J. Anim. Sci. 2004. 82:2169-2174
© 2004 American Society of Animal Science
The toxicosis of Embellisia fungi from locoweed (Oxytropis lambertii) is similar to locoweed toxicosis in rats1
J. McLain-Romero*,
R. Creamer*,2,
H. Zepeda
,
J. Strickland
,3 and
G. Bell
* Department of Entomology, Plant Pathology, and Weed Science and
and
Department of Animal and Range Sciences, New Mexico State University, Las Cruces 88003 and
and
Del Sol Medical Center, El Paso, TX 79925
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Abstract
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Locoweeds cause significant livestock poisoning and economic loss in the western United States. The toxicity of Embellisia sp. fungi isolated from locoweed was compared with locoweed toxicity using the rat as a model. Rats were fed diets containing locoweed, fungus and alfalfa, or alfalfa. Locoweed- and fungus-fed rats consumed swainsonine-containing food at approximately 1.3 mg•kg–1•d–1, gained less weight (P = 0.001) and ate less than controls. Swainsonine is the principal agent responsible for inducing locoism in animals. The concentrations of alkaline phosphatase and aspartate aminotransferase enzymes were greater (P < 0.05) in serum of locoweed- and fungus-fed rats compared with control rats. Similar intracellular vacuolation was observed in renal, pancreatic, and hepatic tissues of rats that consumed either locoweed or fungus. Rats that ate locoweed or Embellisia fungi displayed indistinguishable toxicity symptoms. The Embellisia fungi from locoweed can induce toxicity without the plants. Locoism management strategies need to involve management of the Embellisia fungi.
Key Words: Endophyte • Fungus • Locoweeds • Swainsonine
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Introduction
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Locoweed toxicity is a widespread disease of livestock in the western United States (James and Panter, 1989
). Many plants in the Astragalus and Oxytropis genera are termed locoweeds owing to their ability to cause the chronic neurological disease locoism in horses, cattle, and sheep. Locoweeds contain the indolizidine alkaloid swainsonine (1,2,8-trihydroxyoctahydroindolizidine), which is the principal agent responsible for inducing locoism in animals (Tulsiani et al., 1984). Swainsonine is a potent inhibitor of lysosomal mannosidase (Tulsiani et al., 1984, 1988) and Golgi mannosidase II (Elbein, 1989). Loss of mannosidase activity in both of these cellular organelles ultimately leads to cellular vacuolation and cellular death. The production of lesions and symptoms in rats by locoweed and swainsonine has been shown to be an acceptable model of locoism in livestock (Stegelmeier et al., 1995).
Swainsonine-producing fungal endophytes, Embellisia sp., isolated from locoweeds (Braun et al., 2003), have been correlated with toxicity of locoweeds (Braun et al., 2003; Gardner et al., 2003). Toxic Astragalus and Oxytropis plants lose toxicity when grown without the fungi (Romero et al., 2002). Swainsonine has been found in several other plants and fungi (Watson et al., 2001; Gardner et al., 2003), but those plants have not been tested for the presence of fungal endophytes. The biological activity of the Embellisia fungi in animals is unknown. The objective of this study was to test whether the endophytic fungi Embellisia sp. can induce locoism by comparing symptoms and pathology of rats fed locoweed plants or fungus equalized for swainsonine dose.
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Materials and Methods
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Animals and Diet
Male albino Sprague-Dawley rats (Zivic Laboratories, Pittsburgh, PA), weighing 188 to 258 g, were fed diets containing commercial rat feed (Rat Chow Laboratory Diet 5001; PMI Nutrition, Brentwood, MO) and locoweed, fungus and alfalfa, or alfalfa for 24 d. Locoweed used in the diet consisted of leaves and small stems of Oxytropis lambertii plants from Winston, NM, and fungal mycelium obtained by culturing Embellisia sp. from those plants. Alfalfa hay was used to balance fiber content for all treatments without locoweed. Plants and fungi were dried overnight at 65°C. Locoweed and alfalfa hay were ground in a Walling mill (0.25 horse power; General Electric, Burlingame, CA) using a 40-mesh screen. Fungus and rat feed were ground with a mortar and pestle. Water and molasses were added to the dry material at 45% and 5% (vol/wt), respectively. Molasses was added to overcome an aversion the rats displayed toward eating feed containing locoweed or fungus. The moist feed mixture was compacted into plastic tubes and dried for 3 h at 65°C. The resulting feed pellets were of the same diameter as the commercial rat feed and held together well. The diets for each of the treatments varied less than 1% in crude protein, fat, fiber, moisture, or ash (Table 1; New Mexico Dept. of Agric., State Chemist Office, Las Cruces, NM). Animal use was approved and followed the guidelines of the institutional animal care and use committee.
