detected only in the sample collected from Amajari. The FT-IR spectrum showed a weak signal at 1556 cm1 indicative of 3-nitropropionic acid. The presence of nitro toxins was verified in the use of a colorimetric assay (Williams, 1981) in which a slightly
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Fig. 3. Electron microscopy. Horse poisoned experimentally by I. lespedezioides. A) Frontal cortex. An axon markedly swollen with flocullar axoplasm, absence of organelles, cytoskelektal elements, and axolema is observed (Bar ¼ 1 mm). Inset: Magnification of myelin sheet showing only two lamellae and absence of the axolema (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 the perikarion. D)Thalamus. Lipofuscin granules are observed in the cytoplasm of endothelial cell of capillary and pericyte (black arrow in endothelial cell end white arrow in pericyte) (Bar ¼ 2 mm).
pink solution was observed but the concentration was below the level of quantitation. To confirm the presence of nitro toxins the samples were analyzed using a third method reported by Matsumoto et al. (1961); only the sample from Amajairi was found to contain a detectable level of nitro toxin at a concentration of 2.5 mg/g as 3-nitropropionic acid equivalents. Majak et al. (1992) reported a slightly lower concentration at 1.5 mg/g 3-NPA in a sample of I. linnaei. I. linnaei and I. hendecaphylla also contain indospicine but it has not been shown that this toxin is responsible for the clinical syndrome. In Australia the disease in horses was treated and prevented with arginine or arginine containing substances (Hooper et al., 1971), and it has been suggested that indospicine may competitively interfere with the Table 1 Content of indospicine and nitro toxins in samples of I. lespedezioides. Sample
Indospicine (mg/g)
Nitro toxins (mg/g)a
Bom Fim (2010) Manaus (2010) Amajari (2010) Bom Fim (2008)
263 63 1178 488
n.d. n.d. 2.5 n.d.
a
Measured as 3-nitropropionic acid equivalents.
incorporation of arginine into proteins due to inhibition of arginase activity and nitric oxide synthase (Madsen and Hegarty, 1970; Pass et al., 1996). The presence of indospicine in the three Indigofera species causing nervous signs in horses highly suggests that this amino acid is responsible for the 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 to experimental animals. Anitro toxin has also been suspected as a cause of the disease (Majak et al., 1992), and similar conditions have been observed in other livestock ingesting nitro toxin-containing plants (Shenk et al., 1976; James et al., 1981), in possums and rats dosed with 3-nitropropionic acid (Hamilton and Gould, 1987; Gregory et al., 2000), and in humans with moldy sugar cane poisoning which is considered a 3-nitropropionic acid toxicosis (Liu et al., 1970; Hu, 1992). However, we found the nitro toxins to be either non-detectable or low compared to known nitro toxic plants such as some Astragalus species and would question if these levels would be toxic as Williams (1981) previously suggested and reported. In conclusion, I. lespedezioides causes nervous signs in horses in the state of Roraima. The toxic amino acid indospicine was detected in all samples collected as well as
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a low concentration of nitro toxin in one sample and thus the toxic compound responsible for the poisoning is proposed to be indospicine. However, the presence of nitro toxins might exasperate the toxicological problems encountered with animals grazing I. lespedezioides. Acknowledgments This work was supported by National Institute for Science and Technology for the Control of Plant Poisonings, CNPq, grant 573534/2008-0. Conflict of interest The authors declare that there are no conflicts of interest. References Aylward, J.H., Court, R.D., Haydock, K.P., Stickland, R.W., Hegarty, M.P., 1987. Indigofera species with agronomic potential in the tropics. Rat toxicity studies. Aust. J. Agric. Res. 38, 177–186. Barbosa, I.B., 