(5S)-5-Benzylswainsonines as potent and selective inhibitors of Golgi α-mannosidase II: synthesis, enzyme evaluation and molecular modelling.

Development of novel anti-cancer therapeutics based on Golgi α-mannosidase II (GMII) inhibition is considerably impeded by an undesired co-inhibition of lysosomal α-mannosidase leading to severe side-effects. In this contribution, we describe a fully stereoselective synthesis of (5S)-5-[4-(halo)benzyl]swainsonines as highly potent and selective inhibitors of GMII. The synthesis starts from a previously reported aldehyde readily available from l-ribose, and the key features include an intramolecular reductive amination with substrate-controlled stereoselectivity and a late-stage derivatisation of the benzyl group via ipso-substitution. These novel swainsonine analogues were found to be nanomolar inhibitors of the Golgi-type α-mannosidase AMAN-2 (Ki = 23-75 nM) with excellent selectivity (selectivity index = 205-870) over the lysosomal-type Jack bean α-mannosidase. Finally, molecular docking and pKa calculations were performed to provide more insight into the structure of the inhibitor:enzyme complexes, and a pair interaction energy analysis (FMO-PIEDA) was carried out to rationalise the observed potency and selectivity of the inhibitors.

Plant Secondary Compounds Promote White Adipose Tissue Browning via Modulation of the Gut Microbiota in Small Mammals.

The browning of white adipose tissue (WAT) is a promising area of research for treating metabolic disorders and obesity in the future. However, studies on plant secondary compounds promoting WAT browning are limited. Herein, we explored the effects of swainsonine (SW) on gut microbiota and WAT browning in captive pikas. SW inhibited body mass gain, increased brown adipose tissue (BAT) mass, and induced WAT browning in pikas. The 16S rDNA sequencing revealed a significant reduction in the alpha diversity and altered community structure of the gut microbiota in captive pikas. However, the addition of SW to the diet significantly increased the alpha diversity of gut microbiota and the relative abundance of Akkermansia, Prevotella, and unclassified_f__Lachnospiraceae, along with the complexity of the microbial co-occurrence network structure, which decreased in the guts of captive pikas. Functional profiles showed that SW significantly decreased the relative abundances of energy metabolism, lipid metabolism, and glycan biosynthesis and metabolism, which were enriched in captive pikas. Furthermore, SW decreased deterministic processes of gut microbiota assembly in July and increased them in November. Finally, the genera Prevotella and unclassified_f__Prevotellaceae were positively correlated with BAT mass. Our results highlighted that plant secondary compounds promote WAT browning by modulating the gut microbiota in small mammals.

Analysis of the Mycotoxin Levels and Expression Pattern of SWN Genes at Different Time Points in the Fungus Slafractonia leguminicola.

The fungal plant pathogen Slafractonia leguminicola produces two mycotoxins that affect animals: slaframine, which causes slobbers, and swainsonine, which causes locoism. Slafractonia leguminicola contains the swainsonine-associated orthologous gene clusters, "SWN", which include a multifunctional swnK gene (NRPS-PKS hybrid), swnH1 and swnH2 (nonheme iron dioxygenase genes), swnN and swnR (reductase genes), and swnT (transmembrane transporter). In addition to these genes, two paralogs of swnK, swnK1 (paralog1) and swnk2 (paralog2), are found in S. leguminicola. cDNAs from total mRNA were isolated from the S. leguminicola mycelia grown in the culture plates as well as from leaves inoculated with the fungal mycelia at different time points, and expression pattern of the SWN genes were analyzed using RT-qPCR. The concentrations of swainsonine and slaframine production from this fungus at different time points were also examined using liquid chromatography-mass spectrometry. The timing of gene expression was similar in cultured fungus and inoculated leaves and agreed with our proposed biosynthetic pathway. Substantially more swainsonine was produced than slaframine during time course studies.

Autophagic-lysosomal damage induced by swainsonine is protected by trehalose through activation of TFEB-regulated pathway in renal tubular epithelial cells.

Swainsonine (SW) is the main toxic component of locoweed. Previous studies have shown that kidney damage is an early pathologic change in locoweed poisoning in animals. Trehalose induces autophagy and alleviates lysosomal damage, while its protective effect and mechanism against the toxic injury induced by SW is not clear. Based on the published literature, we hypothesize that transcription factor EB(TFEB) -regulated is targeted by SW and activating TFEB by trehalose would reverse the toxic effects. In this study, we investigate the mechanism of protective effects of trehalose using renal tubular epithelial cells. The results showed that SW induced an increase in the expression level of microtubule-associated protein light chain 3-II and p62 proteins and a decrease in the expression level of ATPase H+ transporting V1 Subunit A, Cathepsin B, Cathepsin D, lysosome-associated membrane protein 2 and TFEB proteins in renal tubular epithelial cells in a time and dose-dependent manner suggesting TFEB-regulated lysosomal pathway is adversely affected by SW. Conversely, treatment with trehalose, a known activator of TFEB promote TFEB nuclear translocation suggesting that TFEB plays an important role in protection against SW toxicity. We demonstrated in lysosome staining that SW reduced the number of lysosomes and increased the luminal pH, while trehalose could counteract these SW-induced effects. In summary, our results demonstrated for the first time that trehalose could alleviate the autophagy degradation disorder and lysosomal damage induced by SW. Our results provide an interesting method for reversion of SW-induced toxicity in farm animals and furthermore, activation of TFEB by trehalose suggesting novel mechanism of treating lysosomal storage diseases.

Impaired Indole Acetic Acid and Reduced Indole-Producing Bacteria Contribute to Swainsonine-Induced Liver Inflammation.

