Hepatogenous Photosensitization Research Articles

Isolation, Characterization, and Quantification of Steroidal Saponins in Switchgrass (Panicum virgatum L.)

Switchgrass (Panicum virgatum L.) has been identified for development into an efficient and environmentally friendly biomass energy crop. A recent 5 year study demonstrated that switchgrass grown for biofuel production produced 540% more energy than what is needed to grow, harvest, and process it into cellulosic ethanol. If switchgrass is grown on a scale useful for a bioenergy source, some of the material could be used by livestock as hay or pasture. Switchgrass has been reported to cause hepatogenous photosensitization in lambs (Ovis aries) and horses (Equus caballus). In this study, a simple extraction and rapid reversed phase high-performance liquid chromatography-electrospray ionization-mass spectrometry method was developed for quantifying the major saponins in switchgrass samples. Differences in the relative concentrations of different saponins were observed between switchgrass cultivars and plant parts.

Natural and Experimental Poisoning of Cattle by Enterolobium contortisiliquum Pods (Fabaceae Mimosoideae) in Central-Western Brazil

Enterolobium contortisiliquum pods are commonly identified as being the cause of abortions and photosensitivity in cattle. This paper describes the clinical and pathological aspects of a natural outbreak of hepatogenous photosensitization by the pods of E. contortisiliquum in Brazil and the results of experimental poisoning in three bovines. The history of natural poisoning was obtained at the site of the outbreak. Clinical examinations were carried out and hepatic enzymes were analyzed. A post-mortem examination was carried out at the outbreak location on a cow that had died. Several samples were collected from the animal's internal organs to carry out histopathological examinations. E. contortisiliquum pods were given to cows in order to reproduce the disease. The natural poisonings were characterized by photosensitization and aborted young. Enterolobium photosensitization was not experimentally reproduced, but the animals fell sick and recovered after 52 days.

Sheep poisoning by Panicum dichotomiflorum in northeastern Brazil

Different species of Panicum, including P. dichotomiflorum,have been reported as a cause of photosensitization in sheep, horses, cattle and goats. An outbreak of hepatogenous photosensitization occurred in 3 flocks of hair sheep in the Brazilian semiarid region. Eighty one out of 365 sheep were affected and 39 died. The main affected animals were nursing lambs and sheep younger than one year old. Donkeys, goats and cattle grazing in the same pasture were not affected. Clinical signs were edema of the head, followed by dermatitis, mainly in the face, ears, and croup, ocular discharge, corneal opacity with blindness, and redness of the coronary band and hoof. At necropsy of one affected lamb the liver was yellowish. Upon histologic examination scattered necrotic hepatocytes were observed in the liver and focal areas of necrosis of myocytes appeared in the heart. Samples of P. dicotomiflorum were analyzed by TLC and those containing saponins were isolated by HPLC using RP-C18 column and eluted with a mixture of MeOH and H2O. The isolated compounds were submitted to ¹H and 13C NMR spectroscopy. Reactions were positive to furostanol saponins with the same Rf of the standard protodioscin (0.21) and methylprotodioscin (0.32). The spectroscopic results indicated a mixture of (25R)- and (25S)-protodioscin isomers in a proportion of 3:1, and methylprotodioscin.

