Residual effects of Mycobacterium avium infection on susceptibility of sheep to copper toxicity and efficacy of treatment with tetrathiomolybdate

Posthaemolytic copper poisoning (post-HCP) in one of six, one-year-old, uninfected sheep (group O) on a Mycobacterium avium experiment prompted an evaluation of copper status and hepatotoxicity in 17 surviving cohorts. Group O had higher mean plasma Cu and δ-glutamyl transferase (GGT) activity and more variable bile acid (BA) concentrations and glutamate dehydrogenase activities (GDH) than two groups infected with M avium soon after birth. Ammonium tetrathiomolybdate (TTM; 3 x 1.7 mg/kg LW) was given subcutaneously over seven days and the pelleted, complete diet replaced by hay, low in copper. Plasma BA immediately declined and was followed by GDH, but erythrocyte superoxide dismutase activity (ESOD) became severely inhibited and took 18 days to recover. Plasma BA and GDH rose sharply after 18 days in uninfected sheep and they became hypercupraemic. TTM treatment was repeated from day 42 and had removed all group differences by day 110 but only after further inhibition of ESOD. M avium infections probably lessened the severity of pre-HCP by reducing copper retention but may predispose grazing livestock to hypocupraemia. The capacity of TTM to reduce liver Cu has probably been overestimated and side effects on cuproenzyme activity underestimated.     

Enhancement of tetrathiomolybdate-induced biliary copper excretion in sheep by general anaesthesia and the effect on copper excretion in urine and bile

To determine whether copper removed from the liver by tetrathiomolybdate (TTM) but not excreted in bile, is excreted in saliva or urine, the effect of an intravenous injection of 100 mg of TTM, as ammonium tetrathiomolybdate, on copper excretion by these routes was measured in conscious sheep and sheep maintained under general anaesthesia. During 4.5 hours the total amount of copper excreted in bile in the untreated, conscious animal was 0.52 +/- 0.04 mumols (mean +/- SE). A dose of 100 mg TTM increased excretion by an average of 11.7-fold to 6.06 +/- 0.29 mumols. Under halothane or barbiturate general anaesthesia copper excretion was increased 3.4-fold to 1.78 +/- 0.30 mumols. In anaesthetised sheep 100 mg TTM produced a 40.7-fold increase to 21.18 +/- 0.95 mumols. Neither anaesthesia nor TTM caused any increase in copper excretion in saliva or urine.     

Use of erythrocyte copper:zinc superoxide dismutase activity and hair or fleece copper concentrations in the diagnosis of hypocuprosis in ruminants

Changes in the activity of superoxide dismutase, a copper-containing enzyme in erythrocytes (ESOD), and changes in copper in hair or fleece were compared with the changes in plasma copper during copper depletion and repletion in steers, lambs and ewes. During depletion the decline in ESOD began later than the decline in plasma copper: the lag varied from 0 to 80 days in individual steers and it was least evident when young rapidly growing lambs were subjected to severe depletion. ESOD activity eventually declined at only one-third to one-seventh of the rate shown by plasma copper, the difference being particularly marked in severely depleted lambs. Repletion of ESOD began after repletion in plasma copper and continued more slowly particularly in growing animals. Hair and fleece copper also responded relatively slowly to depletion and repletion. Low values of ESOD and hair or fleece copper may have diagnostic significance, indicating a more prolonged or intense deficiency of copper and a higher probability of clinical and production responses to copper therapy than low plasma or liver copper values.     