Liver inflammation could be elicited by swainsonine in livestock, affecting the development of agriculture and animal husbandry. Our previous study showed an important role of bile acids (BAs) in swainsonine-induced hepatic inflammation. However, its pathogenesis, particularly the roles of a comprehensive profile of liver and serum metabolites and microbial-derived indole metabolites, has not been clarified. This study aimed to demonstrate the mechanisms linking the indole-producing bacteria and indole metabolites to swainsonine-induced hepatic inflammation by combining Targeted 500 metabolomics and quantitative analysis of indole metabolites. Swainsonine significantly disturbed the liver and serum metabolomes in mice. Genus Akkermansia alleviating inflammation and genus Lactobacillus producing indole metabolites were significantly declined. Indole acetic acid (IAA) was the only reduced aryl hydrocarbon receptor (AHR) ligand in this study. Analogously, some bacteria causing liver damage markedly increased. These findings suggested that indole-producing bacteria and indole metabolites may be potential triggers of swainsonine-induced hepatic inflammation.

A review on mycotoxins and mycotoxicoses in ruminants and Equidae in South America.

Of the mycotoxicoses caused by molds contaminating grains or their byproducts, leukoencephalomalacia of horses and less frequently aflatoxicosis in cattle have been reported in South America. However, the most important group of mycotoxins in the region are those caused by fungi that infect forages and other types of plants and have regional distribution. In this group, ergotism is important, both caused by Claviceps purpurea infecting grains or by Epichloë coenophiala infecting Schedonorus arundinaceus. Other important mycotoxicoses are those caused by indole-diterpenes produced by Clavicipitaceous fungi including Claviceps paspali in Paspalum spp., Claviceps cynodontes in Cynodon dactylon, and by Periglandula a seed transmitted symbiont associated with the tremorgenic plant Ipomoea asarifolia. The latter is an important poisoning in the northeastern and northern Brazil. Other important mycotoxicoses are those caused by swainsonine containing plants. It was demonstrated that swainsonine contained in Ipomoea carnea var. fistulosa is produced by an epibiotic fungus of the order Chaetothyriales whose mycelia develop on the adaxial surface of the leaves. Swainsonine is also produced by the symbiotic, endobiotic fungi Alternaria section Undifilum spp., which is associated with Astragalus spp. in the Argentinian Patagonia causing poisoning. Another form of mycotoxicosis occurs in poisoning by Baccharis spp., mainly B. coridifolia, a very important toxic plant in South America that contains macrocyclic trichothecenes probably produced by an endophytic fungus that has not yet been identified. Pithomycotoxicosis caused by Pithomyces chartarum used to be an important mycotoxicosis in the region, mainly in cattle grazing improved pastures of legumes and grasses. Slaframine poisoning, diplodiosis and poisoning by barley contaminated by Aspergillus clavatus has been rarely diagnosed in Brazil, Uruguay and Argentina.

Identification and distribution of a fungal endosymbiotic Alternaria species (Alternaria section Undifilum sp.) in Astragalus garbancillo tissues.

Plants belonging to the genera Astragalus, Oxytropis, Ipomoea, Sida, and Swainsona often contain the toxin swainsonine (SW) produced by an associated fungal symbiont. Consumption of SW-containing plants causes a serious neurological disorder in livestock, which can be fatal. In this study, a fungal endophyte, Alternaria section Undifilum, was identified in Astragalus garbancillo seeds, using polymerase chain reaction (PCR) followed by direct sequencing. In seeds, the SW concentrations were about 4 times higher than in other parts of the plant. Furthermore, microscopic examination demonstrated that the fungus mycelium grows inside the petioles and stems, on the outer surface and inside the mesocarp of the fruit, in the mesotesta and endotesta layers of the seed coat, and inside the endosperm of the seeds. Our results support the notion that the SW-producing fungus is vertically transmitted in the host plant A. garbancillo.

Transcriptomic Screening of Alternaria oxytropis Isolated from Locoweed Plants for Genes Involved in Mycotoxin Swaisonine Production.

Locoweed is a collective name for a variety of plants, such as Oxytropis and Astragalus L. When these plants are infected by some fungi or endophytes, they will produce an alkaloid (swainsonine) that is harmful to livestock. Chronic toxicity characterized by neurological disorders occurs in livestock overfed on locoweed, and swainsonine (SW) is considered a major toxic component. The mechanism of the SW synthesis of endophytic fungi from locoweed remains unknown. In order to further discover the possible synthetic pathway of SW, in this study, a mycotoxin (SW) producer, Alternaria oxytropis isolate, UA003, isolated from Locoweed plants, and its mutant were subjected to transcriptomic analyses to ascertain the genes involved in the synthesis of this toxin. Mutant strain A. oxytropis E02 was obtained by ethyl methanesulfonate (EMS) mutagenesis treatment, and the strains were sequenced with different culture times for transcriptomic analysis and screening of differentially expressed genes. The results show a highly significant (p < 0.01) increase in SW yield in the A. oxytropis E02 strain obtained by EMS mutagenesis treatment compared to A. oxytropis UA003. A total of 637 differentially expressed genes were screened by transcriptome sequencing analysis, including 11 genes potentially associated with SW biosynthesis. These genes were screened using GO and KEGG data annotation and analysis. Among the differential genes, evm.TU.Contig4.409, evm.TU.Contig19.10, and evm.TU.Contig50.48 were associated with L-lysine biosynthesis, the L-pipecolic acid pathway, and the α-aminoadipic acid synthesis pathway. This study provides new insights to elucidate the mechanism of SW synthesis of endophytic fungi in locoweed and provides data support for further exploration of A. oxytropis genomics studies.

Multiomics Reveals Mechanisms of Alternaria oxytropis Inhibiting Pathogenic Fungi in Oxytropis ochrocephala.