Experimental Procedure
Rats were kept in an air-conditioned room with continuous fluorescent lighting. They were individually caged in 24 x 14 x 13 cm polycarbonate cages (Nalgene, Rochester, NY). Diets for 24 rats were organized as a randomized complete block design of three treatments (locoweed, fungus, and control), with eight replicates per treatment and a block of 12 rats (four rats of each treatment) for each kill day. One additional rat was given feed with a different fungal source of higher swainsonine concentration from a preliminary experiment. Rats were fed between 0900 and 1000 each day and allowed ad libitum access to feed and water for 24 d. Rats and feed were weighed daily on an I-10 balance (Ohaus, Florham Park, NJ). Feed was available ad libitum, and daily feed intake was measured by subtracting the amount of feed remaining from the amount of feed left the previous day. Rat behavior was observed each day while weights were taken. On the final day, rats were killed with CO2 at 1300, and blood samples were collected via cardiac puncture with a 16-gauge needle. Livers, kidneys, spleens, pancreas, and lungs were collected.
Histology
Organs were sliced into approximately 0.3-cm thick pieces and placed into neutral buffered 10% formalin for fixation. Tissues were dehydrated, mounted, sectioned, and stained with hematoxylin and eosin (Prophet et al., 1992). Pancreas and kidneys were examined from all 25 rats, and lungs, spleens, and livers were examined from two fungal-treated, two locoweed-treated, and three control rats.
Swainsonine Analysis of Diets and Blood Sera
Swainsonine (Sigma, St Louis, MO) content was determined for the locoweed and fungal components of the feed and the blood sera of the rats by means of a modified 
-mannosidase enzyme assay (Taylor and Strickland, 2002). For locoweed and fungal components, 50 µL of diluted extract or standards was added to 40 µL of citrate buffer (100 mM, pH 4.5) and 10 µL of jack bean -mannosidase was added to all wells of a 96-well microtiter plate except for blank wells. Plates were incubated at 37°C for 10 min with gentle shaking. Then, 40 µL of p-nitrophenyl –D mannopyranoside substrate (10 mM) was added to all wells, and the plates were incubated for 90 min at 37°C with gentle shaking. To stop the reaction, 120 µL of borate buffer (200 mM, pH 9.8) was added per well. The optical density at 405 nm was determined on a plate reader (Emax; Molecular Devices, Sunnyvale, CA). Samples and standards were assessed in duplicate with corresponding blank wells. A sigmoidal curve was developed from a range of swainsonine (Sigma) standards (0.8, 0.4, 0.2, 0.1, 0.05, and 0.025 mg swainsonine/mL) using Softmax Pro software (1997, Molecular Devices). Values for the unknown samples were determined using the standard curve. Only unknowns that fell within the middle of the curve were analyzed.
Serum swainsonine concentration was determined by modification of the previously mentioned enzyme assay (Taylor and Strickland, 2002). One milliliter of serum was boiled (100°C) for 15 min and centrifuged (10,000 x g) for 30 min. Standards were prepared by spiking swainsonine-free boiled serum supernatant with swainsonine (Sigma). Controls were prepared in fresh swainsonine-free serum and analyzed with the samples. The assay was performed by transferring 25 µL of samples, controls, and standards in triplicate to a 96-well microtiter plate. Serum matrix was added as background control to the standards. Eighty-five microliters of citrate buffer was used and 20 µL of a 20 mM substrate solution was used. Serum swainsonine results were analyzed using DeltaSoft 3 version 2.25 software (Biometalics Inc., Princeton, NJ). All other assay procedures were the same as used on the feed components. The ratio of swainsonine in the plant to swainsonine in the fungus on a weight basis was the same in all three -mannosidase tests. Locoweed was added as 10% of the dry weight of the feed and the fungus was added as 5.5% of the dry weight. Hence, swainsonine was calculated as 15.4 ± 3.7 µg/g and 14.1 ± 6.8 µg/g of the locoweed and fungus feed (DM basis), respectively.
Blood Enzyme Analyses
Aspartate aminotransferase content of the blood serum was determined using Infinity AST reagent procedure No. 122-UV (Sigma). The content of alkaline phosphatase was determined with Sigma kit No. 245-20.
Statistical Analyses
Differences in the data obtained from the control, locoweed, and fungus diets were compared using the SAS (SAS Inst., Inc., Cary, NC) GLM procedure with a block for each kill day. The statistical model tested for treatment x kill day interaction. Means separation tests were performed by least significant differences. Rats served as the experimental unit. The means of all three treatments were compared with each other.
