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This study investigated the effects of intravenous (IV) administration of tetrathiomolybdate (TTM), and α(2)-adrenergic agonist clonidine (CLO) and α(2)-antagonist idazoxan (IDA), alone or in combination with TTM, on sheep fed low (LCu) and high (HCu) copper diets. Effects on bile flow, biliary Cu concentration and excretion, plasma Cu concentration, and lysosomal enzyme β-glucuronidase (β-GLU) activity in bile and plasma were determined. Tetrathiomolybdate alone or with CLO or IDA significantly enhanced biliary Cu excretion most likely by removing Cu from hepatocyte lysosomes as evidenced by a significant increase in β-GLU enzyme activity in bile. A significant increase in plasma β-GLU concentration occurred only in sheep treated with CLO in combination with TTM. Because of the lytic nature of the lysosomal enzymes, caution is advocated in use of drugs, especially α(2)-adrenergic agonists, to further enhance TTM-induced biliary Cu excretion in the treatment of chronic Cu poisoning in sheep., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This study determined the effects of dietary copper (Cu), molybdenum (Mo) and sulphur (S) on urinary Cu and zinc (Zn) excretion in cattle. Four Simmental and four Angus heifers were fed low (L) or high (H) levels (mg/kg DM) of Cu (5,40), Mo (1,10) and S (0.2,0.5%). Initially two of each breed was fed either LCu or HCu (2 mo). Then all eight animals were fed sequentially LCuHS (1.5 mo), HCuHS, HCuHMo and HCuHMoHS (2 mo each). Simmental had a higher urine flow, increased concentration and total excretion of urinary Cu and Zn compared to Angus, but only total Zn excretion was significantly higher. Urinary Cu excretion was greatest with the HCuHMoHS diet. Urinary Zn excretion significantly increased with HS but not HS in combination with HMo and/or HCu. This study, together with previously reported biliary excretion, allows a direct comparison of urinary and biliary Cu and Zn excretion responses to dietary Cu, Mo and S., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The effect of tetrathiomolybdate (TM), disodium calcium ethylenediamine tetraacetate (EDTA), D-penicillamine (PEN), 2-3 dimercapto-1-propanol (BAL) and dimethyl dithiocarbamate (DDC) administration on biliary and urinary excretion of copper (Cu), zinc (Zn) and iron (Fe) was investigated in sheep on a low Cu diet (Group A) and a high Cu diet (Group B). Only biliary Cu excretion increased significantly (P<0.01) with TM treatment. Urinary Cu excretion increased (P<0.01) following PEN treatment. TM, EDTA, PEN, BAL and DDC adminstration increased Cu excretion via bile and urine by 254, 11, 266, 46 and 16%, respectively in Group A sheep, and by 354, 13, 196, 20 and (-) 31% in Group B sheep. Urinary Zn excretion increased (P<0.01) following EDTA and PEN treatments. Only urinary Fe excretion increased (P<0.01) with EDTA treatment. The results show that TM and PEN are the most efficient decoppering agents, but PEN unlike TM also removes Zn. The major routes of excretion of Cu chelates by TM and PEN are different. TM increases Cu excretion significantly (P<0.05 in Group A and P<0.01 in Group B) in bile with only a slight increase in urinary Cu whereas PEN increases Cu excretion significantly (P<0.01) in urine. Therefore from a therapeutic view, a combination therapy of TM and PEN would be useful to maximize Cu removal from the body.

1. Sodium monofluoroacetate (1080), a vertebrate pesticide used in New Zealand, was administered orally to rabbits at two dose levels (sub-lethal and lethal) to determine how long 1080 would persist in plasma, liver, kidney, and muscle so that the risk of consumption of meat from lethally or sub-lethally poisoned rabbits by non-target species could be assessed. 2. The plasma elimination half-life in rabbits receiving a sub-lethal dose was 1.1 h. Retention of 1080 in tissue was greater in rabbits dosed with a lethal dose than in those that received a sub-lethal dose. Irrespective of the dose level, concentration of 1080 in muscle, kidney, and liver was substantially lower than in the plasma. 3. Poisoning of dogs is possible because of their extreme susceptibility to 1080. Poisoning of birds is less likely. The risk of secondary poisoning is reduced as the concentration of 1080 declines in putrefying carcasses.