Endophytic fungi can benefit the host plant and increase the plant resistance. Now, there is no in-depth study of how Alternaria oxytropis (A. oxytropis) is enhancing the ability of inhibiting pathogenic fungi in Oxytropis ochrocephala (O. ochrocephala). In this study, the fungal community and metabolites associated with endophyte-infected (EI) and endophyte-free (EF) O. ochrocephala were compared by multiomics. The fungal community indicated that there was more A. oxytropis, less phylum Ascomycota, and less genera Leptosphaeria, Colletotrichum, and Comoclathris in the EI group. As metabolic biomarkers, the levels of swainsonine and apigenin-7-O-glucoside-4-O-rutinoside were significantly increased in the EI group. Through in vitro validation experiments, swainsonine and apigenin-7-O-glucoside-4-O-rutinoside can dramatically suppress the growth of pathogenic fungi Leptosphaeria sclerotioides and Colletotrichum americae-borealis by increasing the level of oxidative stress. This work suggested that O. ochrocephala containing A. oxytropis could increase the resistance to fungal diseases by markedly enhancing the content of metabolites inhibiting pathogenic fungi.

Metabolomic analysis of swainsonine poisoning in renal tubular epithelial cells.

Locoweed is a poisonous plant widely present in grasslands around the world. Swainsonine (SW), an indole alkaloid that, is the main toxic component of the locoweed. To understand the mechanism of SW-induced toxicity and to delineate the metabolic profile of locoweed poisoning we performed the LC-MS/MS untargeted metabolomic study to analyze metabolites in SW-treated renal tubular epithelial cells (0.8 mg/mL, 12 h) and in order to identify the SW-induced metabolomic changes. The analysis identified 2,563 metabolites in positive ion mode and 1,990 metabolites in negative ion mode. Our results showed that the metabolites were mainly benzenoids, lipids and lipid-like molecules, nucleosides, nucleotides, and analogs, organic acids, and derivatives. The differential metabolites were primarily enriched in pathways involving bile secretion, primary bile acid biosynthesis, riboflavin metabolism, ferroptosis, drug metabolism-cytochrome P450, and primidine metabolism. We have screened out substances such as swainsonine, 3alpha,7alpha-Dihydroxy-5beta-cholestanate, 2-Hydroxyiminostilbene, and glycochenodeoxycholate, which may have the potential to serve as biomarkers for swainsonine poisoning. This study provides insights into the types of metabolomic alteration in renal tubular epithelial cells induced by swainsonine.

Detection of swainsonine and calystegines in Convolvulaceae species from the semiarid region of Pernambuco / Detecção de swainsonina e calisteginas em espécies de Convolvulaceae da região semiárida de Pernambuco

Numerous plant species worldwide including some Ipomoea (Convolvulaceae) and Sida (Malvaceae) species in Brazil cause lysosomal storage disease in herbivores and are known to contain swainsonine and calystegines as the main toxic compounds. The aim of this work was to determine swainsonine and calystegines concentrations in species of Convolvulaceae from the semiarid region of Pernambuco. Seven municipalities in the Moxotó region were visited and nine species were collected and screened for the presence of swainsonine and calystegines using an HPLC-APCI-MS method. The presence and concentration of these alkaloids within the same and in different species were very variable. Seven species are newly reported here containing swainsonine and/or calystegines. Ipomoea subincana contained just swainsonine. Ipomoea megapotamica, I. rosea and Jacquemontia corymbulosa contained swainsonine and calystegines. Ipomoea sericosepala, I. brasiliana, I. nil, I. bahiensis and I. incarnata contained just calystegines. The discovery of six Ipomoea species and one Jacquemontia species containing toxic polyhydroxy alkaloids reinforces the importance of this group of poisonous plants to ruminants and horses in the semiarid region of Pernambuco. Epidemiological surveys should be conducted to investigate the occurrence of lysosomal storage disease associated to these new species.(AU)

Numerosas espécies de plantas em todo o mundo, incluindo algumas espécies de Ipomoea (Convolvulaceae) e Sida (Malvaceae) no Brasil, causam doença de armazenamento lisossomal em herbívoros e são conhecidas por conterem swainsonina e calisteginas como princípios tóxicos. O objetivo deste trabalho foi determinar a concentração de swainsonina e calisteginas em espécies de Convolvulaceae da região semiárida de Pernambuco. Sete municípios na região do Sertão do Moxotó foram visitados, onde foram coletadas amostras das folhas de nove espécies de Convolvulaceae para avaliação da presença de swainsonina e calisteginas utilizando-se cromatografia líquida com espectrometria de massa. A presença e concentração destes alcaloides nas folhas de plantas da mesma espécie e dentre as espécies foram muito variáveis. Seis novas espécies de Ipomoea e uma espécie de Jacquemontia contendo swainsonina e/ou calisteginas são relatadas neste estudo. Ipomoea subincana continha apenas swainsonina. Ipomoea megapotamica, I. rosea e Jacquemontia corymbulosa continham swainsonina e calisteginas. Ipomoea sericosepala, I. brasiliana, I. nil, I. bahiensis e I. incarnata continham apenas calisteginas. A descoberta de novas espécies de Ipomoea e Jacquemontia contendo alcaloides polihidroxílicos tóxicos reforçam a importância deste grupo de plantas tóxicas para ruminantes e equinos na região semiárida de Pernambuco. Pesquisas epidemiológicas devem ser realizadas para investigar a ocorrência de doença de depósito lisossomal associada a essas novas espécies.(AU)

Hypomyelinogenesis associated with transplacental poisoning by Sida carpinifolia (Malvaceae) in fetus and newborn calves / Hipomielinogênese associada à intoxicação transplacentária por Sida carpinifolia (Malvaceae) em fetos e neonatos bovinos

Sida carpinifolia is a plant responsible for poisoning several species of animals. This paper describes Hypomyelinogenesis in fetuses and neonates of cattle that consumed S. carpinifolia. Neonates manifested ataxia and muscle tremors. Two bovine newborns and four fetuses were necropsied and showed no significant gross changes. Histopathologic findings included vacuolation of pancreatic acinar cells, thyroid follicular cells, hepatocytes, cells of renal tubules and neurons of the fetus and the white matter of the telencephalic frontal lobe of the neonates and also revealed axonal spheroids in the brain of the fetuses and neonates. The lectin-histochemical evaluation shoved staining for the lectins Con-A, WGA and s-WGA. The Luxol Fast Blue staining revealed a marked decrease of myelin in the brain of all the fetuses and a moderate decrease in the neonates. Histologic and lectin-histochemic findings indicate that the consumption of S. carpinifolia by pregnant bovine females can cause hypomyelinogenesis in fetuses and neonates.(AU)