1997. Distribuição das chuvas em Roraima. In: Barbosa, R.I., Ferreira, E.J.J., Castellón, E.G. (Eds.), Homem, ambiente e ecologia no Estado de Roraima. INPA, Boa Vista, pp. 326–335. Bell, A.T., Hall, W.T.K., 1952. Birdsville disease of horses: feeding trials with Indigofera enneaphylla. Aust. Vet. J. 28, 141–164. Braga, R.M., 1998. Mata zombando: planta tóxica para equinos em Roraima, vol. 4 (3). Centro de Pesquisa Agroflorestal de Roraima. Informativo, Embrapa, 6 p. Carroll, A.G., Swain, B.J., 1983. Birdsville disease in the Central high-land area of Queensland. Aust. Vet. J. 60, 316–317. FitzGerald, L.M., Fletcher, M.T., Paul, A.E.H., Mansfield, C.S., O’Hara, A.J., 2011. Hepatotoxicosis in dogs consuming a diet of camel meat contaminated with indospicine. Aust. Vet. J. 89, 95–100. Gardner, D.R., Riet-Correa, F., 2012. Analysis of the toxic amino acid indospicine by liquid chromatography-tandem mass spectrometry. Int. J. Poisonous Plant Res. 1, 20–27. Gregory, N.G., Orbell, G.M.B., Harding, D.R.K., 2000. Poisoning with 3nitropropionic acid in possums (Trichosurus vulpecula). New Zeal. Vet. J. 48 (3), 85–87. Hamilton, B.F., Gould, D.H., 1987. Nature and distribution of brain lesions in rats intoxicated with 3-nitropropionic acid: a type of hypoxic (energy deficient) brain damage. Acta Neuropathol. 72 (3), 286–297. Hartley, W.J., Kuberski, G., Legonidec, G., Daynes, P., 1982. The pathology of Gomen disease: a cerebellar disorder of horses in New Caledonia. Vet. Path. 19, 399–405. Hegarty, M.P., Pound, A.W., 1968. Indospicine, a new hepatotoxic amino acid from Indigofera spicata. Nature 217, 354–355.
Hegarty, M.P., Pound, A.W., 1970. Indospicine, a hepatotoxic amino acid from Indigofera spicata: isolation, structure, and biological studies. Aust. J. Biol. Sci. 23, 831–842. Hegarty, M.P., Kelly, W.R., McEwan, D., Williams, O.J., Cameron, R., 1988. Hepatotoxicity to dogs of horsemeat contaminated with indospicine. Aust. Vet. J. 65, 337–340. Hooper, P.T., Hart, B., Smith, G.W., 1971. The prevention and treatment of birdsville disease of horses. Aust. Vet. J. 47, 326–329. Hu, W., 1992. Studies on the epidemiology and etiology of moldy sugarcane poisoning in China. Biomed. Environm. Sci. 5 (2), 161–177. James, L.F., Hartley, W.J., Van Kampen, K.R., 1981. Syndromes of Astragalus poisoning in livestock. J. Am. Vet. Med. Assoc. 178, 146–150. Kelly, W.R., Young, M.P., Hegarty, M.P., Simpson, G.D., 1992. The hepatotoxicity of indospicine in dogs. In: James, L.F., Keeler, R.F., Bailey, E.M., Cheeke, P.R., Hegarty, M.P. (Eds.), Poisonous Plants. Iowa State University Press, Ames, Iowa, pp. 126–130. Liu, X., Luo, X., Madsen, N.P., Christie, G.S., Hegarty, M.P., 1970. Effect of indospicine on incorporation of L-arginine-14C into protein and transfer ribonucleic acid by cell-free systems from rat liver. Biochem. Pharmac. 19, 853–857. Madsen, N.P., Hegarty, M.P., 1970. Inhibition of rat liver homogenate arginase activity in vitro by the hepatotoxic amino acid indospicine. Biochem. Pharmac. 19, 2391–2393. Majak, W., Benn, M., Mcewan, D., Pass, M.A., 1992. Three nitropropanoyl esters of glucose from Indigofera linnaei. Phytochemistry 31, 2393–2395. Matsumoto, H., Unrau, A.M., Hylin, J.W., Temple, B., 1961. Spectrophotometric determination of 3-nitropropanoic acid in biological extracts. Anal. Chem. 33 (10), 1442–1444. Morton, J.F., 1989. Creeping indigo (Indigofera spicata Forsk.) (Fabaceae). A hazard to herbivores in Florida. Econ. Bot. 43, 314–327. Myers, R.K., Zachary, J.F., McGavin, M.D., 2012. Cellular adaptations, injury, and death: morphologic, biochemical, and genetic bases. In: Zachary, J.F., McGavin, M.D. (Eds.), Pathologic Basis of Veterinary Diseases, fifth ed. Elsevier, St Louis, pp. 2–59. Pass, M.A., Hossein, A., Pollitt, S., Hegarty, M.P., 1996. Effects of the naturally occurring arginine analogues indospicine and canavanine on nitric oxide mediated functions in aortic endothelium and peritoneal macrophages. Nat. Toxins 4, 135–140. Pott, A., Pott, V.J., 1994. Plantas Do Pantanal. EMBRAPA-CPAP/SPI, Brasília, 320 p. Riet-Correa, F., Barros, S.S., Mendez, M.C., Fernades, C.G., Pereira Neto, O.A., Soares, M.P., McGavin, D., 2009. Axonal degeneration in sheep caused by ingestion of Halimium brasiliensis. J. Vet. Diagn. Invest. 