Some effects of copper deficiency on leucocyte function in sheep and cattle

Peripheral blood leucocytes from copper-deficient (less than 8 mumol per litre plasma) and copper-sufficient (more than 8 mumol per litre) ewes and lambs were tested for their ability to ingest and kill Candida albicans in vitro. Killing capacity was significantly reduced in both copper-deficient ewes and lambs, although phagocytic capacity was apparently unimpaired. Similar results were obtained with copper-deficient calves, in which it was further shown that copper repletion restored candidacidal activity. Leucocyte and erythrocyte superoxide dismutase (LSOD and ESOD respectively) activities were both significantly decreased in hypocupraemic ewes and there was a linear relationship between their activities in the two cell types: LSOD activities were reduced by approximately 35 per cent and ESOD activities by 67 per cent. Cyanide inhibition tests indicated that the manganese-dependent form of the enzyme contributed little to the total activity of LSOD or ESOD. There was a tendency for leucocytes from copper-deficient ewes to release greater amounts of superoxide anion after stimulation with opsonised zymosan. Their viability was similar to that of leucocytes from control sheep and was unaffected for up to three hours after stimulation.     

Enhancement of tetrathiomolybdate-induced copper excretion in bile of sheep by the alpha 2-agonistic action of xylazine

The effect of intravenous doses of cortisol and xylazine on the quantity of copper excreted in response to 100 and 200 mg doses of tetrathiomolybdate (TTM) was studied in seven sheep. Cortisol alone produced a non-significant 1.4-fold increase and had no enhancing action on the response to TTM. Xylazine produced a significant 2.25-fold increase, doubled the quantity of copper excreted in response to both doses of TTM and reduced bile flow by approximately 35 per cent. The alpha 2 antagonist, idazoxan, prevented both the latter effects showing that they were due to xylazine's alpha 2-agonistic action. It is suggested that the combination of an alpha 2-agonist with the intravenous injection of TTM in the treatment of acute copper toxicity in sheep could reduced by 50 per cent the amount of molybdenum needed.     

The effect of selenium supplementation on immunity, and the establishment of an experimental Haemonchus contortus infection, in weaner Merino sheep fed a low selenium diet

SUMMARY: Immunity in 12 weaner Merino sheep fed a low selenium (Se) diet (low Se sheep) was compared with that in 10 matching sheep fed the same diet but each given an intraruminal Se pellet (high Se sheep), while the sheep were housed in individual, sheltered pens. All sheep were challenged with killed Brucella abortus cells (days 0 and 28), rabbit red blood cells (days 0, 7 and 28) and corynebaclerium pseudotuberculosis toxoid (days 0 and 28), and serum antibody titres were measured weekly for 8 weeks from day 0. The sheep were then experimentally infected with Haemonchus conforfus, and slaughtered 8 weeks later. The mean antibody titre to B. abortus, measured by 4 different tests, was significantly higher in the high Se sheep on occasions during the primary immune response phase (Rose Bengal test - day 21 (p < 0.05), day 28 (p < 0.025); complement fixation - day 7 (p < 0.05); enzyme-llnked immunosorbent assay - day 14 (p < 0.01); serum agglutination - no differences), but not during the secondary phase. The mean antibody titre to rabbit red blood cells, measured by haemagglutination test, was marginally higher in the high Se sheep on day 49 (p = 0.049). The mean antibody titre to C. pseudotuberculois, measured by enzyme-linked immunosorbent assay, was not significantly different between the groups at any time during the trial. In addition, the mean invitro responsiveness of peripheral blood lymphocytes to stimulation with phytohaemagglutinin in the high Se sheep was significantly greater than that in 10 sheep from the low Se group on day 22 (p < 0.01), but not day 50. However, there were no significant differences in the mean number of sheep in which the infection with H. contortus established, time to first shedding of eggs in faeces, abomasal worm burdens at necropsy, or inflammatory response in the abomasal mucosa in the sheep in each group. The results showed that the low Se sheep produced strong overall immune responses that were largely comparable to those in the high Se sheep.     