Sida carpinifolia é uma planta responsável por intoxicar várias espécies animais. Este artigo descreve hipomielinogênese em fetos e neonatos de bovinos que consumiram S. carpinifolia. Os neonatos manifestaram ataxia e tremores musculares. Dois neonatos e quatro fetos bovinos foram necropsiados e não havia alterações macroscópicas significativas. Os achados histopatológicos incluíram vacuolização de células acinares do pâncreas, células foliculares da tireoide, hepatócitos, células renais tubulares e neurônios nos fetos. Nos neonatos havia vacuolização na substância branca do lobo frontal telencefálico, além de esferoides axonais no encéfalo dos fetos e dos recém-nascidos. A avaliação lectino-histoquímica demonstrou marcação para as lectinas Con-A, WGA e s-WGA. A coloração de Luxol Fast Blue revelou diminuição acentuada da mielina no telencéfalo de todos os fetos e diminuição moderada nos neonatos. Os achados histológicos e lectina-histoquímicos indicam que o consumo de S. carpinifolia por fêmeas bovinas gestantes pode causar hipomielinogênese em fetos e neonatos.(AU)

Spontaneous poisoning by Sida carpinifolia (Malvaceae) in horses / Intoxicação natural por Sida carpinifolia em equinos

Sida carpinifolia poisoning causes a chronic neurodegenerative disorder associated with lysosomal storage by indolizidine alkaloids (swainsonine). The epidemiological, clinical, pathological and lectin histochemistry findings of an outbreak of natural poisoning by S. carpinifolia in horses in Rio Grande do Sul state, Brazil, are described. Five horses from a total of 15 that were kept on native pasture with large amounts of S. carpinifolia presented during 90 days clinical signs of progressive weight loss, incoordination, stiff gait and ramble, in addition to exacerbated reactions and locomotion difficulty after induced movement. Four horses died, and one of them was submitted for necropsy. At necropsy, no significant gross lesions were observed. Histological findings observed in the central nervous system were characterized by swollen neurons with cytoplasm containing multiple microvacuoles; these abnormalities were more severe in the thalamus, hippocampus, cerebellum and pons. Using lectin histochemistry, the pons and hippocampus sections stained positive for commercial lectin Con-A, sWGA and WGA. This study aimed to detail S. carpinifolia poisoning in horses to be included in the differential diagnoses of neurological diseases of horses.(AU)

A intoxicação por Sida carpinifolia é uma desordem neurodegenerativa crônica associada ao acúmulo lisossomal pelo alcaloide indolizidínico, denominado swainsonina. Descrevem-se os achados epidemiológicos, clínicos, patológicos e de lectina-histoquímica de um surto de intoxicação natural por S. carpinifolia em equinos no Rio Grande do Sul, Brasil. De um total de 15 equinos, cinco equinos mantidos em campo nativo com grande quantidade de S. carpinifolia apresentaram sinais clínicos de emagrecimento progressivo, incoordenação, andar rígido e deambulação, além de dificuldade de locomoção com reações exacerbadas após estímulos ao movimento em um período de 90 dias de evolução clínica. Quatro equinos vieram a óbito e um foi submetido ao exame de necropsia. À necropsia, não foram observadas lesões macroscópicas. Os achados histológicos observados no sistema nervoso central caracterizaram-se por aumento de tamanho dos neurônios, com citoplasma contendo microvacúolos; tais alterações foram observadas com maior intensidade em tálamo, hipocampo, cerebelo e ponte. Na lectina-histoquímica, fragmentos de ponte e hipocampo marcaram positivamente para as lectinas comerciais Con-A, sWGA e WGA. Este trabalho visa alertar a ocorrência da intoxicação por S. carpinifolia em equinos, a qual deve ser incluída como diagnóstico diferencial dentre as doenças neurológicas de equinos.(AU)

Feeding preferences of experienced and naïve goats and sheep for the toxic plant Ipomoea carnea subsp. fistulosa / Comportamento ingestivo de caprinos e ovinos habituados e não habituados a ingerir Ipomoea carnea subsp. fistulosa

Ipomoea carnea is a toxic plant that grazing goats and cattle may learn to ingest with repeated exposure. The objective of this study was to evaluate the feeding preferences of experienced and non-experienced (naïve) goats and sheep for I. carnea.The study used 3 groups of 5 goats (Group 1, experienced that were previously poisoned by the plant; group 2, naïve; Group 3, experienced eaters, composed of animals adapted to eat the fresh plant) and 2 groups of sheep (group 4, experienced that were previously poisoned by the plant; and group 5, naïve). For the test, the animals were placed daily for 10 minutes and 4 days in a rectangular stall (5x7m) with 4 feeders, each with 200g of a different food (Ipomoea carnea, commercial concentrate food, recently harvested green grass (mainly Brachiaria spp.), and Cynodon dactylonhay. The intake of concentrate food was significantly higher (P<0.05) than the consumption of green grass, hay and I. carnea. In a second 4 day trial, in which the commercial concentrate food was replaced by freshly harvested Amorimia septentrionalis, the ingestion of green grass (Brachiaria spp.) was significantly higher (P<0.05) than the consumption of other foods. In both trials there was no significant difference in food consumption between eaters and naïve animals. The results suggest that experienced or naïve sheep and goats do not prefer I. carnea when it is offered with other foods or forages, suggesting that animals will avoid the plant and not become poisoned if other food options are available.