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Results and Discussion
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Appearance and Behavioral Changes
Rats displayed differences in weight gain and appetite between treatments. Compared with control, feed intake was less in the fungal treatment using all data (Table 2) and less in the locoweed and fungus treatments when the highest-consuming rat of each treatment was eliminated (Table 2). Aversion to feed containing locoweed has been observed previously in sheep and rats (Stegelmeier et al., 1995; Taylor et al., 2000). Weight gain was less (P = 0.001) in the fungal- and locoweed-treated rats compared with control rats (Table 2). Locoweed and fungus-fed rats did not significantly differ in feed consumption or weight gain. The differences in feed consumption and weight gain between control rats and the rats that received the other treatments are unlikely due to differences in the diets because there was little difference in basic macronutrient content among the diets (Table 1
). Stegelmeier et al. (1995) observed decreased appetite and weight gain in locoweed-fed or swainsonine-injected rats and suggested that it may occur due to neurological damage (Marsh, 1909; Ralphs et al., 1990) or toxic effects on intestinal glycosidases (Pan et al., 1993).
The amount of swainsonine consumed was similar between the locoweed and fungus treatments at 1.4 mg•kg–1•d–1 and 1.3 mg•kg–1•d–1, respectively (Table 2). The amount consumed was calculated as the difference between the initial and final feed weights and included feed eaten and feed dropped to the cage bottom. Stegelmeier et al. (1995) observed clinical anxiety, excitability, and slight intention tremors in rats fed locoweed-containing rodent pellets with a dose as low as 0.92 mg•kg–1•d–1. Such symptoms were not observed in the current study. Our calculation of swainsonine consumed is an estimate because some swainsonine was lost in feed that fell to the cage bottom. All rats displayed anxiousness and tremors when they were weighed, but there was no discernible difference in the behavior of control and locoweed- or fungus-treated rats.
Serum Constituents
Swainsonine was detected in the blood serum of four of the locoweed-treated (
= 0.63 µg/mL) and five of the fungus-treated rats ( = 1.38 µg/mL), but not detected in control rats. Swainsonine is cleared from the blood quickly. We found that the terminal half-life of swainsonine clearance from blood in rats is approximately 8 h in preliminary tests (unpublished data). We did not control for the exact time the rats ate. The rats were fed at 0900 and killed at 1300 on the final days. Any rat that did not eat much the morning they were killed would not have had sufficient swainsonine remaining in their blood to be detected with the -mannosidase assay. In addition, the low dose of swainsonine consumed was close to the limit of detection (25 ng/mL) for the -mannosidase assay. Stegelmeier et al. (1995) did not detect swainsonine in the blood of necropsied rats fed locoweed at a similar swainsonine dose.
The concentration of blood alkaline phosphatase was greater (P < 0.05) in fungus- and locoweed-fed rats when each was compared with the controls but not different between fungus- and locoweed-fed rats (Table 2). The concentration of aspartate aminotransferase was significantly different between the control-fed and fungus-fed rats but not between the control-fed and locoweed-fed rats or the fungus-fed and locoweed-fed rats. Increased blood concentrations of these enzymes indicate soft tissue damage induced by swainsonine ingestion in rats and sheep. Van Kampen and James, 1972) observed that increases in aspartate aminotransferase and alkaline phosphatase corresponded to vacuolation in renal, hepatic, and pancreatic tissues of locoweed-fed yearling ewes. Taylor et al. (2000) concluded that increases in serum alkaline phosphatase and aspartate aminotransferase were indicators of subclinical swainsonine toxicosis. The amount of alkaline phosphatase activity in the blood of sheep has been highly correlated with detected levels of swainsonine within the body (Taylor et al., 2000). Thus, these serum clinical enzyme markers are good indicators of swainsonine poisoning in rat and sheep.
Histology
All rats fed either the locoweed or fungus diet displayed pancreatic, renal, and hepatic vacuolation that was not observed in the control rats. Severe renal vacuolation was observed in the proximal convoluted tubules and, to a lesser extent, in the distal tubules (Figure 1a). Glomeruli seemed unaffected. Severe pancreatic vacuolation was observed in the exocrine centro-acinar cells and other acinar cells (Figure 1c) but not in the endocrine islets of Lagerhans. Within pancreatic cells, vacuolation occurred mostly in the middle of the cytoplasm where Golgi apparati would exist (Figure 1c). Normal pancreatic cells appeared granulated with exocrine granules (Figure 1d). Rats in the locoweed and fungus treatments showed liver vacuolation mostly in the limiting plate near the portal veins and to a lesser extent near the central veins (data not shown). There were differences in the degree of vacuolation observed among rats, but there was not any difference in the location or degree of vacuolation between the locoweed- and fungus-fed rats. No significant vacuolation was observed in organs of the control rats (Figure 1b and d), and no gross lesions were observed on any organs. Photomicrographs (Figures 1a through d) are each from a different rat and are representative of the results observed. Our results are similar to the vacuolation observed by Stegelmeier et al. (1995) in rats when fed locoweed or swainsonine of a similar amount.
