Clinical and pathological comparison of Astragalus lentiginosus and Ipomoea carnea poisoning in goats

Journal Pre-proof Clinical and pathological comparison of Astragalus lentiginosus and Ipomoea carnea poisoning in goats Louisiane de Carvalho Nunes, Bryan L. Stegelmeier, Daniel Cook, James A. Pfister, Dale R. Gardner, Franklin Riet-Correa, Kevin D. Welch PII:

S0041-0101(19)30458-1

DOI:

https://doi.org/10.1016/j.toxicon.2019.09.016

Reference:

TOXCON 6211

To appear in:

Toxicon

Received Date: 12 July 2019 Revised Date:

12 September 2019

Accepted Date: 16 September 2019

Please cite this article as: de Carvalho Nunes, L., Stegelmeier, B.L., Cook, D., Pfister, J.A., Gardner, D.R., Riet-Correa, F., Welch, K.D., Clinical and pathological comparison of Astragalus lentiginosus and Ipomoea carnea poisoning in goats, Toxicon (2019), doi: https://doi.org/10.1016/j.toxicon.2019.09.016. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Graphical Abstract

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Clinical and pathological comparison of Astragalus lentiginosus and Ipomoea carnea

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poisoning in goats.

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Louisiane de Carvalho Nunes1, Bryan L. Stegelmeier2*, Daniel Cook2*, James A. Pfister2, Dale

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R. Gardner2; Franklin Riet-Correa3,4, Kevin D. Welch2

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Espírito Santo, Brazil

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2

USDA-ARS Poisonous Plant Research Laboratory, 1150 E. 1400 N., Logan, Utah, USA 84341

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Instituto Nacional de Investigación Agropecuaria, La Estanzuela, Colonia, Uruguay

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Veterinary Hospital, Center for Health and Rural Technology, Patos Campus, Federal

Professor, Department of Veterinary Science, Federal University of Espírito Santo, Alegre,

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University of Campina Grande, Patos, Brazil

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*Corresponding Authors

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Abstract: The indolizidine alkaloid swainsonine, found in some Astragalus and Oxytropis (i.e.,

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locoweed) species, is a potent cellular glycosidase inhibitor that often poisons livestock. Other

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toxic genera such as some Ipomoea species also contain swainsonine as well as calystegines

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which are similar polyhydroxy alkaloids. The toxicity of calystegines is poorly characterized;

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however, they are also potent glycoside inhibitors capable of intestinal and cellular glycoside

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dysfunction. The objective of this study was to directly compare A. lentiginosus and I. carnea

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poisoning in goats to better characterize the role of the calystegines. Three groups of four goats

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each were treated with ground alfalfa (control), I. carnea or A. lentiginosus to obtain daily doses

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of 0.0, 1.5, and 1.5 mg swainsonine/kg bw per day, respectively, for 45 days. Animals were

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observed daily and weekly body weights, serum enzyme activities, and serum swainsonine

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concentrations were determined. At day 45 all animals were euthanized and necropsied. Goats

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treated with A. lentiginosus and I. carnea developed clinical disease characterized by mild

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intention tremors and proprioceptive deficits. Goats treated with A. lentiginosus developed

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clinical disease sooner and with greater consistency. No differences in body weight, serum

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swainsonine concentrations and serum enzyme activity were observed between goats treated

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with A. lentiginosus and I. carnea. Additionally, there were no differences in the microscopic

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and histochemical studies of the visceral and neurologic lesions observed between goats treated

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with A. lentiginosus and I. carnea. These findings suggest that I. carnea-induced clinical signs

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and lesions are due to swainsonine and that calystegines contribute little or nothing to toxicity in

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goats in the presence of swainsonine.

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Keywords: swainsonine; calystegines; Ipomoea; Astragalus; locoweed

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1. Introduction Certain toxic plants induce neurologic disease similar to genetic mannosidosis. The plant

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toxins that produce this disease have been identified as polyhydroxy alkaloids that inhibit

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specific cellular glycosidases. Of these, the indolizidine alkaloid swainsonine is the best

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characterized and is a potent inhibitor of lysosomal α-mannosidase and Golgi mannosidase II

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(Colegate et al. 1979; Dorling et al. 1980; Elbein et al. 1981). Poisoning requires extended

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exposure that continuously inhibits enzymatic function producing intracellular accumulations of

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abnormal oligosaccharides and glycoproteins. Intoxication is best characterized by neurovisceral

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vacuolation with subsequent neurologic disease (i.e., locoism). Poisoning has been identified

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and characterized in grazing animals, experimental dosing studies in livestock with Astragalus

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and Oxytropis plant material, and in rodents using purified swainsonine (James et al. 1970;

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Stegelmeier et al. 1995; 1999a; 2008).

