Proceedings, Western Section, American Society of Animal Science
Vol. 51, 2000.
N. H. Wells, D. M. Hallford, J. A. Hernandez, J. L. Bollinger, M. K. Petersen, and U. McElyea
Department of Animal and Range Sciences, New Mexico State University, Las Cruces, NM 88003
ABSTRACT: Debouillet ewe lambs (n = 31, 6 mo of age, avg BW = 54 kg) were exposed to elevated dietary Cu after being fed pelleted alfalfa from two separate feed mills which contained 33 and 45 ppm Cu. After consuming about 2.5 kg/d of the diet for approximately 50 d, symptoms became apparent (hemoglobinemia, hemoglobinuria) and Cu toxicity was confirmed by a liver Cu concentration of 439 ± 36 ppm in five dead animals. During the course of Cu exposure, eight of 31 ewe lambs died. Serum was collected from eight ewe lambs before Cu consumption and from seven ewe lambs showing extreme symptoms of Cu poisoning. Total, direct, and indirect bilirubin, gamma glutamyl transpeptidase (GGT), and creatine kinase (CK) were elevated (P < 0.05) in ewes consuming Cu. Three extremely affected ewe lambs that had stopped consuming any feed received gelatin capsules (gavage) containing 56 g of a livestock salt/gypsum (76% calcium sulfate) mixture (2:1, wt:wt) daily for 10 d. Before treatment, total bilirubin was 0.6 mg/dL in control ewes and 6.8 ± 1.7 mg/dL in Cu consuming ewe lambs. After 10 d of calcium sulfate treatment, total bilirubin was 0.6 and 0.7 (± 0.1) mg/dL, GGT was 148 and 234 (± 47) IU/L, and CK was 325 and 1,725 (± 666) IU/L in control ewes and those receiving calcium sulfate, respectively (P > 0.10). Neither hemoglobinemia nor hemglobinuria were observed after 10 d. Calcium sulfate administered in gelatin capsules appears to be an effective treatment for acute copper toxicosis in sheep.
Key Words: Sheep, Hemolysis, Copper
Introduction
Copper poisoning can be a serious problem to sheep producers because of the low tolerance sheep have for Cu. The pathogenesis of Cu toxicosis has two distinct stages: a primary phase of Cu accumulation in the tissues and a stage of acute illness resulting from hemolytic crisis (Gopinath et al., 1974). The phase of accumulation is caused by intake of forages or processed feed high in Cu and may take several weeks or months. McCosker (1968) found that during this accumulation or pre-hemolytic phase, liver Cu levels on a dry weight basis, increased from normal ranges of 6 to 279 ppm to a range of 1,000 to 3,000 ppm. Copper toxicosis may remain unnoticed until the animal is stressed by factors such
as transportation, lactation, strenuous exercise, or a declining plane of nutrition (Merck, 1998). The stress results in massive release of Cu from the liver. This leads to a sudden increase in blood Cu concentration and intravascular hemolysis along with severe gastroenteritis, icterus, dehydration, depression of appetite, hemoglobinemia, and hemoglobinuria, (Adamson et al., 1969; Merck, 1998). Necropsy findings show liver concentrations > 150 ppm (wet wt) in chronic poisoned sheep (Merck, 1998). Treatment of the toxicosis may not be very successful because of the sudden, severe hemolysis and resulting detrimental effects. Analgesic drugs, Penicillamine, and calcium verserate may be useful if administered in the early stages of the disease (Merck, 1998). Harker (1976) indicated that addition of ammonium molybdate to the concentrated diet might be a useful method of reducing the risk of nutritional Cu poisoning in sheep. The administration of intravenous tetrathiomolybdate to Cu loaded sheep showed that even though Cu accumulated in the kidneys, less damage was observed (Goonerate et al., 1989). Intravenous and subcutaneous administration of ammonium tetrathiomolybdate have been suggested as effective means of treating the acute phase of Cu toxicosis in sheep (Humphries et al., 1986, 1988). The objective of this study was to determine if oral administration of calcium sulfate could reverse the symptoms of acute Cu poisoning in ewe lambs.