The effects of excess dietary sulphur were studied in sheep supplemented and unsupplemented with thiamine. The diets contained either 0.19 per cent sulphur (LS) or 0.63 per cent sulphur (HS) in combinations with 14 mg kg-1 thiamine (LB1) or 243 mg kg-1 thiamine (HB1). A total of 56 two-month-old lambs were used. Groups consisting of nine, nine, 22 and 16 lambs were fed LS-LB1, LS-HB1, HS-LB1 and HS-HB1 diets, respectively for 14 weeks. Out of 22 lambs fed the HS-LB1 diet, seven lambs developed neurological signs between the third and eighth week of the trial. Two of these lambs died, three that were in extremis were euthanased, and two recovered completely. All clinically affected animals had extensive malacic lesions in the cerebral cortex, midbrain and brainstem. None of the lambs from the LS groups or HS-HB1 group developed clinical signs. Several clinically normal lambs from the HS-LB1 group had necrotic lesions in their brains at gross and microscopic examination. Supplementation with dietary thiamine prevented development of clinical signs, but did not totally prevent development of microscopic brain lesions. Brain thiamine concentration, transketolase activity and thiamine pyrophosphate (TPP) effect were not different (P greater than 0.05) among groups. There was a strong effect (P less than 0.0001) of dietary thiamine supplementation on blood thiamine concentration and TPP effect. Blood thiamine concentration was higher whereas TPP effect was lower in the thiamine supplemented sheep. Blood and tissue thiamine concentrations in sheep exposed to high dietary sulphur did not indicate either systemic or local thiamine deficiency per se. Increased TPP effect in sheep fed the HS-LB1 diet indicated mild to moderate metabolic thiamine deficiency. Thiamine inadequacy may be an effect of an increased requirement for thiamine in animals exposed to excess dietary sulphur.

Semiautomated, microbiological methods for the determination of thiamine in a variety of biological material are presented. Thiamine content was measured in bovine, ovine and rat blood, plasma, bile, urine, duodenal fluid, liver, kidney and brain. Thiamine was extracted from samples by mild acid digestion (trans aconitate pH 4.0) at 115 degrees C for 20 min. Microbiologic assays utilizing Lactobacillus fermenti and Ochromonas danica were performed on 96-well microplates and growth, as monitored by optical density, was directly read on a microplate reader at 650 nm. Particular attention was given to the validation of the two methods in terms of accuracy, reproducibility and recovery. The microbiological methods described here are specific, sensitive, and ensure good reproducibility. The methods can be used to analyse a large number of samples rapidly and at low cost.

Relative efficacy of thiamine (B1) and/or calcium disodium ethylinediaminetetraacetate (EDTA) administration on lead (Pb) excretion via bile and urine was studied in Pb-loaded sheep. The sheep were administered B1 s.c., 75 mg/kg body wt., EDTA i.v., 110 mg/kg body wt., and a combination of B1-EDTA at rates as above. Each treatment was followed by 69 h sampling of blood, bile, and urine. Urinary Pb excretion increased following B1-EDTA greater than EDTA greater than B1 administration. Thiamine and B1-EDTA treatments increased biliary Pb excretion. Overall, B1, EDTA and B1-EDTA administration increased Pb excretion via bile and urine by 72%, 595%, and 842% respectively over basal level (mean +/- SE: 20.1 +/- 2.9 micrograms Pb/h). It has been concluded that thiamine enhances elimination of Pb from the body and this feature may be beneficial in chelation therapy.

Fifty-six female crossbred two-month-old lambs were housed in individual cages, and fed a basic ration of barley (59%), soybean meal (5%), and alfalfa (32%) prepared to meet NRC nutrient requirements. Four percent of the diet contained a standard salt mix to which the factors inorganic sulphur (S) and thiamine (B1) were added. Four treatment groups were used: low sulphur and normal thiamine (0.19% S, 13.7 mg/kg B1) low sulphur and high thiamine (0.19% S, 243 mg/kg B1), high sulphur and normal thiamine (0.63% S, 13.7 mg/kg B1), high sulphur and high thiamine (0.63% S, 243 mg/kg B1). All animals had free access to water and were offered 1 kg/animal/day of diet for 14 weeks, when necropsy was undertaken. Seven lambs fed unsupplemented (normal B1) diets containing added sulphur developed clinical symptoms of polioencephalomalacia (PEM) between the 3rd and 7th week of the trial. Morbidity (P less than 0.013) and mortality (P = 0.08) differences were attributed to S administration. None of the B1 supplemented lambs developed clinical signs of PEM. Body weight and relative organ weights did not differ among treatment groups. Serial sections of all brains were examined grossly and microscopically. Nonparametric statistical analysis revealed sulphur related effects in the cerebrum, midbrain and hindbrain (P less than 0.0001), no thiamine-related effects or interaction between the factors were seen, except in the amygdaloid body. It was concluded that inorganic sulphur was associated with polioencephalomalacia, and that dietary thiamine may decrease the severity of lesions in some affected areas of the central nervous system.