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Swainsonine is reported to occur in three plant families; the Convolvulaceae, Fabaceae,

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and Malvaceae; the genera include some species of Ipomoea, Turbina, Swainsona, Astragalus,

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Oxytropis, and Sida (Colegate et al. 1979; Molyneux and James; 1982; Molyneux et al. 1995;

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Colodel et al. 2002; Cook et al. 2014). Swainsonine is produced by vertically transmitted fungal

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symbionts associated with each respective species (Braun et al. 2003; Cook et al 2013; Grum et

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al. 2013; Cook et al. 2014). Swainsonine concentrations are highly variable among individual

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plants within a population, among different populations of the same species, and among different

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species (Cook et al. 2014; Cook et al. 2015).

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Calystegines are polyhydroxy alkaloids that inhibit glycosidases that have been

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associated with animal poisoning (Asano et al. 1997; Schimming et al. 2005; Eich, 2008).

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Calystegines are reported to occur in three major plant families, the Convolvulaceae, Moraceae,

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and Solanaceae (Asano et al. 1997; Schimming et al. 2005; Eich, 2008). In addition to

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calystegines, some Ipomoea species contain swainsonine; for example, Ipomoea carnea is

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reported to contain swainsonine and mixtures of calystegines (B1, B2, B3 and C1) (Molyneux et

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al. 1995; de Balogh et al. 1999). In vitro studies suggest that many calystegines are potent

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glycosidase inhibitors that could disrupt intestinal glycosidases, lysosomal function and

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glycoprotein processing (Asano et al. 1997; Jockovic et al. 2013). Clinical poisoning from

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Ipomoea carnea has been reported in Brazil, Sudan, India, and Mozambique (Idris et al. 1973;

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Tartour et al. 1973; Tirkey et al. 1987; de Balogh et al. 1999; Armien et al. 2007).

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Experimentally reproduced intoxication has been reported in goats, sheep and cattle (Adam et al.

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1973; Idris et al. 1973; Damir et al. 1987; Armien et al. 2007; 2011). However, I. carnea

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intoxication has been a challenge to definitively characterize, as poisoning is often sporadic as

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the plant is not highly palatable (Oliveira et al. 2015). Toxin concentrations in I. carnea also

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vary between plants and plant populations; for example, swainsonine concentrations in

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individual plants range from not being detected to 0.2% (Cook et al. 2015). The reported clinical

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signs of toxicity and distribution of lesions associated with I. carnea often differ as the

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experimental dose animals received was not standardized in terms of swainsonine or calystegine

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concentrations. For example, animals were dosed with fresh material that was not homogenous,

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or treated animals were group fed thus the resulting dose and duration were poorly defined.

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Ipomoea species as well as Astragalus and Oxytropis species contain swainsonine

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concentrations that are potentially toxic (Armien et al. 1997; Stegelmeier et al. 1999a; Cook et al.

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2014). Little is known about how calystegines from I. carnea contribute to toxicity, or if they

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alter toxicity. More research is needed to better characterize I. carnea-induced disease and to

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clarify the role that calystegines play in poisoning. Thus, the objective of this study was to

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directly compare poisoning in goats dosed with plant material from swainsonine-containing A.

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lentiginosus, and I. carnea containing both swainsonine and calystegines.

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2. Material and Methods

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2.1. Experimental Design

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Animal work was done under veterinary supervision following IACUC protocol 2437 that was

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approved by the Utah State University Institutional Animal Care and Use Committee. Twelve,

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eight-month-old wether Spanish goats weighing 20.9±1.2 kg (mean ± sd) were randomly divided

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into three groups of four animals. The goats were weighed and bled weekly by venipuncture of

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the jugular vein, and whole blood and serum was collected and frozen for later analysis. After a

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three day acclimation period the goats were treated twice a day via intra-ruminal oral gavage

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with finely ground alfalfa, I. carnea, or A. lentiginosus for 45 days. The gavage was done by

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passing a 1.5 cm guarded plastic tube down the esophagus and hand pumping a slurry of the

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plant material mixed with 1 to 1.5 L warm water into the rumen. The amount of ground plant

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was adjusted weekly after the goats were weighed and the individual animal doses were adjusted

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to obtain doses of 1.5 mg swainsonine/kg bw per day for both the A. lentiginosus and I. carnea

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groups. The goats dosed with I. carnea received an estimated dose of 2.6 mg calystegines/kg bw

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per day (quantitated relative to swainsonine). The control group treated with ground alfalfa was

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dosed with similar plant amounts (g/kg BW) as the I. carnea-treated goats. In addition to the

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treatments, all animals had access to both alfalfa hay and water ad libitum. At necropsy the

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entire body, brain, liver, and kidneys were weighed. The brain and spinal cord and portions of

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heart, liver, kidney, thyroid glands, adrenal gland, pancreas, salivary gland, third eyelid with

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lymph and lacrimal tissue, rumen, abomasum, duodenum, jejunum, ileum, cecum, colon, urinary

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bladder and semimembranosus skeletal muscle were collected and fixed in 10% neutral buffered

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formalin.