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Figure 1. a) Photomicrograph of kidney from a rat given Embellisia diet. Note the vacuolation (*) of proximal convoluted tubular epithelium (PT) and lack of change in the glomerulus (G). b) Photomicrograph of kidney from a rat given control diet. Note the lack of vacuolation in the proximal convoluted tubular epithelium (PT). c) Photomicrograph of pancreatic acinus from a rat given Embellisia diet. Note the vacuolation (*) of the cytoplasm of acinar cells. d) Photomicrograph of pancreatic acinus from a rat given the control diet. Note the lack of vacuolation but the presence of exocrine granules. Sections were stained with hematoxylin and eosin. Bar = 50 µm for (a) and (b) and 5 µm for (c) and (d).
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The Embellisia fungi induced toxicosis that was indistinguishable from locoweed toxicosis in the rat tissues examined. Swainsonine toxicity was manifested in the locoweed- and fungus-fed rats as decreased weight gain, increased serum alkaline phosphatase and increased aspartate aminotransferase, and tissue vacuolation compared with controls. These results are not surprising because the Embellisia fungi produce large quantities of swainsonine in culture (Braun et al., 2003). These data suggest that Embellisa fungal ingestion is capable of inducing locoism toxicity in a rat model animal without any plant contribution. Swainsonine has also been detected in the plants Swainsona, Astragalus, Oxytropis, Sida, and Ipomoea and in the fungi Rhizoctonia leguminicola and Metarhizium anisopliae (Watson et al., 2001). The swainsonine pathway has been partially characterized in Rhizoctonia leguminicola (Wickwire et al., 1990a,b) and in Diablo locoweed (Astragalus oxyphysus; Harris et al., 1988). The synthesis of swainsonine seems to follow a similar pathway in both organisms (Harris et al., 1988; Wickwire et al., 1990a,b); however, Diablo locoweed was not tested for the presence of fungal endophytes (Harris et al., 1998).
Evidence presented herein suggests that Embellisia fungi may be necessary for locoweed plants to be toxic. Importantly, the finding that locoweeds contain swainsonine does not definitively prove that the plants produce swainsonine because swainsonine-producing endophytic fungi have been isolated from those plants. Locoweed toxicity is highly variable among locoweed species, populations, individuals, and seasons (Gardner et al., 2003). When Gardner et al. (2003) compared swainsonine concentrations in 16 populations of O. lambertii, they found that high and low levels of swainsonine in plants were highly correlated with endophyte presence or absence, respectively. Braun et al. (2003) found a correlation between in vitro swainsonine production of an Embellisia isolate and the swainsonine content of the locoweed population in the field. Toxic Astragalus and Oxytropis plants lose toxicity after seed coat removal, which eliminates the Embellisia fungi (Romero et al., 2002).
The concept of an endophytic fungus causing the plant to be toxic is new for the locoweed system, but not new for many poisonous plant systems. Alkaloid-producing fungal endophytes are widespread among grasses (Powell and Petroski, 1992) and trees (Tan and Zou, 2001). Legumes have been thought to produce alkaloid toxins themselves (Janzen, 1971). Bovine toxicoses associated with fescue or ryegrass consumption are caused by endophytic fungi within the forage plants (Joost, 1995). The Embellisia fungi have been found in leaves, stems, flowers, and seeds of Astragalus spp. and Oxytropis spp. (Braun et al., 2003). The fungi may be spread solely through plant seeds or they may also be spread as spores. The fungi do not seem to make spores in planta, but little is known about these fungi or plant and fungal interaction. Locoism can be caused by a fungus within the plants; hence, knowledge of fungal presence and activities are important for designing locoweed management strategies.
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Implications
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Locoweeds (Oxytropis lambertii) and fungi isolated from locoweeds were tested for toxicity to rats, which were used as a model of locoweed toxicity in livestock. The fungus-fed rats displayed symptoms of poisoning that were indistinguishable from those of the locoweed-fed rats. Locoism management strategies need to consider that the activity of the toxic fungi within locoweeds partially or wholly causes the locoweed toxicity.
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Footnotes
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1 The protocol for animal use in this research was reviewed and approved by the Institutional Animal Care and Use Committee, New Mexico State Univ., Las Cruces. This work was funded by the New Mexico Agric. Exp. Stn., and the USDA-CSREES Special Grant No. 59-5428-1-327. We thank A. Clayshulte, R. Ashley, M. Siepel, E. Oldrup, H. Hubble, and A. Moya for their assistance. 
3 Current address: Forage-Animal Production Research Unit, ARS, Lexington, KY 40546.
2 Correspondence—phone: 505-646-3068; fax: 505-646-8087; e-mail: creamer{at}taipan.nmsu.edu.
Received for publication December 19, 2003.
Accepted for publication April 9, 2004.
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Abstract
Introduction
