Induction and Transfer of Resistance to Poisoning by Amorimia (Mascagnia) Septentrionalis in Goats

Research Article

Received: 31 October 2012, Revised: 27 December 2012, Accepted: 27 December 2012 Published online in Wiley Online Library: 12 February 2013

(wileyonlinelibrary.com) DOI 10.1002/jat.2860 Induction and transfer of resistance to poisoning by Amorimia (Mascagnia) septentrionalis in goats

Amélia L. L. Duartea, Rosane M. T. Medeirosa, Fabrício K. L. Carvalhoa, Stephen T. Leeb, Daniel Cookb, James A. Pﬁsterb, Valéria M. M. Costaa and Franklin Riet-Correaa*

ABSTRACT: Amorimia septentrionalis contains sodium monoﬂuoroactetate (MFA) and can cause acute heart failure in ruminants when ingested in toxic doses. In this study, we demonstrate that resistance to poisoning by A. septentrionalis can be improved in goats by the repeated administration of non-toxic doses of A. septentrionalis. We also show that increased resistance to poisoning by A. septentrionalis can also be achieved by the transfaunation of ruminal content from goats previously conditioned to be resistant to naïve goats. These methods of improving resistance require further study, but appear to provide potential management solutions to mitigate toxicity problems from A. septentrionalis, and perhaps other plant species containing MFA. Copyright © 2013 John Wiley & Sons, Ltd.

Keywords: Amorimia septentrionalis; goats; monoﬂuoroacetate; monoﬂuoroacetate degrading bacteria; plant poisoning resistance; sudden death

Introduction have not been successful (Silva et al., 2006). In Northeastern Brazil there are numerous anecdotal accounts indicating that In Brazil, there are at least 12 plants that cause acute heart failure animals grazing in areas invaded by A. rigida are more resistant in ruminants, which are responsible for nearly 500,000 cattle to poisoning than animals introduced from areas where A. rigida deaths each year (Tokarnia et al., 2002, 2012). Six of these con- does not occur (Silva et al., 2008). fl tain sodium mono uoroacetate (MFA) as the toxic compound; Therefore the objectives of this study were to determine including Palicourea marcgravii (Oliveira, 1963), P. aenofusca whether resistance to MFA poisoning from A. septentrionalis in (Lee et al., 2012), Amorimia rigida (Mascagnia rigida) (Cunha goats could be induced with repeated administration of non- fl et al., 2012; Lee et al., 2012), A. septentrionalis, A. pubi ora toxic, low doses of this plant and to determine if resistance could fl (Mascagnia pubi ora) (Lee et al., 2012) and Tanaecium bilabiatum be transmitted from plant-induced resistant goats to naive goats (Arrabidaea bilabiata) (Krebs et al., 1994). through transfaunation of ruminal content. In Paraiba. two plants are known to contain MFA and cause acute heart failure, A. septentrionalis, previously reported as Mascagnia rigida, and P. aenofusca (Vasconcelos et al., 2008a,2008b). Experimentally A. septentrionalis caused death in sheep and goats Materials and Methods –1 that ingested 10–20 g kg body weight (bw) (Vasconcelos et al., Amorimia septentrionalis was collected in two locations in the 2008a). Clinical signs were characterized by tachycardia, engorged municipality of São José do Bonfim (S709.47’ W3719.06’; eleva- jugular with venous pulsation, dyspnea and staggering gait pro- tion 305 m) and Teixeira (S712.24’ W3715.11’; elevation 749 m), gressing to sternal recumbence, followed by death. However, some both located in the state of Paraiba, Brazil, in the same locations animals showing clinical signs may recover if they are not disturbed. used by Vasconcelos et al. (2008a) to collect the plant for the In studies conducted by our research group it was observed that experimental reproduction of the intoxication. The mean concen- –1 most animals receiving low daily doses (2–4gkg bw) recovered tration of MFA determined in 20 samples of the plant, 10 from if the administration was stopped immediately after the onset of clinical signs. According to Tokarnia et al. (2012), the clearance time to eliminate the toxin is about 8–14 days. *Correspondence to: Riet-Correa, Franklin, Veterinary Hospital, CSTR, Federal There are no macroscopic lesions at necropsy, but histologi- University of Campina Grande (UFCG), Patos, PB 58700-000, Paraíba, Brazil. cally there is severe vacuolation and pyknosis of the epithelial E-mail: [email protected] cells of the distal convoluted tubules of the kidneys. This is seen a in approximately 50% of the intoxicated animals (Tokarnia et al., Veterinary Hospital, CSTR, Federal University of Campina Grande (UFCG), Patos, Paraíba 58700-000, Brazil 1961; Vasconcelos et al., 2008a, 2008b). Attempts to control Amorimia spp. poisoning by traditional bPoisonous Plant Research Laboratory, Agricultural Research Service, United 220 methods including uprooting the plants or using herbicides, States Department of Agriculture, 1150 E. 1400 N., Logan, Utah 84341, USA