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2.2. Plant Collection and Analysis

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Astragalus lentiginosus Douglas var. diphysus (A. Gray) M.E. Jones was collected in

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Arizona (34° 24′N 109° 13′W; PPRL collection 98-2) and vouchers of that collection are

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cataloged in the Utah State University herbarium (UTC 23793) (Figure 1). Ipomoea carnea

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Jacq. subsp. fistulosa (Mart. ex Choisy) D.F. Austin seeds were collected in April 2011 near the

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veterinary hospital of the University of Campina Grande, Campus of Patos in the city of Patos,

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Paraíba, Brazil (S 7° 04' 02" W 37° 16' 53") (UTC 00260470) (Figure 1). Plants derived from

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the above-mentioned seeds were grown in a greenhouse in Logan, Utah with a 16 hour

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photoperiod and day/night temperatures of 25 °C/ 20 °C. Leaves from the plants were harvested

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and air dried at 40 °C. Alfalfa (Medicago sativa), third crop field dried hay, was used as a

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negative control. All the plant material was finely ground using a Wiley mill to pass through a 2

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mm screen to facilitate dosing. After thorough mixing the plant material was sampled and

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analyzed for swainsonine using previously described techniques (Gardner et al. 2001; Gardner

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and Cook 2011). Plant material was analyzed for calystegines with slight modification to that

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previously reported (Cook et al., 2015) in that calystegines (B1, B2, B3 and C1) were quantitated

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based on peak areas relative to the swainsonine peak area as opposed to castanospermine.

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2.3. Serum Biochemistry and Swainsonine Analysis

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Selected sera samples were analyzed using an automated biochemistry analyzer (Hitachi

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7180, Hitachi High Technologies Inc. Pleasanton, CA) for albumin, total protein, blood urea

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nitrogen, creatinine, sodium, potassium, chloride, carbon dioxide, alanine transaminase (ALT),

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aspartate transaminase (AST), alkaline phosphatase (ALP) and bilirubin using reagents and

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procedures recommended by the instrument supplier. Serum swainsonine concentrations were

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determined using a modification of previously described techniques (Gardner and Cook 2016).

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An aliquot of sera (0.500 mL) was mixed with 0.500 mL of 0.2% H3PO4. Solid phase extraction

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(SPE) tubes (Strata X-C, 60 mg, Phenomenex) were prepared by rinsing with 2 mL methanol, 2

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mL water and 1 mL of 0.1% H3PO4. Samples were loaded under vacuum (slowly) and then

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rinsed with 2 mL 0.2% H3PO4 and then dried under vacuum for 2-3 minutes. SPE columns were

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then rinsed with 2 mL water and 2 mL methanol. The swainsonine was eluted with 3 mL of

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ammoniated methanol and the samples dried under a flow of nitrogen at 60°C. To the samples

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was added 0.200 mL of pyridine and 0.050 mL of BSTFA (N,O-bis(trimethylsilyl)

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trifluoroacetamide) silyation reagent and samples were capped and heated at 60˚C for 30 min.

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After cooling 0.750 mL of DMF (dimethylformamide) was added and sample transferred to

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HPLC autosample vials. Calibration standards were prepared from a 0.100 mL aliquot of a

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standard swainsonine solution (50 ppm) added to a vial containing 3 mL of ammoniated

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methanol. The sample was evaporated to dryness and 0.200 mL of pyridine and 0.050 mL of

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BSTFA silyation reagent was added and sample heated at 60˚C for 30 min. After cooling 1.00

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mL of DMF was added and then a 0.100 mL aliquot was added to 1.90 mL of DMF and serially

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diluted to give standards at 200, 100, 50, 25, 12.5, 6.25 and 3.1 ng/mL. Samples and standards

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were then analyzed by HPLC-HRMS using an Exactive Plus high resolution mass spectrometer

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coupled with a heated electrospray ion source, Ultimate 3000 HPLC and autosampler (Thermo

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Scientific) and Kinetex EVO C18 (50 x 2.1 mm) column. Elution solvents were a gradient

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mixture of 20 mM ammonium acetate (A) and acetonitrile (B) at a flow rate of 0.400 mL/min

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starting at 50% B (0-1 min) with a linear increase to 98% B (1 -10 min). Detection of the

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protonated swainsonine-(SiCH3)3 derivative was from selected ion chromatograms for m/z

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390.2311 with a retention time of approximately 6 minutes under the describe chromatographic

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conditions.