Materials and Methods
Thirty-one Debouillet ewe lambs born during spring 1999 were selected to remain in the breeding herd at the West Sheep Unit on the campus of New Mexico State University. Replacement ewe lambs at this facility are routinely allowed free access to a pelleted alfalfa diet, salt, water, and shade until they are 8 mo of age (mid-November). Over the previous 8 yr, the pelleted alfalfa was supplied primarily by a single feed mill and contained 96% alfalfa, 3% molasses, 0.5% sodium phosphate, and small amounts of vitamin A and terramycin. Average chemical analysis of the diet yielded 18% CP, 40% ADF, 16% ADL, 12% ash, 0.6% P, and 1.6% Ca. In addition, ewe lambs received cracked corn fed at an amount to approximate 30% of their alfalfa intake. In September 1999, ewe lambs were approximately 6 mo old, weighed 55 kg and were eating about 4% of their BW as the pelleted alfalfa. At this same time, a survey trial was initiated in which blood samples were collected twice weekly (Mon/Thur) from each ewe lamb. Blood was collected into serum separator tubes, allowed to stand at room temperature for 30 min and was then centrifuged at 1,000 x g for 15 min at 4°C. Serum was harvested and stored frozen for later hormone analyses as part of a graduate student research project. Also at this time, a new load of feed was received
from the same mill that had supplied pellets for several years.
Results and Discussion
After animals had received this new pellet for about 50 d, one ewe lamb became sick. Visual examination of her serum and urine revealed symptoms of copper (Cu) toxicosis (hemoglobinemia, hemoglobinuria). Within 3 d the ewe died and laboratory examination of serum and whole blood showed elevated bilirubin (7.23 mg/dL) and decreased hematocrit (18.4%). A feed sample was analyzed for Cu (Pope Testing Labs, Dallas, TX) and liver samples from five dead ewes were sent by the New Mexico Department of Agriculture Veterinary Diagnostic Services (Albuquerque, NM) to the Texas Veterinary Medical Diagnostic Laboratory (Texas A&M Univ.) for Cu determinations. The Cu content of the diet was 33 ppm and Cu toxicity was confirmed by liver Cu concentrations of 439 ± 36 ppm (> 150 ppm is diagnostic of Cu poisoning). Ewes were switched to 50% pellet and 50% alfalfa hay and a new load of pellet was ordered from a second feed mill. When the second load of feed arrived, ewes were switched back to receiving all pellet (over a 10-d period). A precautionary sample of the new load was analyzed for Cu (Pope Testing Labs, Dallas, TX) and it contained 45 ppm. At this point, we had unknowingly fed the ewes toxic levels of Cu for about 80 d and several were extremely sick. Ewes were subsequently placed back on all alfalfa hay.
Because livers were already burdened with excess Cu and the dietary source had been removed, supplementation with molybdenum was not felt to be appropriate. Because sodium sulfate is not readily available in southern New Mexico, an experiment was conducted to determine if calcium sulfate could relieve the symptoms of acute Cu poisoning. Livestock feed mix salt (United Salt Corp., Houston, TX) and gypsum (76% calcium sulfate, Kelly Lime and Rock Co., Inc., Newark, MO) were mixed at a ratio of 2 parts salt and 1 part gypsum (wt:wt). Ewes were then allowed free access to this mixture. In addition, any ewe that showed symptoms of Cu toxicosis (off feed, hemoglobinemia, hemoglobinuria) was given the gypsum (calcium sulfate) mix in gelatin capsules (size 12 EL, Torpac Inc., Fairfield, NJ) at a dosage of 56 g/d for 10 d.
As mentioned previously, serum was collected twice weekly from all ewes throughout the Cu episode and this serum was available for examination of serum constituents as markers of both the toxicosis as well as the recovery from Cu poisoning. Samples collected from eight ewes in early Sept. served as controls and samples from seven ewes showing symptoms of extreme Cu toxicosis were used to characterize Cu effects on serum profiles. In addition, serum from three ewes that received the calcium sulfate containing gelatin capsules over a 10-d period was used to monitor treatment effects on symptoms of Cu toxicosis. These samples were analyzed for the constituents shown in Table 1 by a commercial laboratory (Southwest BioLabs, Las Cruces, NM). Statistical comparisons between control and treated ewes were computed using the GLM procedure of SAS (SAS Inst. Corp., Cary, NC).
During the course of Cu exposure, eight of 31 (26%) ewe lambs died. Serum constituents in eight ewe lambs before exposure to elevated Cu and seven ewes showing extreme symptoms of Cu toxicosis (hemoglobinemia, hemoglobinuria, bilirubinuria) are presented in Table 1. These constituents were used to monitor effects of excess Cu on serum profiles and to assist in evaluating the degree of tissue damage induced by Cu poisoning. A number of constituents did not appear to be adversely impacted by Cu toxicosis when compared with control values (P > 0.10) including serum glucose, BUN, creatinine, alkaline phosphatase, serum protein, and alanine aminotransferase.