Proso millet (Panicum miliaceum) poisoning in Iranian fat-tailed sheep

An outbreak of hepatogenous photosensitization and jaundice occurred in a flock of 253 fat-tailed sheep grazing pasture of proso millet (Panicum miliaceum) in Iran. Eighty three animals were clinically diagnosed intoxicated and 41 were found dead and locally grown proso millet was implicated as the cause. The diagnosis of toxicosis was based on clinical signs as well as laboratory and pathological findings. Clinical signs including increase of heart rate (99 bpm), respiratory rate (55 breaths/min) and temperature (40.4°C), photosensitization, severe head swelling, decreasing appetite, and severe icterus in mucosal membranes were observed about 20 days after grazing pastures of millet. Serum biochemical parameters were analyzed through standard biochemical techniques on blood samples collected from 27 intoxicated sheep as well as ten clinically healthy sheep as control group. Serum biochemistry suggested hepatobiliary and hepatoparenchymal damage with impaired renal function. Laboratory findings indicated significant (P < 0.05) increase in the concentrations of creatinine, blood urea nitrogen (BUN), total bilirubin, direct and indirect bilirubin, and the activities of aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT). At post-mortem examination, liver and kidneys were affected. Gross lesions were characterized by pyknosis, karyolysis, kariorrhexis, and cytoplasmic hypereosinophilia in the cells of affected tubules especially proximal tubules, indicating an acute tubular necrosis. Severe centriacinar and periacinar hepatic lipidosis with a lot of cytoplasmic vacuoles in hepatocytes were observed histologically. There is no specific treatment for millet toxicosis; however, feeds can be changed to alfalfa and wheat hay and supportive care with fluid therapy and cathartic administration is recommended.

Steroidal saponin concentrations in Brachiaria decumbens and B. brizantha at different developmental stages

Brachiaria species contain steroidal saponins and are involved in outbreaks of hepatogenous photosensitization. This research presents the levels of a steroidal saponin, protodioscin, in the seeds and aerial parts of B. brizantha and B. decumbens during different developmental stages (growth, bloom, fructification and seed fall). The butanolic fraction of the ethanolic extract of each stage was submitted to thin layer chromatography (TLC) and spectrophotometric analysis through the Ehrlich reagent in 515nm. The chromatograms in TLC of the butanolic fraction of B. brizantha and B. decumbens showed similar spots as the protodioscin standard. The estimated level of protodioscin isomers in B. brizantha and B. decumbens ranged from 0.5% to 2.1%, having the highest level at the end of their developmental stages during seed falling comparison with the previous one. Protodioscin was not detected in the seeds. Outbreaks of Brachiaria spp. poisoning in central Western Brazil are frequently observed in pastures that had been more than 30 days without animals grazing, and also during the growing or blooming stage of the pastures. Other saponin determinations in toxic and non toxic pastures are necessary to determine the saponin concentrations that cause intoxication.

Patogênese, sinais clínicos e patologia das doenças causadas por plantas hepatotóxicas em ruminantes e eqüinos no Brasil

Plantas que causam lesões hepáticas em ruminantes e eqüinos constituem um grupo importante de plantas tóxicas no Brasil. Em geral essas plantas podem ser divididas em três grandes grupos: plantas que causam necrose hepática aguda; plantas que causam fibrose hepática; e plantas que causam fotossensibilização. Em algumas dessas plantas os princípios tóxicos já foram identificados. Das plantas que causam necrose hepática aguda, os carboxiatractilosídeos estão presentes em Cestrum parqui e Xanthium cavanillesi. Os alcalóides pirrolizidínicos estão presentes nas plantas que causam fibrose hepática (Senecio spp., Echium plantagineum, Heliotropum spp. e Crotalaria spp.). Das plantas que causam fotossensibilização hepatógena são conhecidos os furanossesquiterpenos em Myoporum spp., triterpenos em Lantana spp., e saponinas esteroidais em Brachiaria spp. e Panicum spp. O quadro clínicopatológico dessas intoxicações e o mecanismo geral da insuficiência hepática, incluindo meios de diagnóstico, são descritos neste artigo de revisão.

Serum protein concentrations, including acute phase proteins, in calves with hepatogenous photosensitization

One hundred 6- to 12-month-old Nelore calves were allotted into control group (G1; 50 healthy calves) and photosensitization group (G2; n= 50). Blood samples were collected 12 to 24 hours after the onset of dermatitis (M1), and 15 to 30 days after that (M2), at time of resolution of clinical signs. Serum protein electrophoresis was performed by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Eighteen serum proteins with molecular weights ranging from 16,000 to 189,000 daltons (Da) were identified in all calves. In M1 and M2 serum concentrations of proteins with molecular weights of 115,000Da (ceruloplasmin), 61,000Da (a1-antitrypsin), 45,000Da (haptoglobin), and 40,000Da (acid glycoprotein) were significantly increased in calves. In conclusion, measurement of serum acute phase protein concentrations may be useful in monitoring the progression of bovine hepatogenous photosensitization, including guide probable alteration on therapeutic procedures.