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2.4. Microscopic Analysis

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Small sections of the fixed brain, spinal cord, lung, heart, liver, kidney, thyroid, adrenal,

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pancreas, salivary gland, lacrimal gland, rumen, abomasum, small intestine, colon, and

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semimembranosus skeletal muscle were trimmed and embedded in paraffin and sectioned for

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microscopic analysis. Particular care was taken in sectioning the brain with survey samples of

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the parietal lobe of the cerebral cortex, corpus callosum, thalamus and hypothalamus, corpora

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quadrigemina, mesencephalon, medulla oblongata, obex and spinal cord sections of cervical,

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thoracic, lumbar and sacral regions. The cerebellum was sectioned separately making sequential

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4 mm sections beginning at a randomly selected distance (0-3 mm) from the posterior edge of the

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central lobule. This resulted in 3-4 sagittal sections of the lobule that were used for

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morphometric analysis (Weibel 1963). Other than the cerebellum and particularly the central

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cerebellar lobe, the other tissues were examined and subjectively evaluated using basic histologic

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hematoxylin and eosin staining (Carson 1997). However, a modified Bodian silver stain and a

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luxol fast blue/periodic acid Schiff stains were also used on select sections to evaluate axonal

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changes, identify missing neurons and better characterize mild myelin changes (Gato 1987,

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Yamamoto and Hirano 1986).

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Two morphometric studies were undertaken. The first was an estimate of the extent of

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Purkinje cell change. This was done by evaluating 10 random fields of the cerebellar cortex at

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40x magnification and counting the total number of Purkinje cells as well as the number of those

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cells that were pyknotic or vacuolated. The cerebellar lobes examined were randomly selected

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sections of cerebellar cortex that as indicated previously were randomly sectioned. The results

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were reported as the proportion or percentage of vacuolated or pyknotic nucleated Purkinje cells.

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The second study was to determine if there were population differences in cerebellar neuron

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numbers or size between the treated groups. As the central lobe is the most consistently affected

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cerebellar lobe in locoweed poisoning (Stegelmeier et al. 1999a) these studies were done by

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measuring in µm the length of the granular and molecular layer interface in all of the sectioned

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folia of that lobe. The number of Purkinje cells that included an intact nucleus along this

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interface were counted and the number was reported as µm interface per Purkinje cell. The

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second measurement was to count the number of pyknotic Purkinje cells along that same length

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of the central cerebellar lobe. These were also reported as µm per pyknotic Purkinje cell. The

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final measurement was to measure the area of the nucleated Purkinje cells along this same length

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of central cerebellar lobe. This was reported as µm2 per nucleated cell. These measurements

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were done using an Olympus BH1 microscope with a DP73 digital camera and CellSens 1.16

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software (Olympus America Center Valley PA). The system was calibrated using a Zeiss 5

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100/100 mm micrometer (Carl Zeiss Inc, Thornwood NY).

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2.5. Statistical Analysis

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Serum blood chemistry variables, serum swainsonine concentrations, and animal body weights

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were analyzed as repeated measures in a linear mixed model (Proc Mixed, SAS 13.1, SAS Inst.

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Inc., Cary, NC). The model included treatment (Controls vs. I. carnea vs. A. lentiginosus), date,

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and the treatment x date interaction. Animal and date were random factors in the model.

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Generally compound symmetry was the best fitting covariance structure as determined by

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comparing models with Akaike’s Information Criteria (AIC). Least square means were used for

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all comparisons, and the PDIFF procedure in SAS 13.1 was used with preplanned comparisons to

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evaluate the treatment x date interaction. Treatment effects on organ weights and Purkinje

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cellular measurements were compared using analysis of variance (Proc GLM, SAS Inc., Cary,

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NC) with significance determined at P < 0.05. The proportion of pyknotic or vacuolated

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Purkinje cells was compared using a one-way ANOVA to detect treatment differences.

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3. Results and Discussion

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Swainsonine concentrations in A. lentiginosus and I. carnea were 0.16% and 0.04%,

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respectively. Calystegine concentrations in I. carnea were 0.07% and were not detected in A.

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lentiginosus. These concentrations are consistent with what has been reported for field

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collections of each respective species (Haraguchi et al. 2003; Schimming et al. 2005; Cook et al.

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2011; Cook et al. 2015). All goats tolerated dosing well and no animals developed any adverse

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effects relating to the gavage and sampling. No differences in body weight, necropsy weight, or

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organ weights were observed between any of the treatment groups (data not shown). Clinically

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at 28 to 30 days of treatment, two of the four goats treated with A. lentiginosus developed mild

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intention tremors and rear limb proprioception deficits that were most apparent when they were

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forced to cross a 15-cm barrier in their pen. These clinical signs became more severe especially

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in the rear legs as the study progressed making movement more difficult. After 35 to 42 days,

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the other two goats treated with A. lentiginosus, as well as two goats treated with I. carnea,

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developed clinical signs similar to those observed at day 28 to 30 in the two goats treated with A.

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lentiginosus. All goats that developed clinical signs were still mobile and able to cross over a

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15-cm barrier at the end of the study. At the end of the study, all four control goats and two of

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the I. carnea-treated goats appeared to be clinically normal.