21, 478–486. Schoch, T.K., Gardner, D.R., Manners, G.D., Pfister, J.A., Ralphs, M.H., 1998. Fourier transform infrared spectroscopy for rapid analysis of toxic compounds in larkspurs and milkvetches. In: Garland, T., Barr, A.C. (Eds.), Toxic Plants and Other Natural Toxicants. CAB International, Wallingford, UK, pp. 243–254. Shenk, J.S., Wagsness, P.J., Leach, R.M., 1976. Relationship between bnitropropionic acid content of crown vetch and toxicity in nonruminant animals. J. An. Sci. 42, 616–621. Williams, M.C., 1981. Nitro compounds in Indigofera species. Agron. J. 73, 434–436. Wilson, P.G., Rowe, R., 2008. A revision of the Indigofereae (Fabaceae) in Australia. 2. Indigofera species with trifoliolate and alternately pinnate leaves. Telopea 12 (2), 293–307.
Contents lists available at SciVerse ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
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 a
Universidade do Estado do Amazonas, Escola Superior de Saúde, Av. Carvalho Leal, 1777, Cachoeirinha, Manaus, AM 69065-001, Brazil Escola Superior Batista do Amazonas, R. Leonor Teles, 153, Adrianópolis, Manaus, AM 69057-510, Brazil c Veterinary Hospital, CSTR, Federal University of Campina Grande, Patos 58700-310, Paraíba, Brazil d USDA, ARS, Poisonous Plant Research Laboratory, 1150 E. 1400 N., Logan, UT 84341, USA e Regional Diagnostic Laboratory, Federal University of Pelotas, 96100-000 Pelotas, RS, Brazil f Universidade Federal do Pará, Campus de Castanhal, Central de Diagnóstico Veterinário, Maximino Porpino da Silva, 1000, Pirapora, Castanhal 68740-080, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 December 2011 Received in revised form 28 March 2012 Accepted 17 April 2012 Available online 25 April 2012
Poisoning by Indigofera lespedezioides is reported in horses in the state of Roraima, northern Brazil. 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 first nervous signs were observed 44 days from the start of grazing. The animal was euthanized on day 59. No significant gross lesions were observed upon necropsies of the experimental horse as well as one spontaneously affected horse. Upon histologic examination neuronal lipofuscinosis was observed in the brain, cerebellum, and spinal cord. Wallerian-type degeneration was observed on some mesencephalic tracts. Neuronal and axonal degeneration and lipofuscinosis were observed on electron microscopy examination. Indospicine was detected in four samples of I. lespedezioides with concentrations ranging from 63 to 1178 mg/g whereas nitro toxins could be detected in only one of the samples at a concentration of 2.5 mg/g. In conclusion, poisoning by I. lespedezioides is very similar to those poisonings by Indigofera linnaei and Indigofera hendecaphylla. Based on the preponderance of indospince and lack of nitro toxins in the samples it is proposed that indospicine is the toxic compound responsible for the poisoning. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Horses Indigofera Indospicine Lipofuscinosis Neuronal degeneration Toxic plants
The Indigofera genus from the Fabaceae family contains approximately 700 different species (Aylward et al., 1987). Indigofera linnaei (¼ Indigofera dominii, Indigofera enneaphylla) 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 nervous signs in horses. In Florida, I. spicata is now regarded as an incorrect identification of the plant, which is now recognized as Indigofera hendecaphylla (Wilson and Rowe, 2008). I. hendecaphylla contains indospicine (Hegarty and Pound, 1968, 1970). There are no references on the indospicine * Corresponding author. Tel.: þ55 83 34239734; fax: þ55 83 34239537. E-mail address: [email protected] (F. Riet-Correa). 0041-0101/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2012.04.341