The effects of ammonium tetrathiomolybdate intake on tissue copper and molybdenum in pregnant ewes and lambs

Pregnant ewes (d 32 of gestation) were allocated to three treatments and given intraruminal controlled-release devices designed to deliver 0, 20 or 60 mg diammonium tetrathiomolybdate (TTM) per day. Ewes given 20 or 60 mg TTM/d also received an oral drench of 120 or 360 mg TTM twice weekly commencing on d 86 of gestation. Liver and kidney samples were taken from lambs 48 h after birth and from ewes on d 18 postpartum. Trichloroacetic acid soluble Cu, ceruloplasmin and superoxide dismutase activities in the plasma of ewes were decreased (P less than .05) by TTM. Liver Cu concentrations were decreased (P less than .05), but kidney Cu concentrations increased (P less than .05) by 16-fold in ewes given the higher dose of TTM. Liver and kidney Mo concentrations were elevated (P less than .05) 9- and 30-fold, respectively, in ewes given TTM. Plasma glucose concentrations in ewes were decreased (P less than .05) by the highest level of TTM treatment. Lambs of ewes given TTM had a fivefold increase (P less than .05) in liver Mo concentration, but kidney Mo concentration was not affected (P greater than .05) and liver Cu concentration was reduced (P less than .05). In ewes, Mo apparently caused Cu to be mobilized from the liver and a Cu and Mo complex accumulated in the kidney. Some Mo crossed the placenta, but only limited Mo accumulated in the fetal livers. When given to pregnant ewes, TTM reduced liver Cu levels in the lambs but did not affect the concentration of Cu in colostrum.     

Effects of clonidine and idazoxan on tetrathiomolybdate-induced copper and lysosomal enzyme excretion into sheep bile

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.     

Experimental conjunctival infection of lambs with a strain of Chlamydia psittaci isolated from the eyes of a sheep naturally affected with keratoconjunctivitis

Five ram-lambs were inoculated into the left conjunctival sac with the 15R isolate of Chlamydia psittaci, recovered from a sheep with keratoconjunctivitis. A sixth ram-lamb was kept in contact with them. The five lambs developed varying degrees of acute conjunctivitis and 14 days later C psittaci could be recovered from the inoculated eyes, from which Branhamella ovis was also isolated. The eyes were examined regularly for four months; C psittaci could not be re-isolated but the eyes developed varying degrees of follicular conjunctivitis. After four months the sheep were treated with corticosteroids in an attempt to reactivate a latent chlamydial infection but no chlamydiae could be isolated. Five months after the start of the experiment the six lambs were inoculated with 15R into the left conjunctival sacs. Acute conjunctivitis developed which was not as severe as after the first inoculation, but C psittaci could only be recovered from the left eyes of three sheep three days after inoculation. The eyes remained chronically affected by follicular conjunctivitis. Six months after the start of the experiment the left eyes were again inoculated with 15R; on this occasion acute conjunctivitis did not develop and chlamydiae could not be isolated. Chronic follicular conjunctivitis persisted until the experiment was terminated three months later.     

A convenient method for the treatment of chronic copper poisoning in sheep using subcutaneous ammonium tetrathiomolybdate

Effective control of copper poisoning in sheep was obtained by the subcutaneous injection of ammonium tetrathiomolybdate. Three doses, each of 3.4 mg/kg bodyweight, were given on alternate days. This treatment caused a substantial reduction in liver copper content and in liver damage. It also decreased the mortality rate in animals that had developed the haemolytic crisis. The subcutaneous route is as effective as the intravenous route and is more convenient. No adverse side-effects of the treatment were observed.     

Experimental Ostertagia spp. infection of sheep: development of worm populations and lesions resulting from different dose-levels of larvae

Worm-free sheep were dosed with 8 000, 64 000 or 512 000 infective Ostertagia spp. larvae and killed 4, 7, 10, 13, 18, 24 or 66 days later. Up to day 10 the worm counts ranged from 52 to 86% of the infecting dose and results suggested no substantial reduction either in rate of development or in percentage establishment with increasing dose level. However, a reduction of worm counts, which was substantial at the higher dose-rates but comparatively small at the low rate, occurred after day 10.Gross lesions in the abomasum ranged from little more than the development of a few nodules in the low-dose sheep, to a roughened congested surface with diptheritic membranes and severe oedema in the high-dose animals at days 10–24. These effects had subsided by day 66. Histopathological changes are described.The severity and extent of the reaction was related to the parasite dose-level, but not necessarily to the numbers of worms recovered post-mortem. The rate of return to normality was quicker at the lower dose rates.     

