Abstract
Tetrapterys spp. have been reported as a cause of cardiac fibrosis, status spongiosus of the nervous system, and abortion. To study the intoxication by Tetrapterys multiglandulosa, 24 sheep were divided into 4 experimental groups of 4 sheep each and 1 control group. Groups 1 to 3, respectively, received 1, 1.5, and 3 g/kg body weight of the dry plant daily, starting on the 90th day of pregnancy. Group 4 received 1.5 g/kg from the 120th day of pregnancy to the end of gestation. All sheep from groups 2 and 3, except 1 that was submitted to cesarean delivery, aborted between 110 and 134 days of pregnancy. Seven fetuses had anasarca. Seven lambs from groups 1 and 4 were weak and died or were euthanatized. The other 2 were born weak with mild nervous signs but recovered. Cardiac fibrosis and status spongiosus of the nervous system were observed in newborn lambs and fetuses.
Tetrapterys multiglandulosa (Fig. 1) and Tetrapterys acutifolia are lianas or escandecent shrubs of the Malpighiaceae family found in southeastern and central-western Brazil. Poisoning by T. acutifolia occurs in the states of Minas Gerais and Espírito Santo and poisoning by T. multiglandulosa in the states of Rio de Janeiro, São Paulo, 13 and Mato Grosso do Sul. 1 The spontaneous intoxication by these plants in cattle causes abortion, neonatal mortality, and cardiac fibrosis. 1,9,13,14 Clinically, cardiac fibrosis is characterized by cardiac congestive insufficiency with brisket edema, jugular engorgement, jugular pulsation, and cardiac arrhythmia. 14 The clinical manifestation period is subacute or chronic, but some animals die suddenly. Cardiac fibrosis, hydrothorax, hydropericardium, ascites, and nutmeg appearance of the liver are observed at necropsy. Some animals show gait alterations and dullness due to status spongiosus of the central nervous system (CNS) in the grey and white substance of the brain. 14 Cardiac fibrosis is also observed in fetuses and stillbirths from cows ingesting Tetrapterys spp., 9,14 but lesions in the CNS have not been reported. Experimentally, T. multiglandulosa has been demonstrated to cause abortion in goats after the administration of a daily dose of 10 and 20 g/kg, starting on day 30 to 40 of gestation and administrated for 42 to 73 days and 17 to 24 days, respectively. 6 Lesions were not reported in the fetuses. 6
Tetrapterys multiglandulosa. Municipality of Roseira Velha, São Paulo, Brazil.
Reproductive dysfunction related to spontaneous intoxication has not been reported in sheep, but the experimental intoxication in this species causes similar lesions as those reported in cattle, including cardiac insufficiency with cardiac fibrosis and status spongiosus of the central nervous system. 9
In this experiment sheep, were used as an experimental model to test the effects of T. multiglandulosa in fetuses and newborns and to study the mechanisms of abortion and neonatal mortality.
Twenty-four adult Corriedale sheep weighing 32 to 44 kg were divided randomly into 4 experimental groups of 4 sheep each and 1 control group of 8 sheep. The sheep were mated with a ram with a painted chest marker to indentify estrus. The day of mating was considered the first day of pregnancy. Group 1 (sheep 1–4) ingested daily doses of 1.0 g/kg of the dry plant from the 90th to 120th day of gestation. Groups 2 (sheep 5–8) and 3 (sheep 9–12) ingested daily doses of 1.5 and 3 g/kg of the dry plant, respectively, from the 90th day of gestation until the occurrence of the first abortion within the sheep from these groups. At that time the sheep had ingested the plant for 21 to 31 days (day 111 to day 121 of gestation) (Table 1). Group 4 (sheep 13–16) ingested daily doses of 1.5 g/kg of the dry plant from the 120th day of gestation to parturition. Group 5 (sheep 17–24) acted as the control receiving the same amount of concentrate food without the plant. The plant used was collected one month before the start of the experiment in the Municipality of Roseira Velha, state of São Paulo, dried at 50°C, ground, and kept in paper bags at room temperature. During the experiment, all animals received commercial concentrated sheep food in the amount equivalent to 1.5% body weight. The animals were kept in indoor pens, and after the ingestion of the concentrated food, they were moved to a paddock with native pasture for a 4- to 6-hour period. Legal and ethical Brazilian requirements were followed in the animal experiments.