content in I. linnaei, but Hooper et al. (1971) cite a personal communication from Hegarty and Bolton that they detected indospicine in this plant, Hegarty et al. (1988) showed that horses fed I. linnaei accumulated indospicine in their muscle. It has not been fully demonstrated that indospicine is responsible for the clinical signs in horses; it is suspected that a nitro toxin maybe the cause of the disease (Majak et al., 1992). Indospicine is a liver toxin for dogs and has caused secondary poisoning in dogs ingesting meat from horses (Hegarty et al., 1988; Kelly et al., 1992) and camels (FitzGerald et al., 2011) poisoned by I. linnaei. Indigofera lespedezioides has been associated with a neurologic disease in horses in Roraima (Braga, 1998). The plant is also found in wet-lands in Mato Grosso where it is
E.F. Lima et al. / Toxicon 60 (2012) 324–328
suspected of being toxic for cattle (Pott and Pott, 1994) and fish (Braga, 1998). The objective of this paper is to report the poisoning by I. lespedezioides (¼ Indigofera pascuori) (Fig. 1A and B) in horses in the state of Roraima, northern Brazil, and report on the analyses of indospicine and nitro toxins in the plant. Data on the occurrence of the disease were collected during February 2010 during visits to farms in the affected region and in interviews with veterinary practitioners and farmers in the city of Boa Vista. The disease occurs in the northern region of the state of Roraima in at least five counties (Amajarí, Alto Alegre, Normandia, Cantá, and Bom Fim) and has been recognized by the farmers for more than 20 years. The plant is mostly found in the native vegetation (savanna) known as lavrado, mainly in the borders of the forest. The amount of I. lespedezioides was significantly reduced after pastures were planted primarily with Brachiaria spp. and the disease has ceased to occur in those pastures. In this region of the state of Roraima the climate is tropical with yearly rainfalls of 1100 to 1400 mm. The rainy season with monthly rainfalls of 150–300 mm is from April/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 dry season when I. lespedezioides is nearly the only green vegetation available. Typically, up to 10% of the horses can be affected, but in one case a farmer reported 100%
325
mortality in a herd of 30 horses. Cattle and sheep fed the plant were not affected. The main clinical signs are anorexia, sleepiness, unsteady gait, severe ataxia (Fig. 1C and D), weakness, stumbling, and progressive weight loss. Gait alterations are more marked in the hind limbs with the hind hooves dragging and causing excessive wear of the toes. Eye discharge and blindness are also observed. Some farmers have reported corneal opacity in affected horses. Horses of all ages are affected. If the animals are disturbed or forced to move, nervous signs increase and the animals can fall. Abortion is commonly observed in mares. Death occurs 2–4 months after the observation of first clinical signs. If the plant consumption is interrupted, some animals may recover. To induce the disease experimentally, a 7-year-old horse of the Lavradeiro breed was introduced into a small paddock invaded by the plant. First clinical signs were observed 44 days from the start of grazing. The animal was euthanized on day 59. Clinical signs were weight loss, general weakness, ataxia, hind limb dragging, and sleepiness. One spontaneously affected 10-years-old horse and the experimental animal were necropsied. No significant gross lesions were observed. Fragments of liver, kidney, spleen, heart, mesenteric lymph nodes, lung, thyroid, and large and small intestine and the whole brain and spinal cord were collected and fixed in 10% buffered formalin. After fixation, 1 cm thick serial sections were made from the brain and
Fig. 1. A and B) Indigofera lespedezioides. C and D) Horse spontaneously poisoned by I. lespedezioides showing severe ataxia.