Copper poisoning in a flock of sheep. Copper excretion patterns after treatment with molybdenum and sulfur or penicillamine

During an outbreak of chronic copper poisoning, fecal and urinary copper excretion were measured following treatment with molybdenum and sulfur supplementation of the feed (0.1 g ammonium molybdate plus 1 g sodium sulfate/sheep/day) or oral penicillamine (50 mg/kg bodyweight/day) using rams in metabolism cages. Serum glutamic-oxaloacetic transaminase activities and liver levels of molybdenum and copper in sheep that died were also monitored. Within four days of starting molybdenum and sulfur supplementation a highly significant increase in fecal copper excretion was evident and the increase persisted throughout the monitoring period (five weeks — general treatment of the flock continued for another three weeks). There was no effect of the molybdenum and sulfur supplementation on urinary excretion of copper. The molybdenum and sulfur supplementation was very effective, resulting in a rapid marked decrease in mortality. Oral penicillamine treatment induced cupruresis but did not affect fecal copper excretion. The results indicated that, while the cost of penicillamine may be a limiting factor for general treatment of a flock, it may be the drug of choice for the therapy of valuable breeding animals because cupruresis may be accurately and individually controlled. Serum glutamicoxaloacetic transaminase activities were a valuable aid in diagnosing chronic copper toxicosis as well as for monitoring recovery. High initial liver copper levels were gradually reduced following molybdenum and sulfur treatment. However, at the end of the study the liver copper levels of dead sheep varied within wide limits and there were still some sheep with high liver copper levels.     

Copper poisoning of sheep after ad libitum supplementation of rations by a mineral feed

Within 8 weeks 22 of 400 sheep died with symptoms of Cu intoxication. In the liver of 2 dead sheep Cu contents of 491 and 830 mg/kg dry matter were found. The concentration of copper in the concentrate and other feedstuffs (grass, hay, straw, kale, dried sugar beet pulp) could not explain the development of Cu-toxicosis. The mineral-vitamin-supplement contained 86.7 mg Cu/kg. Obviously the Cu intoxication of previously Cu loaded sheep was provoked by offering the mineral supplement ad libitum (with intervals of 3-4 days), the intake of this mixture reached values up to 400 g/animal and day. By renunciation of mineral supplements in the same flock 7 months later lambs died due to Cu deficiency (Cu content in the liver: 13-14 mg/kg dry matter).     

The retention and efficacy of soluble-glass boluses for providing selenium, cobalt and copper to sheep

The efficacy and retention of prototype and commercial (Cosecure) soluble-glass boluses containing selenium, cobalt and copper and having a similar size, density and composition were evaluated in separate 12-month trials with sheep grazing low selenium pastures but with adequate cobalt and copper levels. In both trials, sheep confirmed by X-ray as containing a bolus grew at a significantly greater rate than control sheep. This was attributed to the correction of a selenium deficiency. Although liver copper, and liver and serum vitamin B12 levels were significantly greater in treated sheep than controls on some occasions, the differences were not as great or consistent as with blood selenium levels. The rate of bolus loss was high and in both trials, approximately 40% of the sheep had lost the bolus after 6 months. No prototype boluses were present after 12 months. Intact Cosecure boluses were recovered from 3 sheep out of 30 after 12 months whilst one animal contained a part bolus. The recovered boluses were approximately 45% lighter than when originally administered. Bolus loss did not appear to be due to complete dissolution. In sheep which had lost the bolus, blood selenium levels fell with a half-life of 43 +/- 10 days.     