J. Appl. Toxicol. 2014; 34: 220–223 Copyright © 2013 John Wiley & Sons, Ltd. Induction of resistance to poisoning by Amorimia septentrionalis each location, was 0.002 Æ 0.0009% (Lee et al., 2012). Representa- 6 animals that never ingested the plant, nor had contact with tive voucher specimens sent to Dr William R. Anderson (University animals that had been given the plant. of Michigan Herbarium, Ann Arbor, Michigan, USA) were identi- After the end of the transfaunation and cohabitation phase, fied as A. septentrionalis (Lee et al., 2012). A. septentrionalis was administered to all goats of each group Two experiments using crossbred goats, weighing 20–30 kg, at daily doses of 3 g kg–1 (0.06 mg MFA kg–1 bw) for 20 days, raised in an area free of Amorimia spp. were conducted at the 4gkg–1 (0.08 mg MFA kg–1 bw)for5daysand5gkg–1 Veterinary Hospital of the Federal University of Campina Grande, (0.1 mg MFA kg–1 bw) for 4 days. Plant administration was discon- Campus of Patos, Paraiba, Brazil. tinued for goats that showed clinical signs of poisoning. The experiment was terminated 29 days after the start of the administration, when all goats had shown clinical signs. The resistance to the poi- Experiment 1 soning was determined by the average period for each group to Induction of resistance to poisoning by A. septentrionalis by show clinical signs. The data were analyzed statistically by the administration of non-toxic doses non-parametric Friedman’s test using the software SAS Institute In the first experiment, 12 goats were randomly divided into 2 Inc. SAS/STATTM (1995). The number of goats showing clinical groups of 6 goats each. Group 1 (G1) received increasing daily signs was compared using Fisher’s exact test. doses of green leaves of A. septentrionalis for the following five periods: 1 g per kg body weight (g kg–1) for 10 days; 2 g kg–1 for 5 days; 2 g kg–1 for 5 days; 3 g kg–1 for 5 days; and 3 g kg–1 Results for 5 days. Each period during which plant was administered Experiment 1 was separated by a 15-day washout period with no Amorimia given, thus the total trial lasted 90 days. The plant was offered This experiment was performed to test the hypothesis that the to goats for voluntary consumption. Goats that did not voluntarily administration of repeated non-toxic doses of A. septentrionalis consume their entire dose of the plant were forced to consume increases resistance to the poisoning by this plant. When – the leaves by putting small amounts into their mouths. Group 2 A. septentrionalis was administered at daily doses of 3 g kg 1 bw – (G2) was the control group. Both groups were fed with commer- (0.06 mg MFA kg 1 bw), previously-untreated G2 goats 1, 2, 3, 4, cial feed in an amount equal to 1% of their bw and fresh green 5 and 6 showed clinical signs 6, 6, 7, 4, 6 and 7 days after the start grass, mainly Brachiaria spp. and Echinochloa polystachya. of the administration, respectively. Clinical signs were not Because MFA poisoning occurs from a cumulative effect, observed in any previously-treated goats from G1 by day 7, when goats in G1 were not fed plant material for 15 days to allow a de- the administration of A. septentrionalis was suspended. The pro- toxification period. Fifteen days after the end of A. septentrionalis portion of goats in G2 showing clinical signs differed (P =0.04) administration to G1, the plant was administered to both groups from G1. Clinical signs in G2 goats were apathy, anorexia, reluc- at daily doses of 3 g kg–1 (0.06 g kg–1 MFA) for 7 days. Each day tance to move, sternal recumbence and jugular engorgement prior to administering the plant to G1 goats, each animal was with venous pulse. Goats 4, 5 and 6 showed soft feces or diarrhea. examined for altered behavior, heart and respiratory rates, rumi- After the observation of clinical signs, plant administration was nal motility, and rectal temperature. After the plant was admin- discontinued and all animals recovered within 3–4daysafterthe istered the animals were stimulated to move for approximately last ingestion of A. septentrionalis. Goats 4 and 6 relapsed and 10 min to provoke any clinical signs. Plant administration was showed clinical signs 11 and 17 days after the end of the admin- discontinued for goats that showed clinical signs of poisoning istration, respectively, and died 2 days later. At necropsy both (apathy, anorexia, reluctance to move and jugular engorgement animals showed pulmonary edema. Histologically, pulmonary with a venous pulse). The goats that died were necropsied. edema and severe vacuolation of the cytoplasm and pyknotic Samples of organs of abdominal and thoracic cavities, and nuclei in epithelial cells of distal convoluted tubules of the kidney central nervous system were collected, fixed in 10% formalin, were observed in both goats. processed routinely by embedding in paraffin and subsequently stained with hematoxylin and eosin for histological examination. Experiment 2 This experiment was performed to test if the increased resis- Experiment 2 tance induced by repeated non-toxic doses of A. septentrionalis Transfer of resistance by transfaunation of ruminal contents or can be transferred to naïve goats by transfaunation of ruminal cohabitation with resistant goats contents or cohabitation with resistant goats. The time elapsed Twenty-seven goats were divided into four experimental between the start of administration and the observation of the groups (groups 3–6): Group 3 contained 8 goats with resistance first signs for each animal is presented in Fig. 1. There were no induced using the same protocol as experiment 1, Group 1; and significant differences in the latency to the first clinical signs Group 4, transfauned animals, consisted of 9 goats that received, between G3-resistant (19.4 Æ 8.2 days) and G4-transfauned once a week for 5 weeks, ruminal fluid collected from goats in (19 Æ 7.5 days), or between G5-cohabitation (5.7 Æ 1.3 days) treatment G3 (resistant). The ruminal contents were obtained and G6-control (7.8 Æ 1.9 days). However, for G3 and G4 this by means of a suction pump coupled to a ruminal probe. After latency period was significantly longer than for G5 and G6 collection, ruminal fluid from all animals in G3 was mixed, (P < 0.01) (Table 1). The clinical signs were similar to those divided in equal portions and administered by oral gavage to presented by intoxicated goats in Experiment 1 and the goats each goat in G4; Group 5, cohabitant, composed of four animals recovered in 3–4 days. Goat 5, from G6, died suddenly on the that did not previously ingest leaves of A. septentrionalis, but 9th day of A. septentrionalis administration, 30 minutes after the cohabited for approximately 6 months with resistant animals ingestion of the plant. At necropsy no gross lesions were of Group 1 (Experiment 1); and Group 6, control, composed of observed; however, histologically there were lesions of severe 221