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kept in formalin, for observation of gross lesions. Transverse sections taken from the cervical, thoracic and lumbar spinal cord, medulla oblongata, pons, rostral colliculi, thalamus, internal capsule, cortex, cerebellar peduncles and cerebellum were examined histologically. Longitudinal sections of the spinal cord were also studied. All tissues were embedded in paraffin, sectioned at 4–6 mm, and stained with hematoxylin and eosin and PAS for ceroidlipofuscins. Selected sections of the CNS were also stained with Luxol fast blue for myelin. Within 5–10 min after euthanasia, small fragments of the cerebrum, brain stem, cerebellum, and spinal cord of the experimental horse were fixed in 2% glutaraldehyde with 2% paraformaldehyde in 0.4 M cacodylate buffer (pH 7.4). Blocks were post fixed in 1% osmium tetroxide buffered in 0.4 M sodium cacodylate (pH 7.4), and embedded in Epon 812. Semithin sections were stained with methylene blue. Ultrathin sections were stained with lead citrate and uranyl acetate and examined with an EM 10 Zeiss electron microscope at 60 kV. On histologic examination of the central nervous system of both horses, neurons of the cerebrum, brain stem, spinal cord and cerebellum showed a PAS positive pigment with the characteristics of lipofuscins. Myelin ellipsoids, occasionally with presence of axonal residues and macrophages, suggesting Wallerian-like degeneration were observed in some mesencephalic tracts (Fig. 2). No lesions were observed in other organs examined. In the ultrastructure of the cerebrum, brain stem, cerebellum, and spinal cord different degrees of axonal degeneration characterized by swollen axons, degeneration and disappearance of organelles, neurotubules, and neurofilaments were observed; in some cases the axoplasm was occupied by a flocculent material (Fig. 3A). Concomitantly the number of myelin lamellae decreased and an extraordinary disproportion among the diameter of the axon and the number of lamellae of the myelin sheath was 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) or macrophages (white arrow) are observed in the medial longitudinal fasciculus. HE 200.
the rough endoplasmic reticulum (Fig. 3B); lipofuscin granules ranging from lipoid, membranous and granular appearances (Fig. 3B and C) were also observed. Lipofuscins were also observed in swollen astrocytes, pericytes, and endothelial cells (Fig. 3D). Clinical signs of the neurologic disease observed in horses in Roraima are very similar than those reported in Birdsville disease caused by I. linnaei in Australia (Carroll and Swain, 1983) and in I. hendecaphylla poisoning in US (Morton, 1989). This similarity and the reproduction of the diseases in a horse introduced to a paddock severely invaded by I. lespedezioides after 44 days of grazing confirmed that this is most likely responsible for the poisoning. Gross, histologic, and ultrastructural lesions have not been previously reported in horses poisoned by I. linnaei and I. hendecaphylla. In the poisoning by I. lespedezioides electron microscopy showed neuronal and axonal degeneration. The Wallerian-type degeneration observed in light microscopy (Fig. 2) represents the axonal degeneration observed on electron microscopy. Lipofuscins in different regions of the central nervous system were observed in light microscopy and electron microscopy. Ceroid-lipofuscinosis has been reported as a hereditary lysosomal storage disease of different animal species (Myers et al., 2012). Lipofuscins accumulates in a time-dependent manner in lysosomes of neurons and other cells and are normally observed in old healthy animals (Myers et al., 2012). Lipofuscinosis has been reported in the Purkinje cells in horses with Gomen disease (Hartley et al., 1982). In the poisoning by I. lespedezioides the accumulation of lipofuscins in the central nervous system probably occurs as a consequence of chronic cell injury. Presence of lipofuscins in neurons, astrocytes, and pericytes, and axonal degeneration, are also observed in sheep intoxicated with the plant Halimium brasiliensis (Riet-Correa et al., 2009). One sample of I. lespedezioides collected in the municipality of Bom Fim in 2008, two samples collected in Bom Fim and Amajarí (state of Roaraima) in 