Editor's note: An abbreviated version of this case study appears in the February Bovine Veterinarian. See the full version here.
Chronic copper toxicity has classically been associated with chronic overprovision of copper to sheep and there are few reports of similar cases in cattle. Bradley1 reported a case in dairy cows that were moderately oversupplemented (37.5 ppm copper on a dry basis in total ration) for two years before mortality occurred and Perrin et al2 reported a case also of dairy cows that were greatly oversupplemented (>300 ppm copper on a dry basis in total ration) for nine months before mortality occurred.
Both would seem to imply, as opposed to sheep, some degree of resistance to copper toxicity on the part of cattle. Marta Lopez-Alonso et al3 reported an increasing frequency of bovine copper toxicity. Two cases of presumed copper toxicity in young, growing Holsteins were recently presented to the Wisconsin Veterinary Diagnostic Laboratory.
The first case involved a group of approximately 40, 200-250kg Holstein heifers in a group pen that were fed a ration (fed as individual components) composed of hay, corn, oats and a mineral/trace mineral fortified pellet. Within several weeks of introduction, several animals displayed a vague clinical picture of anorexia and lethargy followed by death, typically within two to three days of onset. Clinical examination revealed only mild icterus and chocolate-colored blood, interpreted as methemoglobinemia. Respiratory distress was not observed, however.
Clinicopathologic findings were not available but reference was made to increased liver enzymes. Treatments with antiobiotics and methylene blue were unsuccessful as were attempts to document nitrate toxicosis. Field necropsies revealed “generalized icterus, yellow-orange liver discoloration and increased, coarse granularity of livers”.
ICP/MS analysis of liver copper performed in our laboratory yielded 1500ug/g dry wt. (440ug/g wet). Toxicity is thought to occur with wet values above 250ug/g4. Routine aerobic and anaerobic cultures of liver, kidney, lung and bile were negative.
Histopathologic examination of formalin fixed, H&E stained sections revealed the following in the liver: There was diffuse vacuolation of hepatocytes with extensive, random, single cell to coalescent hepatocyte necrosis. Portal tracts were markedly expanded by fibrous tissue, often with portal-portal bridging, accompanied by marked biliary hyperplasia. Most tracts contained low to moderate numbers of mononuclear inflammatory cells along with low to moderate numbers of prominent macrophages distended by light brown, coarsely granular, cytoplasmic material that stained brick red with rhodanine, indicative of copper, in this case likely complexed with lipofuscin.
A second, even less well-documented, case involved a group of 25, 4-month-old Holstein steers, two of which died after similar, brief, non-specific illness. No clinicopathologic or ration data was available. Field necropsies revealed generalized icterus, extreme pallor with watery blood, orange colored liver, “gun metal” kidneys and dark red urine. Liver copper was 320ppm (wet basis). Bacteriologic and virologic testing was negative.
Histopathologic findings in the liver included diffuse macro and microvesicular lipidosis (indicative of liver damage as opposed to the flooding with fat that occurs in postpartum dairy cows) with random hepatocyte necrosis and marked bile stasis. Many portal macrophages were distended by brown, granular material that stained positive with rhodanine and PAS (indicative of lipofuscin).
In the kidneys, many cortical tubules exhibited lost, necrotic or regenerative epithelia and iron positive luminal contents (hemoglobin). Some tubules contained casts of necrotic cellular debris. Many medullary tubules contained some combination of proteinaceous and cast material. This damage is likely due to some combination of hemoglobin toxicity and hypoxia.
In the heart, the myocardium exhibited low numbers of small (50-100um diameter) foci of necrotic myofibers along with low numbers of scattered neutrophils.
These cases strongly suggest that growing Holsteins are more susceptible to chronic copper toxicity than previously thought and, likely, at dietary levels lower than those that induce toxicity in adult Holsteins. Both cases appear to have involved simple but unappreciated, overprovision of dietary copper. That said, chronic copper toxicity should be included on a differential diagnosis list for bovine cases exhibiting the gross lesions of methemoglobinemia, hemoglobinuria, pallor, icterus and orange-brown liver discoloration. The most recent Dairy NRC lowered the maximum tolerable ration copper level from 100ppm dry weight down to 40ppm dry weight based on reports of toxicity.
Copper is a trace element essential to all cells but, like iron, must remain properly sequestered to prevent toxicity. Accordingly, serum copper is bound to albumin and ceruloplasmin and liver copper is bound to the metal binding protein metallothionein. Under some circumstances, excess copper can catalyze the formation of highly toxic hydroxyl radicals via the Fenton reaction, resulting in lipid peroxidation of membranes and damage to various other molecules. The extent to which this occurs in affected livers and erythrocytes is not known.