Ipomoea carnea é uma planta tóxica para caprinos e bovinos, que podem desenvolver o hábito de ingeri-la de forma compulsiva. O objetivo deste estudo foi avaliar o comportamento ingestivo de caprinos e ovinos habituados e não habituados a ingerir I. carnea. O estudo utilizou três grupos de cinco caprinos (Grupo 1, animais previamente intoxicados pela planta; Grupo 2, controle; Grupo 3, animais adaptados a ingerir a planta fresca) e dois grupos de cinco ovinos (Grupo 4, animais previamente intoxicados pela planta; e Grupo 5, controle). Para o teste, os animais foram introduzidos por 10 minutos, durante 4 dias consecutivos, em uma baia retangular (5x7m), com 4 caixas de alimentação, cada uma com 200g de um alimento diferente (Ipomoea carnea; concentrado comercial; capim verde recém colhido, principalmente Brachiaria spp.; e feno de Cynodon dactylon). A ingestão de concentrado foi significativamente maior (P<0,05) do que o consumo de capim verde, feno e I. carnea. Em um segundo ensaio de 4 dias, em que o concentrado foi substituído por Amorimia septentrionalis recém-colhida, a ingestão de capim verde (Brachiaria spp.) foi significativamente maior (P<0,05) do que o consumo dos outros alimentos. Em nenhuma das duas espécies, houve diferenças no comportamento ingestivo entre animais habituados e não habituados a ingerir I. carnea. Os resultados sugerem que ovinos e caprinos habituados e não habituados a ingerir I. carnea não preferem a planta quando é oferecida com outros alimentos ou forragens, o que sugere que os animais irão evitar a planta e não haverá intoxicação se outras opções de alimentos estão disponíveis.

Conditioned food aversion to control poisoning by Ipomoea carnea subsp. fistulosa in goats / Aversão alimentar condicionada para o controle da intoxicação por Ipomoea carnea subsp. fistulosa em caprinos

Ipomoea carnea is a toxic plant often ingested by livestock in Brazil. Three experiments were conducted to determine if conditioned food aversion was effective in reducing goats' consumption of I. carnea. In the first experiment, 10 mildly intoxicated goats that had been eating I. carnea were averted using LiCl (175 to 200mg kg-1 body weight). These intoxicated goats did not develop an aversion to I. carnea, demonstrating that the technique is not effective in goats that are already accustomed to consuming the plant. In the second experiment, 14 naïve goats were placed in a pasture with I. carnea, and averted after they ingested the plant. In this group the aversion persisted until the end of the experiment, 2 years and 8 months after the initial aversion. In another experiment, 20 goats were placed in a pasture with I. carnea, and after consuming the plant were averted with LiCl. The averted goats were transferred to Marajo Island and periodically observed over a 2 year period at 2-3 month intervals to determine if they were still averted. The averted goats did not ingest the plant while grazing in the pasture, whereas in 6 neighboring goat farms the prevalence of intoxication from I. carnea poisoning was estimated to be about 40%. These results demonstrated the efficacy of conditioned food aversion to avoid ingestion of I. carnea in formerly naïve goats that had only recently begun to ingest the plant.

Para testar a técnica de aversão alimentar condicionada como método de controle para a intoxicação por I. carnea, foram realizados 3 experimentos administrando cloreto de lítio (LiCl) na dose de 175-200mg kg-1 após a ingestão da planta por caprinos. No primeiro, foram induzidos à aversão 10 caprinos que tinham o hábito de ingerir a planta e com sinais clínicos da intoxicação. Apesar da realização de diversos tratamentos aversivos, após os animais ingerirem a planta, a aversão não foi eficiente, demonstrando que a técnica não é eficiente em caprinos que já estão habituados a ingerir a planta. No segundo experimento, 14 caprinos foram adaptados a ingerir a planta na pastagem e, após ingerirem a planta a campo, foram induzidos à aversão com LiCl. Neste grupo, a aversão persistiu até o fim do Experimento, 2 anos e 8 meses após a aversão. Em outro experimento, 20 caprinos foram adaptados a consumir I. carnea e, em seguida, induzidos à aversão com LiCl. Esses animais foram transferidos para uma propriedade na Ilha de Marajó, onde foram realizadas 9 visitas com intervalos de 2-3 meses para verificar a duração da aversão. Após 2 anos de observações, nenhum animal voltou a ingerir a planta na pastagem e não foram observados casos de intoxicação, enquanto que, em 6 propriedades vizinhas, a doença foi observada com uma prevalência de até 60%. Esses resultados demonstram a eficiência da aversão alimentar condicionada para evitar a ingestão de I. carnea em caprinos recém adaptados a ingerir a planta, nas regiões invadidas por esta planta e nas condições naturais da Ilha de Marajó.

Aversão alimentar condicionada a Ipomoea carnea var. fistulosa em ovinos / Conditioned food aversion to Ipomoea carnea var. Fistulosa in sheep

Quinze ovinos foram adaptados ao consumo de Ipomoea carnea por 36 dias. Posteriormente, foram divididos ao acaso em três grupos de cinco ovinos cada e avertidos com cloreto de lítio (LiCl) (Grupo 1) e Baccharis coridifolia (Grupo 2). O Grupo 3 permaneceu como controle. Os ovinos foram desafiados ao consumo da planta na pastagem e em baias individuais até o 74° dia após o primeiro dia da aversão. Nos dias 14 e 19, o número de ovinos que consumiu I. carnea no Grupo 3 foi significativamente maior que o número de ovinos que consumiu a planta nos Grupos 1 e 2 (P=0,004 e P=0,0004, respectivamente). No dia 24, o número de ovinos que consumiu I. carnea foi significativamente maior no Grupo 3 do que no Grupo 1 (P=0,004). A partir do desafio do dia 29, não houve diferença significativa (P>0,05) no consumo da planta entre os três grupos. A quantidade de I. carnea consumida pelos ovinos do Grupo 3 durante o período do experimento no dia 7 após o primeiro dia da aversão foi significativamente maior do que o consumo das ovelhas dos Grupos 1 e 2 (P=0,0002 e P=0,01, respectivamente). Nos desafios seguintes, não houve diferença na quantidade de I. carnea ingerida pelos ovinos. Na avaliação do comportamento dos ovinos na pastagem infestada por I. carnea, o tempo destinado ao consumo da planta foi de no máximo 2,4%±1,6% do total de tempo de pastejo, não havendo diferença entre os três grupos. I. carnea nunca foi consumida avidamente pelos ovinos experimentais e a aversão incompleta induzida pelos dois agentes utilizados como aversores (LiCl e B. coridifolia) sugere que não seriam eficientes para reduzir ou eliminar o consumo de I. carnea pelos ovinos.