Candidacidal and phagocytic tests were performed on 34 female sheep fed high sulphur diets containing varied concentrations of thiamine, copper, and molybdenum for 14 weeks. Tests were conducted at weekly intervals for five weeks during the latter half of the experiment. The ability of polymorphonuclear leucocytes from sheep on diets unsupplemented with thiamine to kill phagocytosed Candida albicans was lower than those on diets supplemented with thiamine. The ability of polymorphonuclear leucocytes to phagocytose C albicans was lower in animals on low copper diets than in animals on high copper diets. Factorial analysis showed that thiamine supplementation significantly increased (P less than 0.0001) candidacidal but not phagocytic activity. By contrast, copper supplementation significantly increased (P less than 0.0005) the phagocytic activity with no effect on candidacidal activity. The observed detrimental effect of high sulphur intake on in vitro polymorphonuclear leucocyte function can mean that ruminants in areas where large quantities of sulphur are taken in with water and feed have compromised immune function due to lower copper and thiamine status and hence are at risk of increased susceptibility to infections.

Brain stem auditory-evoked response (BAER) is a noninvasive technique used for detecting neurophysiological abnormalities of the brain stem along the auditory pathway. Brain stem auditory-evoked response recordings were obtained from subcutaneous skin electrodes from two control sheep and 22 other sheep fed high sulfur (S) diets with low or high concentration of thiamine (B1), copper (Cu), and molybdenum (Mo). At least four peaks (I,II,III,IV) of varied amplitude were observed in all animals. Neurophysiological abnormalities due to decreased conductivity and/or excitability of nerve fibers along the auditory pathway were found on the BAER recordings of sheep fed high S diet. Abnormalities of peaks and interpeak latencies within BAER recordings were related to histopathological observations of brain stem lesions. Lesions in the areas of the cochlear nuclei and lateral lemniscus were seen in conjunction with altered BAER components. However, abnormalities in BAER recordings and lesions in the brain stem also occurred in the absence of overt clinical signs. Analysis of interpeak latencies between peaks I and IV revealed significant differences among dietary groups. Sheep given diets low in Cu, Mo, and B1 were affected most. Factorial analysis indicated B1 and interactions among Cu, Mo, and B1 as significant factors influencing interpeak latencies.

The distribution of elements in the cells of the liver and kidney of normal sheep, of sheep chronically intoxicated with copper and in sheep given copper and thiomolybdate was studied by atomic absorption spectrophotometry, electron microscopy and electronprobe X-ray microanalysis. Copper concentrations were increased in the liver and kidney during the pre-haemolytic and haemolytic stages of the disease. In addition iron concentrations were markedly increased in the kidney during haemolysis. Copper and molybdenum concentrations were increased in the kidney of sheep receiving copper and thiomolybdate or thiomolybdate alone. By electronprobe X-ray microanalysis at the pre-haemolytic and haemolytic stages, copper together with small amounts of calcium and chromium were found in lysosomes of liver cells. In addition, in animals killed during haemolysis, high concentrations of iron were found in apical lysosomes of kidney proximal tubule cells. Copper and molybdenum were found in apical lysosomes of kidney tubule cells in animals given copper and thiomolybdate or thiomolybdate alone. The accumulation of copper with molybdenum and sulphur in the lysosomes of thiomolybdate treated copper intoxicated animals was demonstrated for the first time and may indicate the sequestration within lysosomes of a copper-molybdenum-protein complex.

Twenty-seven sheep were used in two experiments to study the distribution of copper (Cu), zinc (Zn), and iron (Fe) in the kidney cytosol of control, Cu-loaded, and thiomolybdate (TM)-treated sheep. A comparison was made of the patterns of distribution on Sephadex G-75 of Cu, Zn, and Fe from the cytosol of fresh and frozen kidneys and after thawing of frozen cytosol. In both Cu-dosed and TM-treated sheep, the absolute level of Cu increased in the cytosol, but the percent of Cu decreased. The percent recovery of Cu from the frozen kidney was comparable to that from fresh kidney, but the extraction of Cu from the supernatant of frozen cytosol was approximately 10% less. This was due to a loss of Cu in a precipitate that formed when the frozen cytosol was thawed. Most of the Cu in the cytosol from the kidney of Cu-loaded sheep was in a metallothionein (MT)-like protein fraction and was trichloroacetic acid (TCA) soluble. In contrast, that from the cytosol of TM-treated sheep was mostly in a high molecular weight fraction that was TCA insoluble. The chromatograms obtained from cytosol derived from frozen kidneys, or cytosol that had itself been frozen, contained a similar distribution of Cu, Zn, and Fe, but the peak heights were lower in the latter samples.