Total, direct, and indirect serum bilirubin were all greatly elevated (P < 0.01) in ewe lambs exhibiting symptoms of excess Cu. Total bilirubin was 0.6 mg/dL in serum from control ewes and 10.7 (± 1.9) mg/dL in Cu-affected animals (P = 0.002). Major elevations in serum bilirubin in sheep are usually observed only in cases of hemolytic crisis, and increased direct bilirubin in sheep usually results from hepatic disease and(or) obstruction of bile ducts (Cornelius, 1989). The chronic hemolytic disease induced by Cu toxicosis often induces hepatic necrosis which is generally accompanied by bilirubinuria (Cornelius, 1989). These data suggest that elevations in the various forms of serum bilirubin may serve as effective markers of Cu poisoning.
Gamma glutamyl transpeptidase (GGT) concentration in serum from control ewes was 148 IU/L compared with 404 ± 64 IU/L in ewes consuming excess Cu (P = 0.02). Likewise, aspartate aminotransferase (AST) was elevated (P = 0.03) in Cu consuming ewes compared with controls (2297 and 533 ± 532 U/L, respectively). A chapter on liver function by Cornelius (1989) suggested that GGT may be a valuable marker of hepatic disorders and Lewis et al. (1997) suggested that both GGT and AST could assist in diagnosis and treatment of hepatic problems in sheep. Creatine kinase (CK) concentration in ewes exposed to Cu was 9086 IU/L compared with 325 (± 1550) IU/L before ewes were exposed to dietary Cu. This large elevation in serum CK likely resulted from Cu induced hemolysis. Goonerate and Howell (1980) suggested that CK values would decline at the conclusion of a hemolytic crisis. Based on data shown in Table 1, serum bilirubin and CK were used as indicators of the effectiveness of dietary calcium sulfate in reversing the symptoms of copper poisoning.
Table 2 presents serum bilirubin and CK concentrations at 4 times over a 10-d period during which treated ewes received (gavage) calcium sulfate daily. The control values shown in Table 2 are the same as those in Table 1. On d 0 (extreme symptoms of Cu toxicosis present), the various forms of bilirubin and CK were greatly elevated (P < 0.02) in ewes suffering from excess Cu. Likewise, constituents remained elevated (P < 0.06) after 3 d of calcium sulfate treatment. Similarly, bilirubin and CK remained greater (P < 0.05) in treated ewes than controls after 1 wk of treatment but all values were much lower on d 7 than they had been on d 3. After 10 d of calcium sulfate treatment, total, direct, and indirect bilirubin were similar (P > 0.10) to values observed in control ewes. Total bilirubin on d 10 was 0.6 mg/dL in controls and 0.7 (± 0.1) mg/dL in treated ewes and serum CK was 325 and 1725 (± 656) IU/L in the two respective groups (P = 0.70). Likewise, after 10 d, treated ewes had regained their appetite and hemoglobinemia, hemoglobinuria, and bilirubinuria were no longer observed.
Implications
These data demonstrate that copper toxicosis induces marked changes in serum concentrations of a number of constituents including bilirubin and creatine kinase. Treatment of affected ewes with 56 g/d of a calcium sulfate mixture over a 10-d period appears to be effective in reversing the symptoms of copper poisoning as evidenced by return of serum bilirubin and creatine kinase to control
values.
Literature Cited
Adamson, A. H., D. A. Valks, M. A. Appelton, and W. B. Shaw. 1969. Copper toxicity in housed lambs. Vet. Rec. 85:368-374.
Cornelius, C. E. 1989. Liver Function. In: J. J. Kaneko (ed.) Clinical Biochemistry of Domestic Animals. Academic Press, San Diego, CA.
Goonerate, S. R., and J. M. Howell. 1980. Creatine kinase release and muscle changes in chronic copper poisoning in sheep. Res. Vet. Sci. 28:351-361.
Goonerate, S. R., J. M. Howell, J. M. Gawthorne, and J. S. Kumaratilake. 1989. Subcellular distribution of copper in the kidneys of normal, copper-poisoned, and thiomolybdate-treated sheep. J. Inorg. Biochem. 35(1): 23-36.
Gopinath, C., G. A. Hall, and J. M. Howell. 1974. The effect of copper poisoning on the kidneys of sheep. Res. Vet. Sci. 16:47.
Harker, D. B. 1976. The use of molybdenum for the prevention of nutritional copper poisoning in housed sheep. Vet. Rec. 99(5):78-81.