Fifteen sheep were adapted to consume I. carnea for 36 days. Subsequently sheep were randomly divided into three groups of five sheep each. Group 1 was averted with LiCl, group 2 was averted with B. coridifolia, and Group 3 was the control group. The sheep were periodically tested by exposing to I. carnea in the pasture and in individual pens up to the 74th day after the first day of aversion. On the 14th and 19th days the number of sheep in Group 3 that consumed I. carnea was significantly higher than the number of sheep that consumed I. carnea in Groups 1 and 2 (P=0.004 and P=0.0004, respectively). On day 24 the number of sheep that consumed I. carnea was significantly higher in Group 3 than Group 1 (P=0.004). After the challenge on the 29th day no significant difference (P>0.05) was observed in the consumption of the plant among the three groups. On day 7 of the aversion period control sheep (group 3) consumed more I. carnea than did the sheep from Groups 1 and 2 (P=0.0002 and P=0.01, respectively). After this period there was no difference in the amount of I. carnea ingested by the sheep in other individual challenges. The maximum time spent by the sheep grazing I. carnea was 2.4%±1.6% of the total grazing time and no difference was observed among the groups. I. carnea was never consumed avidly by the experimental sheep, and some LiCl animals did not learn to avoid the plant on a consistent basis. Conditioning an incomplete aversion indicates that neither LiCl or B. coridifolia will work with sheep in field settings to reduce or eliminate consumption of I. carnea.

Intoxicação por plantas que contêm swainsonina no Brasil / Poisoning by swainsonine-containing plants in Brazil

No Brasil, as plantas que contêm swainsonina compõem um grupo muito importante de plantas tóxicas, incluindo Ipomoea carnea subsp. fistulosa, Ipomoea riedelii, Ipomoea sericophyla, Ipomoea verbascoidea, Turbina cordata e Sida carpinifolia, que causam armazenamento de oligossacarídeos em caprinos e, com menor frequência, em bovinos, equinos, ovinos e cervídeos. Uma característica das plantas que contêm swainsonina é que os animais que iniciam a ingerir essas plantas desenvolvem o hábito de ingeri-las compulsivamente e, por facilitação social, induzem a outros animais da mesma espécie a ingeri-las. Os animais intoxicados apresentam pêlos arrepiados, perda de peso e sinais nervosos associados, principalmente, a lesões cerebelares e do tronco encefálico. Infertilidade, abortos, nascimento de animais fracos e maior susceptibilidade aos parasitas gastrintestinais são descritas tanto em casos agudos quanto em animais que deixaram de ingerir a planta e permanecem com sinais, mesmo que discretos. Atrofia cerebelar pode ser observada macroscopicamente em animais cronicamente afetados. As alterações histológicas caracterizam-se por vacuolização de neurônios, células epiteliais do pâncreas e dos túbulos renais, células foliculares da tireoide, hepatócitos e macrófagos de órgãos linfóides. Para o controle da intoxicação, os animais devem ser retirados imediatamente do local onde ocorre a planta. A única medida profilática consiste em evitar a ingestão da planta, eliminando a mesma das áreas infectadas ou mediante aversão alimentar condicionada.

Swainsonine-containing plants comprise a very important group of toxic plants in Brazil, including Ipomoea carnea subsp. fistulosa, Ipomoea riedelii, Ipomoea sericophyla, Ipomoea verbascoidea, Turbina cordata and Sida carpinifolia, which cause a glycoprotein storage disease in goats, and with less frequency in cattle, horses, sheep, and deer. A characteristic of swainsonine poisoning is that the animals that eat these plants develop the habit of eating them compulsively and, by social facilitation, induce other animals of the same species to eat them. Clinical signs of the poisoning are rough hair coat, weight loss, and nervous signs associated mainly with cerebellar and brain stem lesions. Weight losses or decreased weight gains still occurring also in animals with chronic clinical signs removed from the areas where the plants occur. Reproductive losses are also reported. Chronically affected animals, also without still ingesting the plant, are more susceptible to gastrointestinal parasites. Cerebellar atrophy has been observed grossly in chronic cases. Main histologic lesions are vacuolation of neurons, epithelial cells of the pancreas and kidney, thyroid follicular cells, hepatocytes, and macrophages of lymphoid organs. To control the poisoning animals must be removed immediately from the areas invaded by the plant. The only preventive measure to prevent the ingestion of the plant is their elimination from invaded areas or through conditioned food aversion.