Daily and total doses of dry plant administered to experimental sheep; period of administration; day of abortion, parturition, or cesarean delivery; and outcome of the disease in lambs.
∗ Lamb found dead in the morning in a period up to 8 hours after birth. All lambs had coagulum in the umbilical blood vessels and pulmonary aeration, evidence that the death was after parturition.
A caesarian section was performed on sheep 7 (group 2) and sheep 24 (control group) on day 128 of gestation to evaluate the fetuses. Both the sheep and the fetuses were euthanatized after cesarean delivery. Sheep 5, 6, 8, 9, 10, and 12 (groups 2 and 3) aborted single fetuses between 111 and 134 days of gestation. These fetuses, named with the same number as the sheep, and also fetus 7 obtained by cesarean delivery on day 128 of gestation had anasarca (Fig. 2). Sheep 11 (group 2) aborted a mummified fetus on day 118. Table 1 shows the daily doses of dry plant fed to the sheep from the 4 groups, the administration period, the total doses, the day of abortion or parturition, and the outcome of the intoxication in the lambs. At necropsy, fetuses 5, 8, 9, 10, and 12 had anasarca and severe autolysis. No significant lesions other than anasarca were observed in fetus 6 aborted on day 134 and fetus 7 obtained by cesarean delivery.
Fetus from sheep 3. Anasarca is observed after cesarean delivery.
All sheep from groups 1 and 4 delivered live lambs between 142 and 147 days of gestation (Table 1). Only sheep 4 delivered twins (named 4.1 and 4.2); all others delivered only 1 lamb (named with the same number as their mothers). Lambs 2, 14, and 16 were hypothermic, depressed, and unable to stand. The 3 lambs were euthanatized between 3 and 24 hours after birth. Lambs 1 and 3 were found dead less than 8 hours after birth. Both had coagulum in the umbilical blood vessels and pulmonary aeration, evidence that death occurred after parturition. Sheep 4 delivered 2 weak lambs, which did not stand up and died a few minutes after parturition. Lambs from sheep 13 and 15 had moderate depression, could stand up, and after 12 to 24 hours were apparently normal. Necropsy of lambs 1, 2, 3, 4.1, 4.2, 14, and 16 revealed cardiac enlargement with a pale surface of the right ventricle (Fig. 3). On transverse cut, the ventricular walls were thick and firm with myocardial pallor. Those lesions were more severe in the walls of the right ventricle and septum (Fig. 3). The liver was congested with some red dark areas and other areas with increased lobular pattern. Lamb 18 also had subcutaneous edema of the limbs. Lamb 9 had 10 ml of yellow liquid in the abdominal cavity. Sheep 17 to 23 from the control group delivered healthy lambs (Table 1).
Heart, lamb 18. On the left, the heart is enlarged and the wall of the right ventricle is pale. On the right, transverse sections show thickened ventricular walls and septum and myocardial pallor, which are more severe in the walls of the right ventricle and septum.
At necropsy, samples of liver, kidney, heart, lung, lymph nodes, spleen, thyroid, adrenal, forestomachs, abomasum, small and large intestine, and skeletal muscles were fixed in 10% buffered formalin, embedded in paraffin, and sectioned at 4 to 6 µm. The entire CNS was also fixed in 10% buffered formalin, and transverse sections of the cervical, thoracic and lumbar spinal cord, medulla oblongata, pons, rostral colliculi, thalamus, internal capsule, cortex, cerebellar peduncles, and cerebellum were examined histologically. All tissues were stained with HE. Selected sections of the heart were stained by Masson trichrome for fibrous tissue.