Toxicity testing of saponin-containing Yucca schidigera Roetzl. juice in relation to hepato- and nephrotoxicity of Narthecium ossifragum (L.) Huds

Yucca schidigera juice in doses of 1.5 g (63 mg sapogenin) and 3.0 g (126 mg sapogenin) per kg live weight was administered intraruminally to 30 lambs for 21 days to investigate whether the saponins in Y. schidigera were toxic to lambs and whether they could cause hepatogenous photosensitisation. Twelve lambs died or had to be euthanised. The main pathological findings in the diseased lambs were acute tubular necrosis in the kidneys, dehydration and watery content in the gastrointestinal tract. Fifteen lambs were euthanised at the end of the study, and the main pathological findings in dosed animals were accumulation of homogeneous pale PAS-positive material in the hepatocytes. There was a rise in serum creatinine and urea concentrations in the lambs with renal lesions the day before they died. Major Y. schidigera-related saponins were found in the liver and kidney samples from all lambs that were dosed with Y. schidigera juice. The results of the present study demonstrate that un-hydrolysed saponins can be absorbed from the gastrointestinal tract. The possible role of saponins in causing nephrotoxicity is discussed.

Sapogenin Levels in Narthecium ossifragum Plants and Ovis aries Lamb Faeces during Two Alveld Outbreaks in Møre og Romsdal, Norway, 2001

The proposal that saponins produced by the lily bog asphodel (Narthecium ossifragum) may be the direct cause of the hepatogenous photosensitization disease alveld seen in Norwegian lambs was investigated by comparing sapogenin levels in two control and two toxic pastures, and in faeces from lambs grazing the four pastures in the Halsa and Surnadal municipalities, Møre og Romsdal county, Norway. Generally similar levels of sapogenins, determined after hydrolysis of parent plant saponins, were found in Narthecium leaves collected in June/July 2001 from the two alveld outbreak areas and two nearby control areas. Differences in the median sapogenin levels determined for leaf samples in outbreak and control areas were not statistically significant. The total level of free and conjugated sapogenins in faeces recovered from the rectums of lambs grazing the outbreak and control pastures areas varied greatly. The results obtained do not support the hypothesis that a dose-response relationship exists between Narthecium saponin levels and the occurrence of alveld outbreaks.

Crystal-associated cholangiopathy in sheep grazing Brachiaria decumbens containing the saponin protodioscin

An outbreak of hepatogenous photosensitization is reported in a flock of 28 sheep grazing Brachiaria decumbens in Mato Grosso do Sul State, Central-Western Brazil. Seven lambs and an adult sheep were affected and 6 of them died. Two surviving affected lambs and one lamb without clinical signs had increased serum values of gamma glutamyltransferase, bilirubin, and cholesterol. In two adult unaffected sheep those parameters were within normal values. An adult sheep submitted to necropsy presented moderate body condition, unilateral corneal opacity, drying of the muzzle, moderate jaundice, increased lobular pattern of the liver, and a distended gallbladder. Histological lesions were epithelial degeneration, necrosis, and hyperplasia of small bile ducts. Mild amounts of foamy macrophages were observed, mainly in the centroacinar zone. Diffuse swelling and vacuolation were observed in hepatocytes. Crystal negative images were found within bile ducts, foamy macrophages, and the lumen of some renal tubules. The heart showed multifocal areas of degeneration and necrosis of the muscle fibers. Pasture samples (Brachiaria decumbens) contained 2.36% of protodioscin. No Pithomyces chartarum spores were found in the pasture. Samples from a similar neighboring B. decumbens pasture grazed by cattle without photosensitization contained 1.63% of protodioscin isomers. Outbreaks of photosensitization caused by Brachiaria spp. are common in cattle in the Brazilian Cerrado (savanna) with about 51 million hectares of Brachiaria spp pastures. Sheep farming has been recently developed in this region, and the number of sheep is increasing significantly. Because sheep are more susceptible than cattle to lithogenic saponins, poisoning by Brachiaria should be an important limiting factor for the sheep industry.