Conditioned food aversion to Ipomoea carnea var. fistulosa induced by Baccharis coridifolia in goats / Aversão alimentar condicionada de Ipomoea carnea var. fistulosa induzida por Baccharis coridifolia em caprinos

Baccharis coridifolia is a plant that induces strong conditioned food aversion in ruminants. This research aimed to induce a conditioned food aversion to Ipomoea carnea var. fistulosa in goats, using B. coridifolia as an aversive agent, and to compare the aversion induced by this plant with the aversion induced by lithium chloride (LiCl). Thirteen goats were allotted into two groups: Group 1 with six goats was averted with 175mg/kg of body weight of LiCl and Group 2 with seven goats was averted with 0,25g/kg of bw of dried B. coridifolia. All goats were averted on day 1 after the ingestion of I. carnea. The aversion procedure with LiCl or B. coridifolia in goats from Groups 1 and 2, respectively, was repeated in those goats that again consumed the plant during tests on days 2, 3, and 7. The goats of both groups were challenged in pens on 23 and 38 days after the last day of aversion and challenged in the pasture on days 11, 15, 18, 20, 22, 25, 27 and 29 after the last day of aversion. After this period goats were challenged every 15 days on pasture until the 330º day after the last day of aversion (7th day). Two goats from Group 1 ingested I. carnea on the first day of the pasture challenge, 4 days after the last day of aversive conditioning in the pen. In addition, another goat in Group 1 started to consume the plant on day 18, and other two goats ate it on day 20. One goat in Group 1 that had never eaten I. carnea died on day 155. One goat from Group 2 started to ingest I. carnea on the first day of the pasture challenge, and a second goat started to consume this plant on day 182. At the end of the experiment, on day 330, the other five goats averted with B. coridifolia remained averted. These results suggest that B. coridifolia or an active compound from the plant could be used to induce aversion to toxic plants. Using B. coridifolia would be cheaper and, particularly in flocks with large number of animals, possibly easier than using...

Baccharis coridifolia é uma planta tóxica que possui forte poder aversivo em ruminantes. Os objetivos deste trabalho foram induzir aversão condicionada a Ipomoea carnea var. fistulosa em caprinos utilizando B. coridifolia como agente aversivo e comparar a eficiência desta aversão com a aversão induzida por cloreto de lítio (LiCl). Treze cabras foram divididas em dois grupos: o Grupo 1 com seis cabras foi avertido com 175mg/kg de peso corporal (pc) de LiCl e o Grupo 2, com sete cabras, foi avertido com 0,25g/kg de pc de B. coridifolia seco. Todas as cabras foram avertidas no dia 1 logo após a ingestão de I.carnea. A aversão foi repetida nos dias 2, 3 e 7 nos caprinos que ingeriram qualquer quantidade de I. carnea, utilizando-se o mesmo procedimento do dia 1. Os caprinos de ambos os grupos foram desafiados nas baias nos dias 23 e 38 após o último dia da aversão e desafiados na pastagem nos dias 11, 15, 18, 20, 22 , 25, 27 e 29 após o último dia da aversão. Posteriormente os caprinos foram desafiados a cada 15 dias na pastagem até o 330º dia após o último dia da aversão (7º dia). Duas cabras do Grupo 1 ingeriram I. carnea no primeiro dia do desafio na pastagem, quatro dias após o ultimo dia da aversão nas baias. Além disso, outra cabra do mesmo grupo reiniciou a ingestão da planta no 18º dia e outras duas no 20º dia. Uma cabra do Grupo 1 que nunca havia ingerido a planta após a aversão morreu no 55º dia. Uma cabra do Grupo 2 começou a ingerir I. carnea no primeiro dia de desafio na pastagem e uma segunda reiniciou o consumo da planta no 182º dia. No final do experimento no 330º as cinco cabras avertidas com B. coridifolia permaneciam sem ingerir a planta. Estes resultados sugerem que B. coridifolia ou algum princípio ativo dessa planta pode ser utilizado para induzir aversão condicionada a plantas tóxicas. A utilização de B. coridifolia como agente aversivo é aparentemente mais barato e mais fácil de ser utilizado do que o LiCl, o qual requer o uso de ...

Doença de depósito lisossomal induzida pelo consumo de Ipomoea verbascoidea (Convolvulaceae) em caprinos no semiárido de Pernambuco / Lysosomal storage disease induced by the consumption of Ipomoea verbascoidea (Convolvulaceae) in goats at northeastern Brazil

The aim of this paper was to reproduce the poisoning of Ipomoea verbascoidea in goats and describe the epidemiological, clinical and pathological aspects of spontaneous poisoning by this plant in Pernambuco. For this, we studied the epidemiology of the disease in seven municipalities in the semiarid region of the State. Three spontaneously poisoned goats were examined and then euthanized and necropsied (Group I). To reproduce the disease, the dried leaves of I. verbascoidea containing 0.02% swainsonine were supplied at doses of 4g/kg (0.8mg swainsonine/kg) to two groups of three animals. The goats in Group II received daily doses of the plant during 40 days and were euthanized on the 41st day of the experiment. Goats from Group III received daily doses of the plant during 55 days and were euthanized on the 120th day of the experiment. Other three goats constituted the control group (Group IV). In experimental groups, the brain lesions were evaluated by histopathology; additionally the cerebellar lesions were evaluated by morphometry, by measuring the molecular layer thickness, the number of Purkinje cells and the area of the cell bodies of these cells. The main clinical signs and microscopic lesions in goats poisoned were similar to those reported by swainsonine containing plants. In goats of GII and GIII, the first nervous signs were observed between 22th and 29th days; clinically, the disease developed by these animals was similar to the spontaneous cases. The goats of GIII did not recover from the neurologic signs. These results show that the consumption of the plant by 26-28 days after observation of the first clinical signs is enough to cause irreversible damage. By morphometric analysis, the molecular layer of the cerebellum of the goats of Group I and III were thinner than those of goats in the control group, and Purkinje neurons were atrophic. It is suggested that these changes are responsible for the neurological picture observed in goats that stop eating the plant and have sequelae of poisoning.