Humphries, W. R., C. F. Mills, A. Grieg, L. Roberts, D. Inglis, and G. J. Halliday. 1986. Use of ammonium tetrathiomolybdate in the treatment of copper poisoning in sheep. Vet. Rec. 119(24):596-598.
Humphries, W. R., P. C. Morrice, and I. Bremner. 1988. A convenient method for the treatment of chronic copper poisoning in sheep using subcutaneous ammonium tetrathiomolybdate. Vet. Rec. 123(2):51-53.
Lewis, N. J., A. H. Fallah-Rad, and M. L. Connor. 1997. Copper toxicity in confinement-housed ram lambs. Can. Vet. J. 38:496-498.
McCosker, P. J. 1968. Observations on blood copper in the sheep. II. Chronic copper poisoning. Res. Vet. Sci. 9:103.
Merck. 1998. The Merck Veterinary Manual. 8th ed. Whitehouse Station, NJ.1 Research supported by New Mexico Agr. Exp. Sta., Las Cruces.
2 Dept. of Anim. and Range Sci. Appreciation is expressed to L. Matuska and M. Hallford for clerical assistance.
Table 1. Serum constituents in Debouillet ewe lambs prior to exposure to elevated dietary copper and when extreme symptoms of copper toxicosis were observed(a)
Symptoms of Cu Toxicosis ITEM NONE EXTREME SE OSL(b) Glucose, mg/dL 144 99 19 0.11 Blood urea nitrogen, mg/dL 40 79 22 0.21 Creatinine, mg/dL 1.5 2.2 1.1 0.67 Alkaline phosphatase, IU/L 423 304 103 0.41 Total bilirubin, mg/dL 0.6 10.7 1.9 0.002 Albumin, g/dL 1.8 1.8 0.2 0.95 Total protein, g/dL 11.5 14.3 1.8 0.25 Alanine aminotransferase, U/L 64 308 105 0.11 Aspartate aminotransferase, U/L 533 2297 532 0.03 Direct bilirubin, mg/dL .23 4.9 .97 0.004 g-glutamyl transpeptidase, IU/L 148 404 64 0.012 Creatine kinase, IU/L 325 9086 1550 0.001 Globulin, g/dL 9.7 12.6 1.6 0.2 Albumin/globulin, ratio 0.18 0.27 0.07 0.44 Indirect bilirubin, mg/dL 0.4 5.1 0.9 0.003 P, mg/dL 12.6 5.5 2.4 0.05
(a)Extreme symptoms included hemoglobinemia and hemoglobinuria. Serum was collected from eight ewe lambs before Cu consumption and from seven ewes showing extreme symptoms of Cu toxicosis.
(b)Observed significance level.
Table 2. Serum constituents in control ewe lambs and those in which calcium sulfate was administered daily for 10 d after ewes exhibited extreme symptoms of copper toxicosisa
Treatment Item Control(b) Calcuim sulfate(c) SE OSL(d) Day 0(e) Total bilirubin, mg/dL 0.6 6.8 1.7 0.02 Direct bilirubin, mg/dL 0.2 3.2 0.9 0.02 Indirect bilirubin, mg/dL 0.4 3.6 0.8 0.01 Creatine kinase, IU/L 325 8477 1636 0.002 Day 3 Total bilirubin, mg/dL 0.6 5.4 1.5 0.03 Direct bilirubin, mg/dL 0.2 1.7 0.3 0.01 Indirect bilirubin, mg/dL 0.4 3.7 1.3 0.06 Creatine kinase, IU/L 325 2199 479 0.01 Day 7 Total bilirubin, mg/dL 0.6 1.7 0.3 0.02 Direct bilirubin, mg/dL 0.2 0.9 0.2 0.01 Indirect bilirubin, mg/dL 0.4 0.8 0.1 0.05 Creatine kinase, IU/L 325 2199 479 0.01 Day 10 Total bilirubin, mg/dL 0.6 0.7 0.1 0.24 Direct bilirubin, mg/dL 0.2 0.3 0.1 0.13 Indirect bilirubin, mg/dL 0.4 0.4 0.1 0.70 Creatine kinase, IU/L 325 1725 656 0.70
(a)Extreme symptoms included hemoglobinemia and hemoglobinuria.
(b)Control values were obtained from eight ewe lambs before Cu consumption began.
(c)Serum was obtained from three ewes that received (gavage) 56 g/d of a livestock salt:gypsum (76% calcium sulfate) mixture (2:1; wt:wt) in gelatin capsules.
(d)Observed significance level.
(e)Extreme symptoms were observed and treatment with calcium sulfate was initiated.