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Serum biochemistry variables (Table 1) and serum swainsonine concentrations (Figure 2)

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were similar among all the animals treated with A. lentiginosus and I. carnea. A day x treatment

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interaction (P < 0.01) was observed for the serum enzymes ALP and AST, as both enzymes

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increased and remained greater than the control group for the length of the study (Table 1). The

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serum enzyme ALT showed no day x treatment interaction (P = 0.2) indicating no treatment

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effects over time. Serum swainsonine concentrations increased within days of all animals

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receiving the first dose of A. lentiginosus and I. carnea and remained at a similar concentration

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throughout the study (Figure 2). As expected, the control group differed from A. lentiginosus-

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and I. carnea-treated groups (P < 0.04). At some time points, the mean serum swainsonine

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concentrations in the I. carnea-treated group were numerically greater than the A. lentiginosus-

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treated group, nonetheless no statistical differences were observed between the A. lentiginosus-

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and I. carnea-treated groups (P > 0.20). Swainsonine is highly soluble resulting in rapid

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absorption, distribution, and elimination (Bowen et al. 1993, Stegelmeier et al. 1998; 1999b).

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The lack of differences in serum swainsonine concentrations between the A. lentiginosus and I.

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carnea groups suggest that the toxicokinetics of swainsonine are not altered by the calystegines.

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In addition, no differences were observed in serum biochemistry variables and serum

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swainsonine concentrations between goats dosed with I. carnea that developed clinical signs and

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those that did not.

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Histologically, the primary neurologic lesion in the A. lentiginosus- and I. carnea-treated

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goats was neuronal microvesicular degeneration and necrosis. In most neuronal populations the

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changes were minimal with the exception of the cerebellar Purkinje cells and select neurons in

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the cerebellar granular cell layers and basal ganglia. Morphometric studies supported these

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observations as both the I. carnea and A. lentiginosus-treated goats had increased numbers of

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both vacuolated and degenerative Purkinje cells compared to the control (P≤0.003) (Table 2).

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No differences were observed between the A. lentiginosus- and I. carnea-treated goats (Table 2).

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The lesions in the Purkinje cells were characterized by extensive accumulations of fine

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cytoplasmic vesicles that often marginate the nucleus (Figure 3). Silver stains demonstrated that

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the affected Purkinje cells often had fewer associated axons and there were also small numbers

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of empty baskets and pyknotic Purkinje cells (Figure 4). (Table 2). This trend of degeneration,

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necrosis and loss of Purkinje cells in the A. lentiginosus- and I. carnea-treated goats was

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supported numerically as there were fewer Purkinje cells and increased numbers of pkynotic

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Purkinje cells in both the I. carnea and locoweed treated groups (Table 2). Though not

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statistically different, the locoweed treated goats tended to have fewer Purkinje cells (increased

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% pyknotic cells and increased µM/cell) than the I. carnea treated goats (Table 2). Other

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neurologic changes identified in A. lentiginosus- and I. carnea-treated goats included increased

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numbers of dystrophic axons, specifically spheroids and pyknotic neurons with minimal gliosis

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in the nucleus gracilis and cuneatus of the medulla (Figure 5).

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The visceral changes in the A. lentiginosus- and I. carnea-treated goats were also similar.

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The most consistent change for both groups was pancreatic exocrine glandular epithelial

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vacuolation (Figure 6). The pancreatic islet cells (endocrine pancreas) were spared and appeared

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normal. Other lesions included thyroid follicular epithelium vacuolation (Figure 7). Less severe

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vacuolation was also present in the lacrimal gland and salivary gland epithelia of both groups.

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The renal proximal tubular epithelium was also minimally vacuolated in both groups. No

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differences were observed in the microscopic lesions between the goats that developed clinical

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signs that were dosed I. carnea and those that did not develop clinical signs. Lastly, no

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significant microscopic lesions were identified in any of the control animal tissues and the

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remaining tissues from the A. lentiginosus- and I. carnea- treated goats.

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As mentioned previously, A lentiginosus contains the glycosidase inhibitor swainsonine

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while I. carnea contains swainsonine as well as calystegines. The objective of this study was to

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directly compare locoweed represented by A. lentiginosus, and I. carnea poisoning in goats

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dosed with plant material to obtain equal swainsonine doses to thus assess the relative role of

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calystegines. The results from this study suggest that the neurologic disease associated with I.

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carnea poisoning is principally due to swainsonine as the serum biochemistries, swainsonine

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concentrations, and pathological changes were similar in the A. lentiginosus- and I. carnea-

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treated goats. This conclusion is further supported by rodent studies where purified swainsonine

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and relatively high doses of purified calystegines B2 and C1 were dosed to mice (Stegelmeier et

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al. 2008). In these studies, the swainsonine-treated mice developed typical swainsonine-induced

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visceral and neurologic lesions similar to those observed in these goats while the calystegine-

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treated mice were mostly unchanged even when dosed at rates 10 times higher than what the

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goats received herein, and doses reported in other experimental studies (Armien et al. 2007).