The livestock photosensitizer, phytoporphyrin (phylloerythrin), is a substrate of the ATP-binding cassette transporter ABCG2

Hepatogenous photosensitization occurs in livestock following damage to the liver or biliary apparatus that results in impaired excretion of phytoporphyrin (phylloerythrin), a photosensitizer. Based on earlier observations that porphyrin-based photosensitizers are substrates of the ATP-binding cassette transporter ABCG2, we examined the ability of the hepatic transporters ABCB1 (P-glycoprotein) and ABCG2 to transport phytoporphyrin. Transport of phytoporphyrin was blocked by the ABCG2-specific inhibitor fumitremorgin C (FTC) in human embryonic kidney cells transfected with full length human ABCG2, while no transport by cells transfected with human ABCB1 was noted. FTC-inhibited transport of phytoporphyrin was also demonstrated in ABCG2-expressing LLC-PK1 pig kidney cells, consistent with the idea that the pig orthologue, like human ABCG2, transports the photosensitizer. ABCG2 expression was confirmed by immunohistochemistry in the hepatocytes of cow, pig and sheep livers. We conclude that phytoporphyrin is a substrate for ABCG2 and that the transporter is likely responsible for its biliary excretion.

Pithomycotoxicosis (facial eczema) in ruminants in the Azores, Portugal

Outbreaks of pithomycotoxicosis (facial eczema), a hepatogenous photosensitisation caused by the mycotoxin sporidesmin, have affected ruminants in the Azores Islands of Portugal after warm, humid periods during late summer and...

Spectrofluorometric analysis of phylloerythrin (phytoporphyrin) in plasma and tissues from sheep suffering from facial eczema

AIMS: To study the increase in phylloerythrin concentration in plasma and the disposition of phylloerythrin in skin and other tissues of sheep in which the hepatogenous photosensitisation, facial eczema, had been experimentally induced by dosing with the mycotoxin, sporidesmin. Spectroscopic differences between plasma and skin measurements of animals kept inside and outside after dosing were also studied in order to establish whether phylloerythrin undergoes photodegradation when exposed to sunlight. METHODS: Twenty-six Romney x Polled Dorset (25-30 kg) weaned female lambs were purchased from a commercial flock in the Waikato region, New Zealand. Twenty-two of these lambs were dosed with 0.25 mg sporidesmin/kg liveweight on each of two consecutive days (Days —1 and 0); the remaining four lambs served as controls. Both sporidesmin-dosed lambs and controls were randomly divided into two penned groups, one group housed inside in a darkened room and the others outside, exposed to natural sunlight. The lambs were fed green lucerne pellets and lucerne chaff ad libitum for 10 days prior to dosing and until Day 12 after the first dose; thereafter, all the lambs were fed fresh, cut grass (mainly ryegrass) ad libitum, until the end of the experimental period on Day 26. Plasma samples collected on Days —2, 7, 10, 12, 14, 17, 20 and 25 were analysed for gamma glutamyltransferase (GGT) activity, bilirubin concentration, and the fluorescence spectrum of phylloerythrin. Spectrofluorometric analysis of phylloerythrin in skin was performed in vivo on the same days, using an external fibre-optic probe. RESULTS: Eight of 11 lambs (73%) kept outside after sporidesmin dosing became photosensitised during the experimental period. None of the sporidesmin-dosed animals kept inside showed clinical signs of photosensitisation. The GGT activity in plasma increased exponentially during the experimental period in all sporidesmin-dosed animals until it reached a plateau. All plasma obtained from sporidesmin-dosed sheep had spectral characteristics similar to those of phylloerythrin, namely a peak in the excitation spectrum at 422 nm and strong emission band at 650 (SE 1) and 709 (SE 1) nm. The fluorescence under excitation at 422 nm of phylloerythrin added to plasma from control lambs had identical peaks. Emission spectra obtained from plasma from healthy sheep without addition of phylloerythrin showed either no fluorescence or minor fluorescence at around 671 nm. Fluorescence in skin of sporidesmin-dosed animals had similar spectra to that in plasma. The appearance of the phylloerythrin-like spectra occurred 2–3 days later in the skin than in plasma, and phylloerythrin in sunlight-exposed skin did not suffer photodegradation during the course of the study. CONCLUSION: Plasma concentrations of phylloerythrin in healthy sheep were <0.1 µmol/l, and clinical signs of photosensitisation were not evident until concentrations exceeded 0.3 µmol/l. Plasma concentrations of phylloerythrin rose as high as 4.9 µmol/l in some animals. The concentration of phylloerythrin in skin began increasing 2–3 days later than that in blood. Hepatogenous photosensitisation can be diagnosed by analysis of plasma phylloerythrin concentrations using a spectroscopic method.

Measurement of phylloerythrin (phytoporphyrin) in plasma or serum and skin from sheep photosensitised after ingestion of Narthecium ossifragum

b>AIM:> To establish a method for measuring phylloerythrin in plasma or serum and skin from lambs photosensitised after ingestion of the plant, Narthecium ossifragum, which induces an hepatogenous photosensitisation similar to the disease known as facial eczema in sheep. METHODS: For two successive summers, lambs were grazed on uncultivated pastures containing N. ossifragum. Clinical photosensitisation was deemed to have occurred when symptoms such as restlessness, scratching, oedema and reddening of the skin were observed. Sixteen lambs that exhibited signs of photosensitisation were included in this study in the first year and five in the following year. A total of 16 clinically healthy lambs served as controls. Fluorescence emission and excitation spectra of phylloerythrin were measured in plasma or serum samples from the 21 photosensitised and 16 non-photosen-sitised lambs. In the first year of the study, skin samples were collected post mortem from the ear, lip, neck, nose, leg, belly, udder, back, vulva and perineal region, from all photosensitised and from seven non-photosensitised lambs, and examined by fixing them between two glass plates (each of 1 mm thickness) and placing them at a fixed angle in front of a fluorescence spectrofluorometer. RESULTS: All plasma or serum samples obtained from the photosensitised lambs exhibited strong phylloerythrin-like fluorescence of identical spectra; maximum fluorescence was at 650 and 711 nm, and maximum excitation at 425 nm. Emission spectra obtained from plasma or sera from non-photosensitised sheep grazing the same N. ossifragum-containing pastures exhibited either no or only minor fluorescence. Phylloerythrin concentration in plasma or serum exceeded 0.3 µg/ml before clinical photosensitisation occurred, whereas the concentration in samples from clinically healthy lambs was <0.05 µg/ml. Fluorescence from skin samples from the photosensitised lambs showed emission peaks at 650, 670 and 711 nm, whereas the phylloerythrin emission peaks at 650 and 711 nm were not observed in skin from clinically healthy lambs. CONCLUSION: Plasma concentrations of phylloerythrin in healthy sheep were <0.05 µg/ml. Clinical signs of photosensitisation were not observed until the concentration of phylloerythrin in plasma exceeded 0.3 µg/ml. This is the first reported spectroscopic method for analysis of phylloerythrin and the only one which does not involve exposure of the analyst to hazardous chemicals. It has the additional benefit of distinguishing between hepatogenous and primary photosensitisation.

Experimental Tribulus terrestris poisoning in sheep: clinical, laboratory and pathological findings.

Eleven native sheep, 1-2 years old, of both sexes were randomly divided into two groups, 6 sheep being allocated to the experimental group and 5 serving as controls. The sheep in the experimental group were fed 80% Tribulus terrestris and 20% alfalfa hay and wheat straw, while the control sheep were given a mixture of 40% alfalfa hay and 60% wheat straw. Clinical signs of hepatogenous photosensitivity were observed from day 11, including reddening and crust formation on the muzzle, nose, ears and eyelids, depression, weight loss, icterus, conjunctivitis, and yellow discoloration of the urine. Laboratory findings on weekly samples indicated significant differences (p < 0.05) in white blood cell count, total plasma protein and fibrinogen, total and direct bilirubin, blood urea nitrogen and creatinine concentrations, and aspartate aminotransferase and alkaline phosphatase activities. There were no significant differences in the packed cell volume, in the neutrophil, lymphocyte or eosinophil counts, or in the serum calcium, phosphorus, potassium, sodium or chloride concentrations. At necropsy of the experimental animals, there were various degrees of generalized icterus and the livers were swollen and discolored by bile pigment. Histopathological examination revealed varying amounts of crystalloid material in the bile ducts and renal tubules, hepatocellular degeneration, biliary fibrosis and proliferation, renal tubular necrosis and focal necrosis of cardiac muscle.

Intoxicação espontânea por Senecio brasiliensis (Asteraceae) em ovinos no Rio Grande do Sul

Descreve-se a ocorrência de um surto de intoxicação espontânea por Senecio brasiliensis em ovinos em um estabelecimento do município de Mata, Estado do Rio Grande do Sul, em meados de janeiro de 1997. De um total de 94 ovinos, 51 (54,25%) animais adoeceram e 50 (53,2%) morreram. Esse rebanho permaneceu durante aproximadamente 7 meses (de junho de 1996 a janeiro de 1997) em piquetes de pastagem nativa onde havia grande quantidade de S. brasiliensis. O quadro clínico manifestado pelos animais afetados consistia em fotossensibilização, emagrecimento progressivo, apatia, fraqueza, perturbações neurológicas como depressão, andar a esmo e desequilibrado, icterícia e hemoglobinúria. Houve melhora das lesões de pele naqueles ovinos que desenvolveram fotossensibilização hepatógena depois que foram retirados do sol. As principais lesões macroscópicas observadas em 9 dos 10 ovinos necropsiados incluíam fígado diminuído de tamanho, firme, difusamente marrom amarelado ou esverdeado, com quantidades variáveis de nódulos de 1-3 mm de diâmetro, bem circunscritos, salientes na cápsula, amarelados, distribuídos aleatoriamente por todo o parênquima. A vesícula biliar estava repleta e preenchida por bile verde escura e espessa. Havia também derrames cavitários (hidropericárdio e ascite). Crise hemolítica aguda fatal associada à intoxicação crônica hepatógena por cobre foi observada em cinco ovinos. Além das lesões hepáticas macroscópicas já mencionadas, foi observada icterícia generalizada da carcaça, rins tumefeitos, friáveis, difusamente escurecidos ou com fino pontilhado enegrecido; a urina era marrom escura (hemoglobinúria). As principais lesões microscópicas foram observadas no fígado e consistiam em hepatomegalocitose, proliferação de ductos biliares (hiperplasia ductal) e fibrose periportal moderada acompanhada de infiltrado inflamatório mononuclear. Macrófagos carregados de pigmento acastanhado formavam aglomerados nas tríades portais ou estavam dispersos entre os hepatócitos remanescentes. O material armazenado no citoplasma desses macrófagos correspondia a ceróide e cobre, positivo nas técnicas de PAS e rodanina, respectivamente. Nos rins de cinco animais, havia nefrose hemoglobinúrica caracterizada por degeneração e necrose do epitélio tubular, presença de hemoglobina e hemossiderina no citoplasma das células epiteliais dos túbulos contorcidos e cilindros de hemoglobina na luz tubular. Evidência morfológica de encefalopatia hepática incluía degeneração esponjosa (status spongiosus) da substância branca do encéfalo. Achados ultra-estruturais no fígado incluíam graus variáveis de degeneração hepatocelular caracterizada pelo acúmulo de numerosas gotas lipídicas no citoplasma das células hepáticas e presença de lisossomos carregados de material eletrodenso que, na maioria dos casos, correspondia à lipofuscina-ceróide. Adicionalmente, havia discreta dilatação do retículo endoplasmático rugoso e moderada hiperplasia do retículo endoplasmático liso em algumas regiões do citoplasma dos hepatócitos. No epitélio dos túbulos contorcidos proximais do rim foi observado edema intracelular e diversas alterações mitocondriais de caráter degenerativo que incluíam tumefação, desorganização e ruptura das cristas, matriz finamente granular, acúmulo de gotículas de gordura e ruptura das membranas em alguns casos. Lisossomos contendo material fortemente eletrodenso foram observados em muitas células tubulares renais. O exame laboratorial de fragmentos de fígado e rim dos ovinos afetados revelou níveis elevados de cobre que variaram respectivamente de 369 ppm a 1248 ppm e 152 ppm a 687 ppm com base na matéria seca. O diagnóstico de intoxicação por Senecio brasiliensis baseou-se em dados epidemiológicos, clínicos, de necropsia, histopatológicos e laboratoriais.

Metabolism of diosgenin-derived saponins: implications for hepatogenous photosensitization diseases in ruminants

Disposition of sapogenins in the gastrointestinal tract of a lamb was determined after daily dosing of a diosgenin-derived saponin extract during late spring. The saponin (26.1 g) was obtained by ethanolic extraction of powdered Costus speciosus rhizomes followed by butanol–water partitioning. Upon completion of dosing, samples of gut contents and internal organs were subjected to chemical analysis and histological examination. No clinical symptoms, changes in serum chemistry, or microscopic changes were observed. Dosing of a saponin extract revealed that ruminal metabolism resulted in conversion of diosgenin to epismilagenin, smilagenone, smilagenin and tigogenin. Electrospray mass spectrometry of selected ethanolic ruminal extracts showed the absence of diosgenin-derived saponins 1 h after dosing, indicating that rapid hydrolysis of saponins by ruminal micro-flora occurred. Epismilagenin was the dominant conjugated sapogenin detected in the bile sample, no free sapogenins were identified. Three distinct zones of metabolic activity were identified in the gastrointestinal tract of the dosed lamb. The rumen appeared to be a region where the ingested diosgenin-derived saponins underwent hydrolysis and reduction and were converted to tigogenin, smilagenin, smilagenone and epismilagenin. The duodenum, jejunum and ileum formed an absorption and secretion zone where there appeared to be variation in both the concentrations of free and conjugated sapogenins. Sapogenins appeared to undergo continued epimerization in the caecum and colon. The plant saponins administered in this study were readily metabolized, but did not appear to induce crystal-associated hepatogenous photosensitization in the dosed lamb.

Fotossensibilização hepatógena em bovinos no sul do Rio Grande do Sul

São descritos aspectos epidemiológicos e patológicos de 19 surtos de fotossensibilização observados em bovinos na região sul do Rio Grande do Sul, no período entre 1984 e 1997. A doença ocorre, principalmente, nos meses de outono e primavera com morbidade de 0,08% a 64% e mortalidade de 0 a 14%. Todas as categorias são afetadas e as áreas onde ocorrem os surtos se caracterizam por campos planos e baixos, freqüentemente utilizados para o plantio de arroz irrigado ou pastagens de azevém, trevo e aveia ou, ainda, em áreas similares com vegetação nativa. A enfermidade caracteriza-se, clinicamente, por depressão, anorexia, salivação intensa e fotossensibilização observada nas áreas de pele despigmentadas e desprovidas de pêlos como o focinho, ao redor dos olhos, virilha, úbere e vulva. Nos casos severos, observam-se lesões caracterizadas por ressecamento, rachaduras e desprendimento da pele, principalmente do dorso e membros, deixando à mostra superfície ulcerada. As lesões macroscópicas são características de insuficiência hepática, observando-se icterícia, fígado aumentado de tamanho com coloração amarelada, aumento de tamanho da vesícula biliar, presença de líquido nas cavidades e, em alguns casos, edema do tecido subcutâneo e peritônio. As lesões histológicas caracterizam- se, principalmente, por lesões degenerativas do parênquima hepático, observando-se tumefação e vacuolização difusa ou zonal dos hepatócitos. Observa-se, também, apoptose ou focos de necrose distribuídos aleatoriamente pelo parênquima, presença de megalócitos e, ainda, fibroplasia dos espaços porta e proliferação de células de ductos biliares. A enfermidade é caracterizada como fotossensibilização hepatógena relacionada a dano primário ao parênquima hepático, de etiologia não determinada, uma vez que foram descartadas as principais causas conhecidas de fotossensibilização de origem hepática em bovinos.

Catalase gene is associated with facial eczema disease resistance in sheep.

Facial eczema (FE) is a hepatogenous photosensitization disease of ruminant animals, particularly in sheep which vary widely in their susceptibility to the disease. The liver damage is caused by the mycotoxin, sporidesmin. There is evidence that the toxicity of sporidesmin is due to its ability to generate 'active oxygen' species. We evaluated the catalase gene, which encodes an enzyme with antioxidant functions, as a candidate for determining the susceptibility of sheep to the disease. Two microsatellite markers, OarSHP3 and OarSHP4, which flank the sheep catalase gene, were isolated from a Yeast Artificial Chromosome (YAC) clone. These markers mapped the catalase locus by linkage to ovine chromosome 15. Eleven informative markers spaced throughout chromosome 15, inclusive of the catalase marker OarSHP4, gave no significant linkage with the disease traits when analysed in four outcross resource pedigrees. However, OarSHP3 and OarSHP4 allele frequencies showed significant differences between FE resistant and susceptible selection-lines. Comparison of sequences of catalase cDNAs from sheep of resistant and susceptible lines showed only two silent mutations. A single nucleotide polymorphisms (KP1) in exon 6 of the catalase gene also showed significant differences in allele frequencies between the selection lines. The lack of evidence for linkage in outcross pedigrees, but the significant association in the genetic lines, implies that catalase is involved in determining the susceptibility of sheep to facial eczema, and that the candidate gene's effect is probably recessive or minor.

Urinary excretion of immunoreactive sporidesmin metabolites in sheep in relation to factors influencing susceptibility to sporidesmin intoxication

Aim. To study the urinary disposition of orally administered sporidesmins A and D in sheep and identify factors influencing their kinetics, particularly the influence of breeding for resistance and susceptibility to sporidesmin, the mycotoxin responsible for the hepatogenous photosensitisation, facial eczema. Methods. A competitive ELISA was used to monitor urinary output of immunoreactive metabolites after the intraruminal administration, tofemale Romney sheep,of either sporidesmin A or sporidesmin D, the nontoxic analogue. Preliminary characterisation of metabolites was carried out using HPLC with fractions monitored by ELISA. Results. Maximum urinary excretion rates of immunoreactive metabolites occurred 2–8 h after dosing with sporidesmin D and 15–30 h after dosing with sporidesmin A. Sporidesmin D caused no liver injury, as detected by changes in serum enzyme activity, while the liver injury caused by sporidesmin A was greatest for the sheep with the highest cumulative output of metabolite. When sporidesmin D was administered in two separate doses to sheep bred for either resistance or susceptibility to facial eczema, the variability of metabolic output between sheep within groups was much less after the second dose. The mean urinary metabolite excretion was greater for the susceptible than the resistant sheep but the difference was not significant. Potentiation (caused by pre-administration of small doses of sporidesmin A) resulted in a more severe reaction to the dosed sporidesmin A. Urinary output of metabolite was less in the potentiated than in the unpotentiated sheep. When resistant and susceptible sheep were dosed with sporidesmin A after potentiation there was no difference between them in their cumulative totals or excretion rates of immunoreactive metabolites. However, the volume of urine produced by the susceptible sheep was lower and less variable than the resistant sheep and consequently the concentration of their urinary metabolites was higher. Preliminary ELISA examination of HPLC-fractionated urine from a sheep dosed with sporidesmin A indicated the presence of several metabolites of sporidesmin. Conclusion. Sporidesmin A and metabolites are rapidly excreted in urine but not as rapidly as sporidesmin D and its metabolites. Only minor differences between sheep bred for resistance and susceptibility were seen. Potentiation caused a more severe reaction to sporidesmin A and less urinary excretion of the sporidesmin and its metabolites. Clinical relevance. This work is part of a programme with the aim of identifying FE-resistant animals without the need for sporidesmin dosing.