O objetivo deste trabalho foi reproduzir a intoxicação por Ipomoea verbascoidea em caprinos e descrever os aspectos epidemiológicos, clínicos e histopatológicos da intoxicação espontânea por essa planta no Estado de Pernambuco. Para isso, realizou-se o acompanhamento da epidemiologia da doença em sete municípios do semiárido pernambucano. Três caprinos espontaneamente intoxicados foram examinados e, em seguida eutanasiados e necropsiados (Grupo I). Para reproduzir experimentalmente a doença, as folhas secas de I. verbascoidea contendo 0,02% de swainsonina, foram fornecidas na dose de 4g/kg (0,8mg de swainsonina/kg) a dois grupos de três animais. Os caprinos do Grupo II receberam a planta diariamente por 40 dias e foram eutanasiados no 41º dia de experimento. Os caprinos do Grupo III receberam a planta diariamente por 55 dias e foram eutanasiados no 120º dia de experimento. Outros três caprinos constituíram o grupo controle (Grupo IV). Nos grupos experimentais, as lesões encefálicas foram avaliadas por histopatologia e adicionalmente avaliaram-se as lesões cerebelares por morfometria, mediante mensuração da espessura da camada molecular, do número de neurônios de Purkinje e da área dos corpos celulares dessas células. Os principais sinais clínicos e lesões microscópicas foram semelhantes aos previamente reportados em animais intoxicados por plantas que contem swainsonina. Nos caprinos do GII e GIII, os primeiros sinais clínicos foram observados entre o 22º e 29º dia de experimento; clinicamente a doença desenvolvida por esses animais foi semelhante aos casos espontâneos. Nenhum dos caprinos do GIII se recuperou dos sinais neurológicos. Esse resultado evidencia que o consumo da planta por 26-28 dias após a observação dos primeiros sinais clínicos é suficiente para provocar lesões irreversíveis. Pela análise morfométrica, a camada molecular do cerebelo dos caprinos do Grupo I e III eram mais delgadas que às dos caprinos do grupo controle, e os neurônios de Purkinje estavam atróficos. Sugere-se que essas alterações sejam responsáveis pelo quadro clínico neurológico observado nos caprinos que deixam de ingerir a planta e apresentam seqüelas da intoxicação.

Aversão alimentar condicionada no controle de surtos de intoxicações por Ipomoea carnea subsp. fistulosa e Turbina cordata em caprinos / Conditioned food aversion to control outbreaks of poisoning by Ipomoea carnea subsp. fistulosa and Turbina cordata in goats

A aversão alimentar condicionada é uma técnica que pode ser utilizada em animais para evitar a ingestão de plantas tóxicas. A técnica foi utilizada em uma fazenda para controlar a intoxicação por Turbina cordata e em outra para Ipomoea carnea subsp. fistulosa. Os caprinos eram presos à noite, e na manhã do dia seguinte lhes era ofertada a planta verde, recém-colhida, por dez minutos. Os caprinos que ingerissem qualquer quantidade da planta eram identificados, pesados e tratados com LiCl na dose de 175mg/kg peso vivo através de sonda esofágica. No rebanho da fazenda na que havia T. cordata a técnica foi aplicada a cada dois meses durante o período em que a planta é encontrada. Durante todo o experimento, de dezembro de 2009 a abril de 2011 não ocorreu nenhum novo caso de intoxicação no rebanho e diminuiu gradualmente o número de animais avertidos e a quantidade de planta que ingeriam os mesmos durante o processo de aversão. Na fazenda na que ocorria intoxicação por I. carnea a maioria de rebanho foi avertido em dezembro de 2010, 15-20 dias antes do início das chuvas, e os animais não ingeriram a planta espontaneamente no campo até setembro-outubro de 2011, durante o período da seca, quando havia extrema carência de forragem e iniciaram a ingerir a planta no campo. Posteriormente, apesar de três tratamentos aversivos com 21 dias de intervalo, os animais continuaram a ingerir a planta e ocorreram casos clínicos. A técnica de aversão alimentar condicionada demonstrou ser eficiente e viável para o controle da intoxicação por T. cordata. Para a intoxicação por I. carnea a técnica impediu a ingestão da planta somente durante a época de chuvas, mas não durante a seca, quando há pouca disponibilidade de forragem. A diferença nos resultados com as duas plantas é, aparentemente, resultante das condições epidemiológicas diferentes nas que ocorrem as intoxicações. T. cordata desaparece durante a maior parte do período de seca. A planta rebrota e fica verde durante o fim de seca, quando diminui a oferta de forragem, por curto espaço de tempo, permanecendo verde durante a época de chuvas. I. carnea, por crescer próximas as fontes de água, em áreas húmidas, permanece verde durante todo o período da seca, quando é maior a escassez de forragem, favorecendo desta forma a ingestão da planta pelos animais.

Conditioned food aversion is used to train livestock to avoid the ingestion of toxic plants. This technique was used to control Turbina cordata poisoning in goats in one farm, and to control Ipomoea carnea subsp. fistulosa poisoning in another farm. The goats were penned at night and the next morning the green plants were offered for 10 minutes. Goats that ingested any amount of the plant were treated through a gastric tube with 175mg of LiCl/kg body weight. In the flock in which the poisoning by T. cordata was occurring, the goats were averted every two months during the period that the plant was found in the pastures. During the experiment, from December 2009 to April 2011, new cases of poisoning were not observed, and there was a progressive decrease in the number of goats that ingested the plant and were averted. In the farm where I. carnea poisoning was occurring, most of the goats were averted in December 2010, 15-20 days before the first rains. The goats of this flock did not ingest the plant spontaneously in the field until September-October 2011, when, due to the dry season, there was a severe forage shortage, and the goats started to ingest the plant in the field. Later, despite three aversive treatments with 21 days intervals, the goats continued to ingest the plant and some animals became poisoned. In conclusion, conditioned food aversion was effective in to control intoxication by T. cordata. The technique was also effective in conditioning goats to avoid consuming I. carnea during the rainy season, but not during the dry season, with low forage availability in the field. The differences in these results seem to be due to the epidemiology of both poisonings: T. cordata is senescent and unavailable during most of the dry period, and green biomass is typically available either at the very end of the dry season, for a short period of time, and during the rainy season when there is no shortage of forage. In contrast, I. carnea grows in wet areas near water sources, and stays green during the dry period when there is a lack of other forage.