Boluses of controlled release glass for supplementing ruminants with copper

A controlled release glass was formulated into boluses weighing approximately 17 g or 75 g and administered to 19 lambs and 20 steers respectively. The animals were at pasture during the summer months. The lambs were slaughtered between 57 and 219 days after dosing when the mean concentration of copper in their livers had increased to 107.8 +/- 33.4 mg copper/kg fresh weight compared with 55.9 +/- 23.0 mg copper/kg fresh weight in undosed controls. The steers were slaughtered more than 140 days after dosing; the mean concentrations of copper in plasma had increased and the mean concentration of copper in liver was significantly greater than in undosed control steers (14.1 +/- 4.8 mg copper/kg fresh weight liver in dosed steers, 4.7 +/- 1.4 mg copper/kg in control steers) and was similar to the concentration in steers which had received 200 mg copper as copper calcium edetate (18.2 +/- 4.2 mg copper/kg fresh weight). In sheep the minimum rate of release of copper into the reticulorumen was 2.1 mg/day and in steers 11 mg/day.     

Immune cell kinetics in the ovine abomasal mucosa following hyperimmunization and challenge with Haemonchus contortus

Sheep were sensitized by repeated infection with Haemonchus contortus L3, followed by a 12 week rest period, and an abomasal cannula was surgically implanted in all sheep. Seven of the sensitized sheep were subsequently challenged with 50 000 H. contortus L3 while 4 control sheep were challenged with saline. Biopsy samples were taken using a fibreoptic endoscope on days 0, 1, 2, 3, 5, 7 and 28 after challenge and leukocyte subpopulations quantified by (immuno)histology. Differential blood cell counts were performed on the same days. At the end of the trial, sheep showed significantly reduced worm burdens compared to unsensitized control sheep, confirming their resistance status. Both blood and tissue eosinophils, as well as tissue γδ TCR⁺ cells were rapidly elevated by day 1 post L3 challenge (pc), peaking at day 3 pc. There was a slight increase in tissue CD4 T cells at day 2 pc, peaking at day 3 pc while no significant changes in CD8 T cells were observed. B cells (CD45R⁺) increased later into challenged tissues with a peak at 5 days pc. All tissue lymphocyte subpopulations as well as tissue and blood eosinophils were reduced by day 7 pc before increasing again at day 28 pc, suggesting separate responses to larval and adult antigens. In contrast, globule leukocytes and mucosal mast cells only showed one peak at day 5 pc and 28 pc, respectively. Unexpectedly, globule leukocytes correlated significantly with tissue eosinophils but not mucosal mast cells. The results are consistent with an early eosinophil-mediated killing of L3, possibly recruited by IL-5 produced by γδ T cells. In contrast to post-mortem studies, abomasal cannulation allowed sequential analysis of both early and late time points in the same animal, providing a more complete picture of cellular interactions at both peripheral and local sites, and their correlation with the different stages of parasite development.     

Effectiveness of a non-surgical alternative to the Mules operation in sheep

Objective To measure changes to the perineal bare area, local tissue reaction and healing responses of young sheep, following intradermal administration of cetrimide and polyvinylpyrrolidone (PVP), with and without ethanol, to the breech and tail. Method A needle‐less injector was used to deposit formulations containing 40 g/L cetrimide and 30 g/L PVP (group 2) or 20 g/L cetrimide, 30 g/L PVP and 15 g/L ethanol (group 3), within the dermis of the tail and the region surrounding the perineal bare breech area of groups (N = 8) of Merino weaner sheep. The dimensions of the perineal bare area (length, width and diagonal distances left and right) and tail width were recorded before and at intervals after treatment for 60 days. Observations of swelling and bruising and scab formation at the treatment sites were recorded for up to 35 days after treatment. Rectal temperatures were monitored for up to 35 days after treatment and bodyweight for up to 60 days after treatment. An untreated control group (group 1) was included. Results Comparison of day ‐3 and day 35 measurement data showed that both treated groups had significantly (P < 0.05) wider breech bare areas compared to the untreated controls and that group 2 sheep had significantly (P < 0.05) longer breech bare areas compared to group 3 sheep or to the untreated controls, which were not significantly different. At this time scabs were still firmly in place on many treated sheep. At day 35 there was no increase in tail bare area caused by either treatment. By day 60 there was no significant difference between the treated and control groups in either the breech or tail regions indicating that the changes present at day 35, were not permanent. Mean weight gain in the groups throughout the 60‐day interval was unaffected by treatment. Intradermal treatment was associated with a significant elevation in body temperature. This effect lasted for 3 days and was associated with signs of discomfort and depressed appearance in at least some of the treated sheep. Bruising was mild to severe in all treated sheep within two days of treatment but was not evident in any sheep by day 21. Mild to moderate swelling was also associated with treatment but was not uniform across sheep in the groups. The tail of one sheep was severely swollen for several days. Swelling remained obvious in most treated sheep until day 14 but was not present at day 21. Conclusion Under the conditions of this study intradermal injection of cetrimide had no permanent effect on bare area measurements on the breech or the amount of wool‐bearing skin on the tail. It also caused signs of discomfort and pain that raise welfare concerns.     