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Similar results were reported by Hueza et al. (2005) where rats were administered calystegine

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and swainsonine extracts from I. carnea.

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In general, the clinical signs, serum biochemistries, and the pathological lesions described

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for locoweeds (swainsonine-containing Astragalus and Oxytropis species) and I. carnea are

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similar to what has been reported in cases where these plants have been dosed individually

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(Armien et al. 2007, 2011; Stegelmeier et al. 1999 a, b). However, there are some studies that

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indicate that I. carnea poisoning can present with a severe and progressive disease that often

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culminates in a fatal neurologic disease. In some locations, livestock producers identify Ipomoea

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carnea-induced disease as “mata cabra” or goat killer. Reported observed mortality can be high

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and can occur as early as 15 days after the onset of clinical signs (Armien et al. 2007). Such fatal

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disease is not seen with locoweed poisoning and it did not occur at the dose and duration used in

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the present study. The I. carnea-treated goats in this study appeared to respond very similar to

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that observed in locoweed-poisoned animals. Clinical and experimental studies indicate that

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locoweed poisoning generally requires several weeks of continuous ingestion for overt

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neurologic signs to develop. As poisoning progresses animals lose condition and develop

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weakness, trembling, proprioceptive deficits, altered gait, anxiety, changes in demeanor and

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reluctance to stand or move. Later in the course of disease, locoweed-poisoned animals become

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emaciated, dehydrated and recumbent. Most affected animals are euthanized due to loss of

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condition or lack of production or die from accidents or misadventure (Stegelmeier et al. 1999a,

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b). This highlights that any comparisons from field cases regarding the clinical signs of toxicity

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and distribution of lesions associated with each respective plant are problematic as the conditions

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of poisoning are different. Furthermore, previous experimental studies with I. carnea are

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difficult to compare, as these studies used varying doses of plant material given for different

300

durations; further, animals were typically group fed, plant materials fed over time were not

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homogenous, and the daily dose of plant toxins was poorly characterized or unknown (de Balogh

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et al. 1999; Armien et al. 2007, 2011).

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Some clinical reports suggest severely Ipomoea-poisoned animals have seizures and die

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suddenly. Though the exact cause of these Ipomoea carnea-associated deaths has not been

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identified, it has been suggested that it is directly due to combined swainsonine and calystegine

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induced-neurologic disease (Armien et al. 2007). As previously suggested, this relative high

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mortality differs from the clinical presentation and disease of chronic locoweed poisoning. A

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complicating factor may be due to animal susceptibility as goats may be uniquely susceptible to

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I. carnea toxicity. Goats are generally not kept on locoweed-infested ranges and reports of

310

naturally occurring poisonings are rare (Stegelmeier et al, 2001). As seen in this work, goats

311

respond differently than other livestock species to poisoning. For example, A. lentiginosus and I.

312

carnea treated goats developed rear limb proprioceptive deficits with neurologic lesions in the

313

gracilis and cuneatus nuclei. This is similar to the extensive dorsal column lesions described in

314

mule deer (Odocoileus hemionus) poisoned by locoweed (Stegelmeier et al. 2005). These nuclei

315

contain second-order neurons of the medial lemniscus pathway that are key players in control of

316

fine touch and proprioception. Such lesions could produce the accentuated intention tremors and

317

proprioceptive deficits in the rear limbs seen in these goats. It may be that with extended

318

durations or higher doses, neurologic damage might progress to involve other systems that

319

eventually may produce seizures or sudden death.

320

Calystegines may play a role in the toxicity of plants that do not contain swainsonine;

321

however, this hypothesis remains to be experimentally verified. Recently Salinas et al. (2019)

322

reported that goats in Nicaragua ingesting I. trifida and I. carnea developed a neurologic disease

323

with neuronal micro vacuolation and degeneration that were restricted to cerebellar Purkinje cells

324

and basal ganglia neurons with minimal secondary changes of gliosis and axonal dystrophy. No

325

vacuolation was observed in glandular tissues of the exocrine pancreas, thyroid follicular

326

epithelium, or renal tubular epithelium. The associated plants were analyzed and found to

327

contain no swainsonine, but they did contain calystegines at a concentration of 0.03 to 0.06%.