Upon histologic examination of fetuses 6 and 7 and lambs from groups 1 and 4, the myocardium had disorganized fibers with separation by fibroblasts and collagen. The myocytes were swollen with pale cytoplasm, especially around the nuclei (Fig. 4). The nuclei were enlarged and were either rounded or rectangular. Multifocal areas of fibrosis (Fig. 5), associated with mononuclear infiltration and necrosis of myofibers, were also observed. In the areas adjacent to necrosis, the myofibers had vacuolated cytoplasm and enlarged nuclei with irregular outline. In the CNS of fetuses 6 and 7, there was mild to moderate status spongiosus of the cortex and subcortical white matter. Severe status spongiosus was also observed in the white matter of the cerebellum (Fig. 6) and cervical spinal cord. Areas of malacia were observed in the thalamus and temporal cortex in fetus 7. Diffuse vacuolation of hepatocytes was observed in the liver. All lambs from groups 1 and 4 had moderate to severe status spongiosus in the subcortical white matter and white matter of the cerebellum and cerebellar peduncles. In lamb 14, the vacuolation also affected the deep layers of the cortical grey matter. Mild to moderate status spongiosus was also observed in basal nuclei in lambs 1, 4.1, and 16; in the thalamus in lambs 2, 3, and 14; and in the rostral colliculi in lambs 2, 4.1, and 14. Vacuolation of the centrilobular hepatocytes were observed in lambs 4.1 and 14. Lambs 1 and 4.2 had diffuse vacuolation of hepatocytes. In the vacuolated areas, some astrocytes were reactive, with swollen and vesicular nuclei.
Heart, lamb 15. The myocytes are enlarged and swollen with pale cytoplasm, especially around the nuclei. The nuclei are enlarged and rounded. HE.
Heart, lamb 15. Disorganized myocytes and multifocal areas of fibrosis. HE.
Cerebellum, fetus from sheep 3. Severe vacuolization (status spongiosus), cerebellar white matter. HE.
In the 2 fetuses obtained by cesarean delivery (sheep 7 from group 2 and sheep 20 from control group) and in lambs 16 (group 4) and 24 (control group), small fragments of the myocardium and CNS (cerebellum, cortex, and subcortical white matter) were collected within 5 to 10 minutes of euthanasia and fixed in 2% glutaraldehyde with 2% paraformaldehyde, in 0.4 M cacodylate buffer (pH 7.4). Blocks were postfixed in 1% osmium tetroxide, buffered in 0.4 M sodium cacodylate (pH 7.4), and embedded in Epon 812. Semithin sections were stained with methylene blue. Ultrathin sections were stained with lead citrate and uranyl acetate and examined with an EM 10 Zeiss electron microscope at 60 kV. On the ultrastructure of the myocardium, there were cardiac fibers in different degrees of degeneration with abundant glycogen deposits in monoparticulate form. In some fibers, the mitochondria were severely swollen, with disorganization of the cristae. The cisterns of the smooth endoplasmic reticulum were dilated. Other cells showed edema of the sarcoplasm, swollen mitochondria with lyses of the cristae, and dilatation of the smooth endoplasmic reticulum. The sarcomeres were disorganized, with rupture of the myofibrils (Fig. 7). On electron microscopy of the nervous system, the vacuoles observed on the histology corresponded to intramyelinic vacuolation formed by splitting of the myelin sheaths at the intraperiod lines (Fig. 8). No significant histologic or ultrastructural lesions were observed in the control lambs.
Heart, fetus from sheep 3. Electron microscopy. Cardiac fibril with sarcolema indicated by arrowheads. In the sarcoplasm, there are abundant deposits of monoparticulate glycogen (asterisk). Some mitochondria are severely swollen, and the sarcomeres are partially disorganized at the periphery. Cisterns of the smooth endoplasmic reticulum are dilated (arrow). Bar = 2 µm.
Cerebellum, fetus from sheep 3. Electron microscopy. Intramyelinic edema with vacuole formation. The axoplasm is well preserved. Bar = 1 µm. Inset. Detachment on the myelin lamella. Bar = 200 nm.
Sheep 5 from group 2 was euthanatized 60 days after the end of the plant ingestion; sheep 11 and 12, from group 3, were euthanatized 60 and 39 days after the end of ingestion, respectively; and sheep 17 from group 5 was euthanatized at the end of the experiment. Those animals, as well as sheep 7 and 24 (which were euthanatized after cesarean delivery), were necropsied and studied histologically. Sheep 5 and 11 had pale firm areas in the left ventricle and interventricular septum, increased lobular pattern in the liver, hydropericardium (sheep 5), and ascites (sheep 11). Sheep 7 and 11 had only whitish areas on myocardium of the left ventricle. Microscopically the myocardium had multifocal areas of fibrosis associated with infiltration by mononuclear cells. In other areas, the fibers were separated by fibroblasts and collagen. In the brain of sheep 7 and 11, there was moderate status spongiosus of the subcortical white matter and also in the adjacent grey matter. Mild status spongiosus was also observed in the cerebellar white matter, pons, and internal capsule. Mild centrilobular congestion and vacuolization of hepatocytes were also observed. Lung congestion was observed in sheep 5. No significant lesions were observed in other organs. Sheep 24 from the control group had no macroscopic or histologic significant lesions. The placenta of the sheep from the 3 groups had no significant lesions on macroscopic and histologic examination.