Figure 1. Experiment 2. Graphic representation of the period (days) between the start of the administration and the ﬁrst signs in individual animals in the treatment groups: resistant (G3), transfauned (G4), cohabitants (G5) and control (G6) goats.

Table 1. Latency (mean number of days) from initial dosing until ﬁrst observation of clinical signs when goats were dosed with Amorimia septentrionalis in different experimental groups during Experiments 1 and 2

Experiment Experimental groupa (number of goats) Latency to first signs (days) Experiment 1 G1 resistant (6 goats) 6Æ1.91 G2 control (6 goats) 0.0b b Experiment 2 G3 resistant (8 goats) 19.4Æ8.21 G4 transfauned (9 goats) 19Æ7.51 G5 cohabitants (4 goats 5.7Æ1.32 G6 control (6 goats) 7.8Æ1.92 aG1-resistent goats that had been previously dosed in phased periods for 90 days; G2 control; G3-resistant goats that had been previously dosed with A. septentrionalis in phased periods for 90 days; G4-goats that had been given rumen fluid from resistant goats to transfer rumen microflora; G5-goats that had been penned with resistant goats; G6-controls that had no previous exposure to A. septentrionalis or to animals that bad been exposed to the plant. bGroups 1 and 2 received the plant during 7 days. Coefficient of variation for experiment 2 = 42.65% Means followed by the same superscript numbers do not differ between experimental groups when submitted to Friedman’s test (P < 0.01).

vacuolation and pyknosis of the epithelial cells of the distal con- can up regulate these rumen microflora favoring their multipli- voluted renal tubules. cation and increasing the resistance of animals to poisoning (Camboim et al., 2012b). Another possibility to explain the higher resistance of G1 and G3 groups is that monofluoracetate- Discussion degrading bacteria were administered to the animals with the The results of experiments 1 and 2 (Table 1 and Fig. 1) demonstrate green leaves of A. septentrionalis during the process of resistance that repeated ingestion of non-toxic doses of A. septentrionalis induction. In the second experiment, it was demonstrated that increase the resistance of goats to poisoning by this plant and that the resistant microflora can be transferred by transfaunation of this resistance can be transferred from resistant-induced goats to ruminal contents from a resistant to a susceptible animal. naïve goats by transfaunation of ruminal fluid. However, cohabita- Ruminal detoxification is a well-known mechanism for the tion of resistant goats with naive goats did not increase time from control of some plant poisonings. Ruminants that ingest increas- treatment to first clinical signs. Amorimia septentrionalis contains ing amounts of oxalate-containing plants become resistant to MFA (Lee et al., 2012) and this resistance is probably because of the poisoning owing to the development of microbial detoxifica- changes in populations of rumen microflora that degrade MFA. tion in the rumen (Craig & Blythe 1994). Microbial detoxification In Paraíba, Brazil, aerobic bacteria that degrade MFA have has been used with success in the control of Leucaena leucocephala been isolated from soil, leaves and flowers of A. septentrionalis poisoning. The plant contains the amino acid mimosine which in (Camboim et al., 2012a), and from the rumen of goats reared in the rumen is converted to 3-hydroxy-4(IH)-pyridone (3,4-DHP) areas where the plant does not occur (Camboim et al., 2012b). and 2,3-DHP. It was demonstrated that in the rumen 3,4-DHP is The fact that these bacteria were isolated from the rumen of converted to non-toxic compounds by Synergistes jhonnei,an animals raised in places without plants containing MFA, suggests anaerobic ruminal bacteria (Allison et al., 1992), and resistance to that they occur naturally in the rumen of the animals and the the poisoning can be induced by the transference of this bacte- 222 administration of low level non-toxic doses of A. septentrionalis rium (Cheeke, 1998). In Australia, ruminal inoculation of strains of

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