328

The authors suggested that calystegines may be responsible for the neurologic disease. Similar

329

cerebellar degenerative diseases have been described in livestock species poisoned by some

330

Solanum species (Pienaar et al. 1976; Riet-Correa et al. 1983; Barros et al. 1987; Burrows and

331

Tyrl, 2001; Verdes et al. 2006). Later, some of these Solanum species were shown to contain

332

calystegines which were proposed to be responsible for poisoning (Nash et al. 1993; Molyneux

333

et al. 1994; Burrows and Tyrl, 2001). These cases are often associated with long term ingestion

334

of the respective plant and at very high doses, as animals consume the plants under conditions of

335

forage scarcity and drought. More research is needed to determine if these plants that do not

336

contain swainsonine, but contain calystegines are toxic, and to determine the dose and duration

337

of exposure necessary for intoxication.

338

In conclusion, goats treated with A. lentiginosus and I. carnea developed clinical disease

339

characterized by mild intention tremors and proprioceptive deficits. The presentation of clinical

340

disease occurred sooner in goats treated with A. lentiginosus and all the locoweed-treated goats

341

showed clinical signs compared to two goats treated with I. carnea. No differences in body

342

weight, serum swainsonine concentrations and serum enzyme activity were observed between

343

goats treated with A. lentiginosus and I. carnea. Lastly, there were no differences in microscopic

344

and histochemical studies of the visceral and neurologic lesions; and only minor or no

345

differences were found in the morphologic comparisons of Purkinje cell size, number, and

346

degeneration. These findings suggest that I. carnea-induced clinical signs and histologic lesions

347

are due to swainsonine and that calystegines contribute little or nothing in the presence of

348

swainsonine.

349

350 351 352 353

Acknowledgements. - We thank Kermit Price, Edward Knoppel, Joseph Jacobson, and Scott Larsen for their technical assistance. Louisiane de Carvalho Nunes was funded by a fellowship from CAPES.

354

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489

Table 1. Select serum biochemical data (mean and standard deviation) for goats (n=4 goats per group) dosed with ground alfalfa (Controls), Ipomoea carnea (Ipomoea), and Astragalus lentiginosus (Locoweed). Ipomoea carnea and Astragalus lentiginosus were dosed to obtain a swainsonine dose of 1.5 mg/kg/day for 45 days. Day of study1

Analyte/Treatment 0

4

11

18

25

32

39

46

360 (109)

348 (123)

309 (132)

299 (146)

326 (104)

362 (62)

321 (106)

340 (76)

Ipomoea

344 (45)

637 (116)

1269 (377)

772 (339)

1002 (424)

1203 (192)

1118 (327)

1156 (225)

Locoweed

377 (117)

935 (338)

1037 (564)

632 (293)

674 (415)

950 (424)

895 (338)

930 (328)

Aspartate Transaminase (IU) Control

81 (8)

82 (7)

73 (9)

67 (19)

77 (14)

87 (15)

75 (13)

86 (10)

Ipomoea

86 (5)

180 (26)

307 (97)

218 (101)

299 (111)

367 (71)

309 (94)

361 (63)

Locoweed Alanine Transaminase (IU) Control

80 (16)

187 (60)

257 (118)

180 (52)

263 (30)

355 (105)

337 (114)

367 (93)

24 (4)

26 (3)

21 (7)

21 (7)

23 (6)

26 (1)

24 (7)

28 (3)

Ipomoea

30 (5)

30 (4)

25 (7)

16 (7)

22 (8)

26 (4)

23 (7)

28 (2)

Locoweed

24 (6)

25 (8)

20 (10)

14 (4)

17 (4)

21 (7)

22 (8)

23 (6)

Alkaline Phosphatase (IU) Control

1

There was a day x treatment interaction for both alkaline phosphatase and aspartate transaminase (P < 0.02). For both serum variables, there was no difference on day 0 (baseline, P > 0.6); on each study day thereafter both Astragalus lentiginosus and Ipomoea carnea treatment groups differed (P < 0.03) from Controls, but did not differ from one another (P > 0.4). Alanine transaminase activities did not differ by treatment nor was there a day x treatment interaction (P > 0.1).

Table 2: Morphometric comparisons of the cerebellar Purkinje cell changes in goats (n=4 goats per group) treated with ground alfalfa, Ipomoea carnea (Ipomoea), and Astragalus lentiginosus (Locoweed) for 45 days. The I. carnea and A. lentiginosus groups were dosed with ground plant to obtain a swainsonine dose of 1.5 mg/kg/day. Vacuolated and pyknotic variables show the incidence of lesions (mean and standard deviation for proportion of cells) in randomly sectioned and selected cerebellar cortex.1 The remaining variables illustrate Purkinje cell number, size, and degeneration in the cerebellar central lobe (mean and standard deviation of the length- µM linear granular interface per cell number).2

Variable

Control

Ipomoea

Locoweed

Vacuolated cells (%)

0.1 (0)

28 (12)

40 (9)

Pyknotic cells (%)

0.1 (0)

27 (8)

32 (8)

Purkinje Cells (µm/Nucleated Cell)

169 (47)

259 (67)

298 (99)

Pyknotic Cells (µm/Pyknotic Cell)

8593 (8387)

2472 (1167)

4980 (3092)

Purkinje Cell Size (Area µ 2)

1367 (336)

1630 (312)

1641 (226)

1

There were differences in the proportion of vacuolated and pyknotic cells with the controls differing from I. carnea and A. lentiginosus treatments (P≤0.003); the I. carnea and A. lentiginosus treatments did not differ (P≥0.18)

2

There were no differences (P > 0.09) in Purkinje cell measurements (µm or area).