The copper (Cu), zinc (Zn), and iron (Fe) excreted daily in the bile of three sheep were estimated at 0.20, 0.10 and 0.36 mg respectively. Intravenous administration of tetrathiomolybdate (TM) increased bile Cu excretion and reduced liver Cu concentration. Tetrathiomolybdate had no significant effect on the excretion of Zn and Fe. These findings indicate that at least one of the mechanisms by which TM induces hypocuprosis is by increasing biliary Cu excretion. Hence it has a possible use in the treatment of Cu storage diseases in man and animals.

In sheep given copper (Cu) at the level of 10 ml of a 0.2% solution of CuSO4.5H2O/kg body weight, the volume density of nuclei and cytoplasm of hepatocytes increased and the volume density of the sinusoids and the space of Disse decreased. These changes were interpreted as an indication of cellular swelling. There was a significant increase in volume density, number, and absolute volume of lysosomes when Cu was given. The greatest increase in number occurred during the pre-hemolytic period (mean = 48 days), and the highest increase in volume occurred thereafter. Thus, the most extensive proliferation of lysosomes occurred in livers of the pre-hemolytic sheep, and the hemolytic sheep had the largest and heaviest lysosomes. The liver necrosis in sheep undergoing hemolysis was possible caused by hydrolytic enzymes released by the rupture of the enclosing lysosomal membranes. The significant increase in volume density of mitochondria observed in livers of sheep given Cu was due to an increase in volume (swelling) rather than an increase in number.

The effect of high dietary sulfur (S) supplementation on blood thiamine (B1) concentration, biochemical indices of liver, muscle and kidney damage and selected plasma electrolytes was studied in six sheep. Three of these sheep received an additional 230 mg thiamine/kg diet (Group 2). After approximately 2.5-3 weeks on this diet, all three sheep in the non-B1-supplemented group (Group 1) showed loss of appetite and developed mild neurological signs: depression, intermittent signs of excitation and head pressing. Increases in blood B1 concentration and plasma creatine kinase (CK) and aspartate aminotransferase (AST) were observed during this time in all affected animals. Clinical signs lasted only for two to five days. Sheep in group 2 were clinically normal throughout the experiment, but all of these animals also had elevated blood B1 concentrations and plasma CK activity at the 3 wk sampling. Plasma magnesium concentrations of group 1 sheep were elevated at the 2.5-3 wk and 6 wk samplings but they declined significantly (p less than 0.05) to low normal levels thereafter. Magnesium concentrations of group 2 sheep were low at the beginning but progressively increased during the course of the experiment. At necropsy, brain lesions suggestive of polioencephalomalacia (PEM) were observed in all sheep but were most marked in group 1. It is speculated that PEM may be caused by a direct toxic effect of S, S metabolites or B1 antimetabolites in the brain rather than by an in vivo B1 deficiency per se.

Following development of polioencephalomalacia in one of 105 cattle in a farm in southeastern Saskatchewan, a study was initiated to monitor thiamine (B(1)) and copper (Cu) status, and to evaluate interactive nutritional factors which may have been responsible for this occurrence. It was evident that a combination of high sulfur (S) and low Cu intake was responsible for the depletion of blood B(1) and plasma Cu. Supplementation with trace minerals alone resulted in a significant (p<0.05) improvement in both B(1) and Cu status of the herd. We recommend that herds exposed to high intakes of S be supplemented with Cu up to 50 mg/kg feed dry matter to alleviate potential deficiencies of B(1) and Cu.

Histological, ultrastructural and kidney function techniques were used to assess changes in the kidney of sheep given either copper (Cu) or Cu and the Cu complexing agent thiomolybdate (TM), or TM alone. Kidney function was normal in sheep given Cu and TM together or TM alone. In these animals the cells lining cortical tubules accumulated Cu within numerous, large, electron-dense lysosomes. Sheep given Cu alone developed haemolysis, impaired kidney function and a variety of morphological defects including an increase in number of large lysosomes in cells of the cortical tubules. There was a breakdown of the glomerular endothelial lining and fusion of foot processes. Cells of the cortical tubules showed degeneration and necrosis and an increase in microbodies and rough endoplasmic reticulum. Cortical and medullary blood vessels were dilated, with evidence of breakdown of the endothelial lining. Copper appeared to injure kidney tissue at three sites, tubular epithelium, glomerular basal lamina and capillary blood vessels. Changes reported here are similar to the renal lesions in cadmium toxicity.