In these experiments, T. multiglandulosa caused abortion, fetal mummification, and neonatal mortality similar to the spontaneous poisoning by Tetrapterys spp. in cattle 1,9,14 and to spontaneous and experimental intoxication by Ateleia glazioviana, a plant from a different family (Leguminoseae-Papilionoideae), in cattle 2,3,4,12 and sheep. 7,8,11 Cardiac and CNS lesions observed in the fetuses and lambs born with severe clinical signs were similar to those observed in adult sheep 9 and cattle 1,14 intoxicated by the plant, demonstrating that the unknown toxic substance contained in the plant passed through the placenta and caused fetal lesions. Abortion probably occurs due to fetal lesions' causing fetal death or fetal stress. Fetal stress in cattle and sheep due to chronic lesions activates the hypothalamic-pituitary-adrenal axis, causing premature delivery of the fetus. 10
In a previous experiment with pregnant goats, it was suggested that abortion induced by T. multiglandulosa is due to focal placentitis with early involution (apoptosis) and placentary coagulation necrosis, leading to placental detachment and fetal death due to lack of placental support. 6 In that report, the most remarkable fetal lesions were focal or diffuse hemorrhages in the skin, meninges, and visceral serosae. 6 In our experiment, the absence of placental lesions and the presence of severe lesions in the heart and CNS of the fetuses do not support the hypotheses of those authors.
The toxic substances of Tetrapterys spp. are unknown, but the observation of cardiac and nervous lesions in fetuses, similar to those observed in adult animals, suggests that both lesions are caused by the same substance. The experimental poisoning by A. glazioviana in sheep also causes abortion and neonatal mortality with cardiac and nervous lesions in the lambs. 7 The similarities between the intoxications caused by Tetrapterys spp. and A. glazioviana, both causing abortion, cardiac fibrosis, and status spongiosus due to intramyelinic edema in the CNS, suggest that they contain a similar toxic substance.
Sheep from groups 2 and 3 fed daily with 1.5 and 3 g/kg of dry plant, respectively, from day 90 of gestation aborted after 21 to 31 days of ingestion, respectively. In contrast, sheep from group 1 receiving a lower daily dose (1 g/kg) during day 90 to day 120 of gestation did not abort but delivered weak lambs. Neonatal mortality was also observed in sheep from group 4 after the daily ingestion of 1.5 g/kg, from day 120 of gestation until parturition, the same dose that causes abortion when administrated earlier in gestation in group 2. These results suggest that the occurrence of neonatal mortality or abortion depends on the dose ingested and also on the time of gestation during ingestion. Lower doses and late ingestion during pregnancy probably cause weak lambs and neonatal death. In contrast, higher doses ingested earlier in gestation probably cause abortion.
Spontaneous poisoning by Tetrapterys spp., including reproductive dysfunction, has not been reported in sheep, but this species is not raised in the area of distribution of the plant. Experimental intoxication by T. multiglandulosa in sheep causes lesions similar to those reported in cattle, including cardiac insufficiency with cardiac fibrosis and status spongiosus of the CNS, 9,14 suggesting that sheep can be used as an experimental model to study the disease. The toxicity of Tetrapterys spp, in laboratory animals has not been tested, but attempts to intoxicate laboratory animals, such as rabbits, mice, rats, and guinea pigs, with A. glazioviana have failed. 5
Footnotes
Acknowledgements
This work was financially supported by Institutos do Milênio, National Council of Scientific and Technologic Development (CNPq), Grant No. 420012/2005-2 and by the Programa de Apoio a Núcleos de Excelência (PRONEX), Grant No. 001/04, CNPq, FAPESQ, MCT.