Figure Legends Figure 1. (A) Spotted locoweed (Astragalus lentiginosus) (photo courtesy and copyright Al Schneider, www.swcoloradowildflowers.com) is a perennial legume that includes various subspecies that grow in western North America. The plant contains the indolizidine alkaloid, swainsonine the locoweed toxin. (B) Ipomoea carnea is a flowering perennial plant that originates from North, Central and South America. The plant contains swainsonine and calystegines B1, B2, B3 and C1. Figure 2. Mean serum swainsonine concentrations ± standard deviation from control goats (n=4), goats treated with ground Ipomoea carnea (n=4) to obtain swainsonine doses of 1.5 mg swainsonine/kg bw/day, and goats treated with ground Astragalus lentiginosus (n=4) to obtain swainsonine doses of 1.5 mg swainsonine/kg bw/day at days 4, 11, 18, 25, 32, 39, and 46. Figure 3. Photomicrographs of cerebellar Purkinje cells from (A) control goats that were dosed with ground alfalfa at volumes similar to the other treated groups for 45 days; (B) goats treated with ground Ipomoea carnea to obtain swainsonine doses of 1.5 mg swainsonine/kg bw/day for 45 days; and (C) goats treated with ground Astragalus lentiginosus to obtain swainsonine doses of 1.5 mg swainsonine/kg bw/day for 45 days. Notice the swollen and finely granular vacuolation (*) within Purkinje cells. The vacuolation often expands one margin of the cell displacing the nucleus to the margin. The oligodendroglia and small granular neurons of the cerebellum are not visibly affected at this dose and duration. Figure 4. Silver stained photomicrographs of cerebellar Purkinje cells from (A) goats that were dosed with ground alfalfa at volumes similar to the other treated groups for 45 days; (B) goats treated with ground Ipomoea carnea to obtain swainsonine doses of 1.5 mg swainsonine/kg bw/day for 45 days; and (C) goats treated with ground Astragalus lentiginosus to obtain swainsonine doses of 1.5 mg swainsonine/kg bw/day for 45 days, Notice the Purkinje cells (*) that in the Astragalus lentiginosus- and Ipomoea-treated goats were swollen with fewer, closely related black stained axonal communications. Also notice the pyknotic Purkinje cell (arrowhead) and empty baskets (arrows) in the Astragalus lentiginosus-treated goat. Figure 5. Photomicrographs of nucleus gracilis from an Astragalus lentiginosus-treated goat to obtain swainsonine doses of 1.5 mg swainsonine/kg bw/day for 45 days. Notice the swollen and dystropic axon (arrowhead). Similar axonal dystrophy or spheroids were also present in the Ipomoea carnea-treated goats. Figure 6. Photomicrographs of pancreas from (A) control goats that were dosed with ground alfalfa at volumes similar to the other treated groups for 45 days; (B) goats treated with ground Ipomoea carnea to obtain swainsonine doses of 1.5 mg swainsonine/kg bw/day for 45 days; and (C) goats treated with ground Astragalus lentiginosus to obtain swainsonine doses of 1.5 mg swainsonine/kg bw/day for 45 days. Notice the swollen and extreme vacuolation (*) within the exocrine pancreatic cells in the Astragalus lentiginosus- and Ipomoea carnea-treated goats Figure 7. Photomicrographs of thyroid gland from (A) control goats that were dosed with ground alfalfa at volumes similar to the other treated groups for 45 days; (B) goats treated with ground Ipomoea carnea to obtain swainsonine doses of 1.5 mg swainsonine/kg bw/day for 45 days; and (C) goats treated with ground Astragalus lentiginosus to obtain swainsonine doses of 1.5 mg swainsonine/kg bw/day for 45 days, Notice the vacuolation of the thyroid follicular

epithelium (arrowheads) that is pronounced in the Astragalus lentiginosus- and Ipomoea carneatreated groups and minimal in the control goats.

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Highlights -Goats were intoxicated with Ipomoea carnea and Astragalus lentiginosus. -No differences in serum swainsonine concentrations and serum enzyme activity were observed. -No differences in pathological lesions were observed. -Calystegines contribute little or nothing to toxicity in goats in the presence of swainsonine.

The submitted work has not been published previously or is not under consideration for publication in any other journal. The manuscript has been reviewed and approved by all authors. The authors declare that there are no conflicts of interest

Thanks, Daniel Cook

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: