Sapogenin Levels in <Emphasis Type="Italic">Narthecium ossifragum</Emphasis> Plants and <Emphasis Type="Italic">Ovis aries</Emphasis> 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.     

Fungi onNarthecium ossifragum leaves and their possible involvement in alveld disease of Norwegian lambs

Spores ofPithomyces chartarum (Berk. & Curt.) M.B. Ellis were only rarely seen on leaves ofNarthecium ossifragum (L.) Hudson collected in summer from five areas in western Norway in which alveld, a photosensitization disease of lambs, is endemic.Cladosporium magnusianum (Jaap) M.B. Ellis was found on all 118 leaf samples collected in the summers of 1990 and 1991. The hypothesis thatP. chartarum contributes to the aetiology of alveld could not be supported, but it is possible thatC. magnusianum may have a role in the causation of the disease.     

Ruminal Metabolism in Sheep of Saponins from Yucca schidigera

Fifty-five mg per kg live weight of crystallized Yucca schidigera saponins, corresponding to 26 mg/kg live weight of sapogenins, was given daily intraruminally to two lambs for 11 consecutive days. Neither of the lambs showed any sign of toxicity throughout the experimental period. One lamb was killed 5 h after the last dose and GC-MS analysis of the free and conjugated sapogenin content samples of liver, and of the contents of the rumen, omasum, abomasum, duodenum, jejunum, ileum, colon and rectum, of faecal samples collected before dosing started, and of parts of the administered Yucca saponin were performed. The Yucca material contained mainly sarsasapogenin and smilagenin saponins. Ingested saponins were quickly hydrolysed in the rumen to free sapogenins and, in part, epimerized at C-3 to afford episapogenins. The absorption of free sapogenins appeared to occur in the jejunum. The metabolism of Yucca saponins was identical to that of Narthecium ossifragum saponins, and it is suggested that Yucca saponins could replace N. ossifragum saponins for toxicity studies on the latter plant.     

Ovine Metabolism of Saponins: Evaluation of a Method for Estimating the Ovine Uptake of Steroidal Saponins from Narthecium ossifragum

A sheep was dosed three times per day over six consecutive days with 70 g Narthecium ossifragum, and once on the seventh day with 70 g N. ossifragum. Additionally, it was dosed once on days 1–7 with 20 mg of [20,23,23-²H₃]sarsasapogenin. After 7 days, the sheep was killed and GC-MS analysis of the free and conjugated sapogenin content in bile, urine, rumen, duodenum, jejunum, colon and rectum samples collected from the sheep, faecal samples collected on days 4–7, and dosed plant material was performed. The N. ossifragum contained mainly sarsasapogenin and smilagenin. Only negligible levels of deuterium-labelled sarsasapogenins were detected in the samples from the animal. Ingested saponins were quickly hydrolysed in the rumen to free sapogenins and, in part, epimerized at C-3 to afford episapogenins. The absorption of free sapogenins appeared to occur in the jejunum. The concentration of sapogenins in faeces reached a plateau 108 h after dosing started.     

Glycogen accumulation and histological changes in the livers of lambs with alveld and experimental sporidesmin intoxication

Alveld is a hepatogenous photosensitization disease seen in lambs grazing Narthecium ossifragum pastures in Norway. Mycotoxins, possibly sporidesmin, have been suspected to cause the liver damage in alveld as in facial eczema. The histological changes in the liver of alveld cases and in lambs photosensitized after experimental sporidesmin intoxication were compared. The liver damage characterized by necrosis in single centrilobular hepatocytes, was of the same type in both conditions. Minor to moderate portal fibroplasia and bile duct proliferation were almost always present. Accumulated glycogen was seen in hepatocytes in the centrilobular areas. This was significantly correlated to the enzymatically measured glycogen content and there was good correlation between parenchymal damage and glycogen accumulation. The glucose-6-phosphatase and glycogen phosphorylase activities were normal. These findings indicate that parenchymal damage, rather than obstruction of the bile ducts, caused the retention of phylloerythrin both in alveld cases and in experimentally sporidesmin-intoxicated lambs. The accumulation of glycogen could not be explained.Abbreviations HE haematoxylin and eoson - PAS periodic acid Schiff - EDTA ethylene diamine tetracetic acid     

Accumulation of Sapogenin Conjugates and Histological Changes in the Liver and Kidneys of Lambs Suffering from Alveld,a Hepatogenous Photosensitization Disease of Sheep Grazing Narthecium ossifragum

Sixteen lambs exhibiting hepatogenous photosensitization (alveld) after grazing pasture containing Narthecium ossifragum and seven nonphotosensitized lambs grazing the same pastures were studied. All the alveld-affected lambs revealed liver damage dominated by single cell necrosis, portal fibroplasia and bile duct proliferation. Crystalloid clefts were demonstrated in the bile ducts of two and in the hepatocytes and Kupffer cells of nine photosensitized lambs. Plasma bilirubin concentration was severely increased in ten of the cases of alveld whereas the activity of aspartate aminotransferase was moderately to severely increased in seven cases. The activity of glutamate dehydrogenase was moderately elevated in one of the photosensitized lambs. The main histopathological findings in the kidneys from the alveld-affected lambs were dilated tubules, often with eosinophilic material in the tubular lumina. Regenerative changes were seen in a large proportion of the renal sections. Elevated plasma concentrations of urea and creatinine, and the renal histopathological changes, suggested that the photosensitized lambs had been through a phase of renal injury. Analysis of the free and conjugated sapogenin content in liver tissue and bile was performed by gas chromatography–mass spectrometry. There were significantly higher concentrations of conjugated episapogenins in both the liver and bile in the alveld-affected lambs than in the nonphotosensitized lambs.     

Alveld-Producing Saponins: II. Toxicological Studies

The phototoxic lamb disease alveld, prevalent in South-Western Norway, is caused by ingestion of Narthecium ossifragum. Earlier studies have shown that peroral administration of large amounts of crude saponins from this plant elicits the disease. Such saponins have now been purified further by 2 different methods (A and B). Two A type preparations resulted in alveld when fed to 2 lambs. The most highly purified preparation (type B) did not cause alveld in the 2 lambs tested. Lambs vary, however, in their susceptibility to the disease. Both types of preparations led to increases in serum aspartate aminotransferase, bilirubin and 5′-nucleotidase in rats when injected intraperitoneally in amounts of 50 or 100 mg/kg body Weight. Cannulation of the bile duct showed that injected saponins reduced both the volume of bile and the amounts of bilirubin and bile acids excreted. Histological changes seen in the light microscope were, except for the most peripheral parts of the liver, hardly noticable. These observations support the view that saponins are the liver-toxic agents responsible for alveld. The possibility is discussed that the effect arises through a change in the lipid environment of carrier-mediated transport systems.     

Tolerance to the Nephrotoxic Component of Narthecium ossifragum in Sheep: The Effects of Repeated Oral Doses of Plant Extracts

Young adult sheep were dosed with extracts of Narthecium ossifragum plants by the oral or parenteral routes and the resulting nephrotoxicity was assessed from the increases in the concentrations of creatinine and urea in the serum. Following single intraruminal or intraperitoneal doses of extracts derived from 30 g N. ossifragum (wet weight) per kg live weight (kg lw), serum creatinine concentrations increased from about 100 mol/L to between 260 and 510 mol/L. The serum urea concentrations increased from about 5–8 mmol/L to between 11 and 66 mmol/L in individual sheep. Daily intraruminal administration of 5–30 g/kg lw to three sheep over a 10- or 15-day period increased creatinine concentrations from 100 mol/L to 300–760 mol/L, and urea concentrations from 5–8 mmol/L to 35 mmol/L. A single intraperitoneal challenge dose of 30 g/kg lw, delivered 7 or 12 days after the final intraruminal dose, did not lead to increased serum creatinine or urea concentrations, indicating that oral treatment had apparently resulted in an increased tolerance to the nephrotoxic principle(s) in N. ossifragum.     

Nephrotoxicity in lambs apparently caused by experimental feeding withNarthecium ossifragum

Seven lambs were fed a mixture of bog plants containing 15 g (wet matter)Narthecium ossifragum per kg live weight for 10 consecutive days. Their serum creatinine concentration increased from day 1 to 4 but then fell to normal by day 6 after feeding started. In the same animals, the serum magnesium concentration was increased on days 3, 4 and 5. Histopathological examination of the kidneys of 2 lambs killed after being fed 25 gN. ossifragum per kg live weight for one day revealed tubular epithelial cell degeneration and necrosis. There were no signs of disease or distress in the experimental animals and their appetite remained normal throughout the experimental period. It was concluded thatN. ossifragum is probably nephrotoxic to lambs.Abbreviations ALP alkaline phosphatase - ASAT aspartate aminotransferase - -HBA -hydroxybutyric acid - CK creatine kinase - d.m. dry matter - -GT -glutamyltransferase - GLDH glutamate dehydrogenase - MCV mean cell volume - RBC red blood cells - SEM standard error of the mean - WBC white blood cells - w.m. wet matter     

Nephrotoxicity and hepatotoxicity in calves apparently caused by experimental feeding withNarthecium ossifragum

Seven calves were fed a mixture of bog plants containing 15 g (wet matter)Narthecium ossifragum per kg live weight for two consecutive days. Their serum creatinine, urea and magnesium concentrations increased, whereas the serum calcium concentration decreased. Histopathological examination of the kidneys of the 5 calves that were killed revealed tubular epithelial cell degeneration and necrosis. There were signs of liver dysfunction in all the calves including increased aspartate aminotransferase (ASAT), glutamate dehydrogenase (GLDH) and -glutamyltransferase activities.All the calves refused to ingestN. ossifragum after 2 days feeding, and their appetite for hay and concentrate was also reduced. It can be concluded thatN. ossifragum is nephrotoxic and hepatotoxic to calves.Abbreviations ALP alkaline phosphatase - ASAT aspartate aminotransferase - -HBA -hydroxybutyric acid - CK creatine kinase - d.m. dry matter - -GT -glutamyltransferase - GFR glomerular filtration rate - GLDH glutamate dehydrogenase - MCV mean cell volume - RBC red blood cells - SEM standard error of the mean - WBC white blood cells - w.m. wet matter     

Microsomal enzymes in lambs and adult sheep,and their possible relationship to alveld

The difference in susceptibility to alveld between lambs and adult sheep may be caused by differences in the microsomal enzyme activities in their livers. There was no difference in NADPH-cytochrome c reductase or 7-ethoxyresorufin-O-deethylase (EROD) activity between ewes, control lambs and phenobarbitone-dosed lambs 3 weeks after dosing ceased. However, aldrin epoxidase activity was at that time significantly highest in the phenobarbitone-dosed lambs and significantly lowest in the ewes. The liver cytosolic glutathione transferase activity was significantly highest in the ewes and significantly lowest in the control lambs at the same time.     

Detection of singlet oxygen in blood serum samples of clinically healthy lambs and lambs suffering from alveld disease

Alveld is a disease in lambs of domestic sheep (Ovis aries L.), characterized by a combination of photosensitivity and liver damage. Generation of singlet oxygen play a major role in phototoxicity reactions. The compound phylloerythrin (phytoporphyrin) is so far assumed to be the main photodynamic agent in hepatogenous photosensitivity diseases in sheep. Phylloerythrin is a potent photosensitizer and an efficient source of singlet oxygen. The compound accumulates in the peripheral circualtion upon liver damage. Liver dysfunction is also likely to cause an increase in the blood level of bilirubin. Formation of singlet oxygen by bilirubin is reported. In the present work the photosensitizing potential of serum has been measured and related to the bilirubin- and phylloerythrin levels in lambs suffering from alveld and in clinically healthy controls. The singlet oxygen level of the serum was taken as a measure of the photosensitizing potential. The observed singlet oxygen values in serum from alveld lambs were significantly higher than the corresponding values observed in clinically healthy control lambs. This indicates that the serum of the alveld lambs contains an elevated concentration of photosensitizer. The singlet oxygen level was not correlated to the concentration of bilirubin or phylloerythrin. The results indicate that the photosensitizing mechanism is quite complex and may involve other sensitizer(s) than phylloerythrin.     

Summer Variation in the Concentration of Steroidal Sapogenins in and the Degree of Fungal Infection on Narthecium ossifragum plants from Møre og Romsdal County,Norway

Thirty-nine leaf samples ofNarthecium ossifragum collected from eight sites in More og Romsdal County, Norway, during June–September 1997 and 41 leaf samples collected at five sites in the same county during June–August 1998 were analysed for the concentrations of steroidal sapogenins using GC-MS. The 1998 samples were also examined for fungal elements (conidia and hyphae) after incubation in a moist chamber for 10–14 days. The highest 1997 and 1998 leaf sapogenin concentrations (4881 and 7115 mg/kg dry matter, respectively) were 13–14 times greater than the lowest sapogenin concentrations found (344 and 531 mg/kg dry matter, respectively). The results did not reveal systematic differences in sapogenin concentrations between the two seasons, or between samples harvested early or late in the same seasons, or between sapogenin concentrations in plants harvested at different sites.Cladosporium magnusianum was the predominant fungus found in the samples. The degree of fungal infection on the samples was in generally low, but the number ofC. magnusianum colonies in the moist chamber preparations and fungal elements (conidia and hyphae) in leaf washings and on leaves tended to increase with time. Factor analysis and multiple regression analysis performed on the chemical and fungal results suggest that sporulation may have occurred in the fungi in response to increase in sapogenin concentrations.     

Investigations of Outbreaks of Contagious Caprine Pleuropneumonia in Eritrea

Mycoplasmas were isolated from freeze-dried lung samples from goats from the western lowlands of Eritrea suspected of being affected by contagious caprine pleuropneumonia. The goats belonged to two herds in which mortality and morbidity rates were high. Mycoplasma capricolum subsp. capripneumoniae was identified in most samples by the polymerase chain reaction and by conventional serological tests. The latex agglutination test detected more positive serum samples in both herds than did the complement fixation test. Following cloning, the isolates of M. capricolum subsp. capripneumoniae were analysed biochemically and shown to be metabolically similar. They oxidized glucose, N-acetylglucosamine, pyruvate and L-lactate with high affinity and mannose, glucosamine and 2-oxobutyrate with low affinity; they were unable to utilize maltose, trehalose, fructose or ethanol. Major improvements were seen in the growth yield of the Eritrean strains with the addition of pyruvate to the medium. Thus, it may be that organic acids are important energy sources for these strains and may be used in addition to or in place of glucose. In contrast to most other strains of the M. mycoides cluster, the Eritrean strains produced large amounts of hydrogen peroxide during the oxidation of NADH by lysed cells. This characteristic had previously been reported for strain M. F38, the type strain of M. capricolum subsp. capripneumoniae, although strain F38 did not metabolize sugars. Hydrogen peroxide has long been considered a pathogenicity factor in mycoplasma infections. This is the first isolation of M. capricolum subsp. capripneumoniae from Eritrea.     

Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in sheep from Mossoró, Rio Grande do Norte, Brazil

Toxoplasma gondii is an obligate intracellular parasite with a variety of hosts, responsible for reproductive problems and economic losses in sheep flocks. Neospora caninum was recently identified and its clinical presentation in sheep is similar to that of toxoplasmosis, which can cause repeated abortions, though less frequently in this species. In order to confirm the prevalence of these agents in the city of Mossoró, Rio Grande do Norte, Brazil, 409 serum samples from adult sheep (364 females and 45 males) were tested by the indirect immunofluorescence antibody test, using cut-off point at a dilution of 1:64 and 1:50 for T. gondii and N. caninum, respectively. From the 35 properties examined, 23 (65.7%) had at least one seropositive animal for T. gondii and six (17.1%) for N. caninum. The prevalence of seropositive animals for T. gondii was 20.7% and for N. caninum 1.8%. There was no association between the presence of the agent’s antibody and gender, reports of reproductive problems and presence of dogs and/or cats in the properties. T. gondii is well distributed and N. caninum has low prevalence in sheep and in the properties of the studied region.     

Prevalence of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> and <Emphasis Type="Italic">Neospora caninum</Emphasis> infections in sheep from Federal District,central region of Brazil

Serum samples from 1028 sheep were collected from 32 herds within Federal District, in the central region of Brazil. The samples were examined by indirect fluorescent antibody test (IFAT) using sera diluted 1:64 and 1:50 as cut-off values for the detection of antibodies against Toxoplasma gondii and Neospora caninum, respectively. The observed prevalence for T. gondii infection was 38.22% (26.81%<CI 0.95<49.62%), and the titers ranged from 64 to 65536. The observed prevalence for N. caninum infection was 8.81% (7.08%<CI 0.95<10.53%). The titers ranged from 50 to 51200. The reactant sera to both pathogens corresponded to 4.67% of the samples. The risk factors were not determined because of the absence of negative herds for T. gondii and the high proportion of positive herds for N. caninum (87.50%). The prevalence for T. gondii infection was significantly higher among males than in females. The present work is the first report on seroprevalence of T. gondii and N. caninum in sheep from Federal District and shows that infection by both parasites is widespread in the ovine population from this region.     

Diversity of swine Bordetella bronchiseptica isolates evaluated by RAPD analysis and PFGE

The degree of genetic diversity in 45 Bordetella (B.) bronchiseptica strains comprised of a vaccine strain (N = 1), reference strains (N = 3) and field isolates (N = 41) was evaluated using random amplified polymorphic DNA (RAPD) fingerprinting and pulsed-field gel electrophoresis (PFGE). Three candidate primers were selected for RAPD analysis after screening 20 random decamer oligonucleotides for their discriminatory abilities. The OPA-07, OPA-08 and OPA-18 primers yielded 10, 10, and 6 distinct fingerprint patterns, respectively. The most common identical RAPD pattern was produced by OPA-07 which was shared by 32 isolates (71.1%), the pattern produced by OPA-08 was shared by 26 isolates (57.8%), and the pattern produced by OPA-18 was shared by 40 isolates (88.9%). The RAPD patterns of the vaccine strain and the 3 reference strains did not match any of the patterns produced by the field isolates when primers OPA-07 and OPA-08 were used. PFGE using the restriction endonuclease XbaI produced a total of 15 patterns consisting of 4 PFGE types (A, B, B1 and C, differing by ≥ 4 bands) and 11 A subtypes (differing by ≤ 3 bands). Most of the field isolates exhibited identical type A and B patterns, suggesting that they were related. The vaccine strain and the three reference strains showed different PFGE patterns as compared to the identical type A strains.     

Sterol composition in polyene antibiotic-sensitive and resistant strains ofMalassezia pachydermatis

The membrane sterol compositions of ten strains ofMalassezia pachydermatis were analysed by gas-liquid chromatography. Six of these were wild-type strains from clinical sources that were sensitive to polyene antibiotics, whereas the other four strains were resistant mutants that had arisen after treatment withN-methyl-N-nitrosoguanidine or UV radiation.The wild-type strains contained 838.8 to 1291.7 µg of sterol per gram of freeze-dried mycelium. The sterols were composed of 79% ergosterol, 19% fecosterol and 2% lichesterol. The mutant strains contained 277.1 to 348.8 µg/g of sterols composed of 76% ergosterol and 24% fecosterol.Abbreviations MIC minimum inhibitory concentration - UV ultraviolet     

Clostridium perfringens epsilon toxin inhibits the gastrointestinal transit in mice

Epsilon toxin produced by Clostridium perfringens type B and D is a potent toxin that is responsible for a highly fatal enterotoxemia in sheep and goats. In vitro, epsilon toxin produces contraction of the rat ileum as the result of an indirect action, presumably mediated through the autonomic nervous system. To examine the impact of epsilon toxin in the intestinal transit, gastric emptying (GE) and gastrointestinal transit (GIT) were evaluated after intravenous and oral administration of epsilon toxin in mice. Orally administered epsilon toxin produced a delay on the GIT. Inhibition of the small intestinal transit was observed as early as 1 h after the toxin was administered orally but the effects were not observed after 1 week. Epsilon toxin also produced an inhibition in GE and a delay on the GIT when relatively high toxin concentrations were given intravenously. These results indicate that epsilon toxin administered orally or intravenously to mice transitorily inhibits the GIT. The delay in the GIT induced by epsilon toxin could be relevant in the pathogenesis of C. perfringens type B and D enterotoxemia.     

Peripheral white blood cell counts throughout pregnancy in non-aborting Neospora caninum-seronegative and seropositive high-producing dairy cows in a Holstein Friesian herd

Pregnancy is characterized by transient changes in the maternal immune system, also evident at peripheral level. The present study analyzes the kinetics and possible factors affecting peripheral white blood cell populations throughout pregnancy in a herd of high-producing dairy cows chronically infected or not with Neospora caninum. We examined 54 pregnant parous cows: 29 Neospora-seronegative and 25 Neospora-seropositive cows. Blood samples were collected on Days 90, 120, 150, 180 and 210 of gestation. General Linear Model (GLM) repeated measures analysis of variance showed that the interaction Neospora-seropositivity × parity significantly affected total leukocyte, neutrophil and monocyte counts with lower levels of total leukocytes, lower neutrophil and higher monocyte counts recorded in primiparous Neospora-seropositive cows. In addition, N. caninum-seropositive cows had significantly increased monocyte counts on Day 180 of gestation compared to seronegative ones. Other factors significantly associated with changes in total and/or differential leukocyte profiles were period of pregnancy, season, twin pregnancy and milk production. In conclusion, a parity-associated effect of chronic N. caninum infection was observed on peripheral blood cell profiles in dairy cattle during gestation.