2010, and one sample collected in Manaus (state of Amazonas) in 2010 were analyzed for indospicine and nitro toxins (typically glycosides of 3-nitropropanol and 3-nitropropionic acid). The sample from Manaus was from plants collected in Roraima that were then introduced one year before in a place where the neurologic disease has not occurred. Samples were analyzed for indospicine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as reported by Gardner and Riet-Correa (2012). Nitro toxins were analyzed by both Fourier transform infrared spectroscopy spectroscopy (FT-IR) (Schoch et al., 1998) and spectrophotometric methods (Matsumoto et al., 1961; Williams, 1981; Majak et al., 1992). Chemical analysis demonstrated the presence of indospicine in all samples of I. lespedezioides analyzed (Table 1). The concentration ranged from a low of 63 mg/g up to 1178 mg/g. In a previous analysis of I. lespedezioides, Aylward et al. (1987) reported an indospicine concentration of 0.02% (200 mg/g). Nitro toxins were detected only in the sample collected from Amajari. The FT-IR spectrum showed a weak signal at 1556 cm1 indicative of 3-nitropropionic acid. The presence of nitro toxins was verified in the use of a colorimetric assay (Williams, 1981) in which a slightly
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Fig. 3. Electron microscopy. Horse poisoned experimentally by I. lespedezioides. A) Frontal cortex. An axon markedly swollen with flocullar axoplasm, absence of organelles, cytoskelektal elements, and axolema is observed (Bar ¼ 1 mm). Inset: Magnification of myelin sheet showing only two lamellae and absence of the axolema (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 the perikarion. D)Thalamus. Lipofuscin granules are observed in the cytoplasm of endothelial cell of capillary and pericyte (black arrow in endothelial cell end white arrow in pericyte) (Bar ¼ 2 mm).
pink solution was observed but the concentration was below the level of quantitation. To confirm the presence of nitro toxins the samples were analyzed using a third method reported by Matsumoto et al. (1961); only the sample from Amajairi was found to contain a detectable level of nitro toxin at a concentration of 2.5 mg/g as 3-nitropropionic acid equivalents. Majak et al. (1992) reported a slightly lower concentration at 1.5 mg/g 3-NPA in a sample of I. linnaei. I. linnaei and I. hendecaphylla also contain indospicine but it has not been shown that this toxin is responsible for the clinical syndrome. In Australia the disease in horses was treated and prevented with arginine or arginine containing substances (Hooper et al., 1971), and it has been suggested that indospicine may competitively interfere with the Table 1 Content of indospicine and nitro toxins in samples of I. lespedezioides. Sample
Indospicine (mg/g)
Nitro toxins (mg/g)a
Bom Fim (2010) Manaus (2010) Amajari (2010) Bom Fim (2008)
263 63 1178 488
n.d. n.d. 2.5 n.d.
a
Measured as 3-nitropropionic acid equivalents.
incorporation of arginine into proteins due to inhibition of arginase activity and nitric oxide synthase (Madsen and Hegarty, 1970; Pass et al., 1996). The presence of indospicine in the three Indigofera species causing nervous signs in horses highly suggests that this amino acid is responsible for the 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 to experimental animals. Anitro toxin has also been suspected as a cause of the disease (Majak et al., 1992), and similar conditions have been observed in other livestock ingesting nitro toxin-containing plants (Shenk et al., 1976; James et al., 1981), in possums and rats dosed with 3-nitropropionic acid (Hamilton and Gould, 1987; Gregory et al., 2000), and in humans with moldy sugar cane poisoning which is considered a 3-nitropropionic acid toxicosis (Liu et al., 1970; Hu, 1992). However, we found the nitro toxins to be either non-detectable or low compared to known nitro toxic plants such as some Astragalus species and would question if these levels would be toxic as Williams (1981) previously suggested and reported. In conclusion, I. lespedezioides causes nervous signs in horses in the state of Roraima. The toxic amino acid indospicine was detected in all samples collected as well as
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