Slightly different versions 5,6,7,8 exist between sources but the pathogenesis of chronic copper poisoning can generally be stated as: Copper is absorbed from the small intestine and transported to the liver via albumin and copper transport protein(s). It is partitioned within the liver into several pools, one of which is a biliary pool involved with resecretion into the gut via bile, constituting the main excretory pathway. A second pool is complexed with ceruloplasmin for export. Yet another pool involves excess accumulation within lysosomes to a point of release with subsequent apoptosis of the host hepatocyte. Increasing copper concentrations result in accelerated apoptosis and hepatocellular degeneration until a point is reached wherein the rate of hepatocellular loss exceeds the capacity of the organ to phagocytose and sequester the debris quickly enough, at which point plasma copper levels will begin to rise.
Excess circulating copper will saturate transport molecules resulting in free circulating copper that leads to lysis of erythrocyte membranes, perhaps by free radical mediated membrane lipid peroxidation. The resulting hypoxia likely results in the centrilobular hepatic necrosis typically seen in poisoned sheep (but not observed in these cases) which serves to release even more copper until a full blown hemolytic crisis ensues, accompanied by liver failure. The released hemoglobin accumulates in renal tubules where it contributes to tubular damage. Affected bovines appear to suffer a greater degree of methemoglobinemia than sheep.
Sheep, as a species, have a limited ability to excrete excess copper, often resulting in a hemolytic crisis and fulminant liver failure while certain breeds of dogs are genetically unable to excrete excess copper resulting in progressive accumulation and culminating with an end-stage liver rather than a hemolytic crisis. The liver lesions described here have much in common with those associated with Wilson disease, the human version of genetically impaired copper excretion that typically becomes evident around 5 years of age and, interestingly, at a liver copper level also around 250ppm wet weight. Another example of genetic copper toxicity involves the Long Evans cinnamon rat, a species maintained for the study of Wilson disease and free radical/oxidative stress mediated disease. A large percentage of these rats die of non-infectious hepatitis at several months of age while survivors typically die of malignant liver tumors at around a year of age.
Though detailed analyses of the ration were not available in either case, it is known in case 1 that a pellet containing 1014ppm copper was being fed. It is well known that bovines have strong dietary predilections and are very adept at “sorting” through feeds to suit their tastes. It is suspected, in this case, that affected animals consumed excessive amounts of the aforementioned, copper fortified pellets and that this would have resulted in a total ration level of around 175-200ppm Cu and perhaps more. It is interesting to note that, in case 1, a group of similar sized Holstein steers in an adjacent pen fed the same ingredients but with a higher proportion of said pellets, incurred far lower mortality.
It seems evident from these cases that copper poisoned, growing Holsteins can exhibit either or a combination of hemolytic anemia and methemoglobinemia and that the massive hepatic necrosis observed in sheep does not necessarily occur. Differential diagnoses for hemolytic anemia with hemoglobinuria in growing Holsteins should include: Leptospirosis, Babesiosis, bacillary hemoglobinuria due to Clostridium hemolyticum and copper toxicity. Differential diagnoses for methemoglobinemia would include nitrate toxicity
An antemortem, preferably noninvasive, test to assess copper accumulation would be desirable but, unfortunately, associations between blood copper or ceruloplasmin and liver copper are too weak to be of diagnostic value so liver biopsy remains the only viable method3.
A second installment of this article will include results of liver copper assays obtained from WVDL case material and a slaughter survey of cull dairy cow livers. The possibility of subclinical, copper induced hepatopathy in dairy cows will also be touched on.
1-Bradley CH: 1993, Copper poisoning in a dairy herd fed a mineral supplement. Canadian Veterinary Journal 34: 287-292
2- Perrin DJ, Schiefer HB, Blakley BR: 1990, Chronic copper toxicity in a dairy herd. Canadian Veterinary Journal 31: 629-632
3- Marta Lopez-Alonso et al: 2006, Assessment of some blood parameters as potential markers of hepatic copper accumulation in cattle. Journal Veterinary Diagnostic Investigation 18: 71-75
4-Puls R: 1990, In Mineral Levels in Animal Health, pg 71. Sherpa International
5-Maxie MG: 2007, In Jubb, Kennedy and Palmers Pathology of Domestic Animals, 5th ed., pp 380-381. Elsevier Ltd.
6- Kaneko JJ: 1989, In Clinical Biochemistry of Domestic Animals, pg 765. Academic Press
7- Gupta RC: 2007, In Veterinary Toxicology, 1st ed. Pg 428. Elsevier Ltd
8- McGavin MD and Zachary JF: 2007, In Pathologic Basis of Veterinary Disease, pg 426-427. Mosby Elsevier
This case study submitted by Doug Lyman, DVM, Dipl. ACVP, Wisconsin Veterinary Diagnostic Laboratory.