Failure of orally administered zinc to prevent experimentally induced footrot in sheep

Two groups of adult Merino sheep, initially grazed on pasture, were dosed daily with zinc sulphate (1 mg Zn/kg LW) or zinc oxide (15 mg Zn/kg LW) for six and seven weeks, respectively. On the 18th day, both groups were transferred to indoor pens together with unmedicated control sheep, and five days later the feet of all animals were infected artificially with a virulent strain of Bacteroides nodosus. By the fourth week after challenge, 85% or more of the feet challenged had developed advanced footrot, and no significant differences in the incidence and severity of lesions between dosed and un-dosed groups were recorded. Plasma zinc concentrations, monitored throughout the experiment, remained at similar levels in sheep receiving the lower dose rate and in the controls. At the higher dose rate, plasma zinc levels increased till Day 23, but thereafter declined to values comparable to those of undosed animals. At post mortem, no evidence of zinc toxicosis was found, and only the kidneys of sheep receiving the higher dosage showed a significant accumulation of zinc compared with the controls.     

The Salmonella excretor incidence of dogs in Hawke's Bay

Two groups of adult Merino sheep, initially grazed on pasture, were dosed daily with zinc sulphate (1 mg Zn/kg LW) or zinc oxide (15 mg Zn/kg LW) for six and seven weeks, respectively. On the 18th day, both groups were transferred to indoor pens together with unmedicated control sheep, and five days later the feet of all animals were infected artificially with a virulent strain of Bacteroides nodosus. By the fourth week after challenge, 85% or more of the feet challenged had developed advanced footrot, and no significant differences in the incidence and severity of lesions between dosed and undosed groups were recorded. Plasma zinc concentrations, monitored throughout the experiment, remained at similar levels in sheep receiving the lower dose rate and in the controls. At the higher dose rate, plasma zinc levels increased till Day 23, but thereafter declined to values comparable to those of undosed animals. At post mortem, no evidence of zinc toxicosis was found, and only the kidneys of sheep receiving the higher dosage showed a significant accumulation of zinc compared with the controls.     

Tear copper and its association with liver copper concentrations in six adult ewes.

Tear and liver copper concentrations from 6 clinically healthy adult mixed-breed ewes were measured by Atomic Absorption Electrothermal Atomization (graphite furnace) Spectrometry and Flame Absorption Spectrometry, respectively, 7 times over 227 d to determine if their tears contained copper and if so, whether tear copper concentrations could reliably predict liver copper concentrations. To produce changes in liver copper concentration, the diet was supplemented with copper at concentrations that increased from 23 mg to 45 mg Cu/kg feed/day/sheep during the study. This regimen raised liver copper for all sheep to potentially toxic hepatic tissue concentration of greater than 500 mg/kg dry (DM) matter (tissue). The results of the study showed that copper was present in the tears of all sheep. The mean tear copper concentration showed a positive correlation with liver copper concentration (P = 0.003), increasing from 0.07 mg/kg DM at the start to 0.44 mg/kg DM at the end of the study, but could not reliably predict liver copper concentration (R2 = 0.222).