Copper, zinc and iron levels were measured in cerebrospinal fluid (CSF) of normal and copper poisoned sheep. Copper values were within the range recorded for humans but zinc levels were six to 20 times and iron levels were 20 to 30 times higher than values reported for humans. The amount of copper, zinc and iron in the CSF did not increase in sheep dosed with copper and hence it is unlikely that the level of copper in the CSF is related to the changes in the nervous system that have been reported in copper poisoned sheep.

Thirteen sheep were dosed repeatedly with copper sulphate in order to induce chronic copper poisoning. Twelve similar sheep acted as controls. Three dosed sheep were killed before haemolysis, six at haemolysis and four after cessation of the haemolytic crisis. The subcellular distribution of Cu, Zn, Fe and lysosomal marker enzymes was investigated in the livers of normal sheep and copper loaded sheep. In control sheep the highest concentration of Cu was in the nuclear fraction. Copper loading increased the concentration of Cu in all fractions. The proportion of Cu in the nuclear fraction continued to increase while that in the heavy mitochondrial fraction decreased. Significant increase in lysosomal enzymes along with electron microscopic studies on pellets of fractions indicated: (a) proliferation of lysosomes, (b) possible localisation of Cu in lysosomes, and (c) sedimentation of the denser lysosomes on N and MH fractions in test sheep with the majority being in the N fraction. Thus the increase in Cu observed in the N fraction is at least partly due to the storage of Cu by lysosomes which sediment with the heavier nuclei. The excessive deposition of Cu in lysosomes and nuclei may lead to cell damage. With increase in hepatic Cu concentration the proportion of Zn and Fe in the cytosol decreased.

Twenty-seven sheep given either copper (Cu) and/or tetrathiomolybdate (TM) were used to study the subcellular distribution of Cu within the kidney and to monitor the location of lysosomes within the subcellular fractions using acid phosphatase (AP) as a marker enzyme. Copper dosing alone increased the Cu content in the liver and the kidneys. The administration of intravenous TM prevented the development of chronic copper poisoning (CCP) in sheep, reduced the rate of accumulation of Cu in the liver of Cu-dosed animals, but increased the Cu content of kidneys in both the control and Cu-dosed sheep. The total amount of Cu that accumulated in the kidneys of sheep given TM appears to depend on several factors: a) liver Cu concentration, b) Cu intake, and c) dosage of TM. Thus, the highest Cu concentration was found in the kidneys of sheep that continued to receive Cu orally at the same time as they were given TM. The intracellular distribution of Cu and AP in the kidneys showed that in the control sheep given neither Cu or TM, the highest proportion of Cu was in the cytosol fraction, and the highest specific activity of AP was in the light mitochondrial (lysosomal) fraction. Dosing with Cu markedly increased the Cu concentration and greatly elevated the total activity of AP in the heavier fractions, i.e., the nuclear (N) and heavy mitochondrial (MH). Thus, the increase in Cu observed in the N and MH fractions was not caused by an accumulation of Cu by nuclei and mitochondria, but was due to an accumulation of Cu by lysosomes that sedimented with the heavier fractions. The intracellular distribution of Cu in the kidneys of TM-treated sheep was similar to that seen in Cu-loaded sheep. Although Cu accumulated readily in the kidneys of animals receiving TM, kidney function tests showed neither glomerular nor tubular functional impairment.

Chronic copper poisoning was induced in sheep by repeated dosing with an aqueous solution of copper sulphate. A sudden rise in the level of plasma creatine kinase (CK) occurred at haemolysis but the level returned to normal once the 'crisis' was over. The level of CK in the cerebrospinal fluid remained normal throughout the experiments. An isoenzyme study showed that the CK was liberated from muscle. Light microscopic studies failed to reveal consistent changes in muscle structure but ultrastructural changes in mitochondria, similar to those described as the earlies changes in muscle fibres in vitamin E deficiency, were seen in sheep killed during and after haemolysis. The elevations of CK levels and the mitochondrial changes were most marked in sheep that did not receive selenium and vitamin E supplements. It is suggested that the CK release at haemolysis may occur because of a transient increase in the permeability of muscle membranes which may be brought about by a number of factors such as: (a) hypoxia, (b) hypercupraemia, (c) decrease in vitamin E and/or selenium content in blood and tissues.