Postparturient Hemoglobinuria

Johanna L. Watson • Gary P. Carlson

A syndrome of intravascular hemolysis, hemoglobinuria, and anemia has been recognized in postparturient dairy cattle around the world.¹⁰ The disease occurs sporadically, and the incidence is relatively low.

Blood transfusion and supportive IV fluids are indicated in valuable cows with severe life-threatening anemia. Correcting hypophosphatemia consists of providing phosphate, initially as sodium acid phosphate (NaH₂PO₄H₂O), 60 g/300 mL of water IV, followed by oral supplementation with 200 to 300 g sodium phosphate salts every 12 hours. Correction of dietary imbalances is indicated.

■ Copper Toxicosis

Johanna L.

Copper is an essential nutrient for domestic animals, but excessive supplementation can result in toxicity. Copper poison­ing is a common intoxication in ruminants. Reviews of the toxicodynamics and clinical syndromes have been published.^1-3 Lambs are most susceptible to the toxic effects of copper, but poisonings have occurred in adult sheep, goats, and cattle; Merino sheep are more resistant than British breeds.⁴ Cattle have been poisoned by ingesting diets containing 37 mg of copper/kg of feed for 2 years.⁵ Cattle fed 12 mg or more of copper/kg of body weight have been reported to develop subclinical hepatic disease.⁶

Elemental copper is an essential trace mineral with a narrow therapeutic window. Dietary requirements for growing sheep range between 4 and 6 parts per million (ppm), yet toxicity can occur whenever sheep are exposed to diets containing as little as 10 to 20 ppm.^7-9 Phytogenous copper poisoning results from concomitant ingestion of copper at 20 ppm of food, molybdenum, and sulfate. Ingested salts of copper are absorbed through enterocytes by carrier proteins and transported to blood in loose complexes with albumin and amino acids.¹⁰ Roughly 20% of total plasma copper is ionized copper. Between 70% and 90% of this ionic copper is internalized by hepatocytes, where it is redistributed to bile, packaged in lysosomes in protein complexes, or used for the formation of ceruloplasmin.¹⁰ Accumulation of copper occurs because daily hepatic biliary copper excretion amounts to less than 1% of ingested copper in ruminants.¹¹ Hepatic storage can buffer high levels of copper intake until the sites become saturated. At that time, hepatocytes die spontaneously or in response to environmental stress or dietary changes. Hepatocyte death causes the release of large amounts of cuprous copper into the blood. Animals that have preexisting hepatitis from pyrrolizidine alkaloids store less copper and are more susceptible to the hemolytic crisis.^4,12

Free inorganic copper is an oxidant and can participate in the Fenton reaction.

The hemolytic phase of copper poisoning is often initiated by noxious stimuli that could include shipping, hierarchal change, administration of oxidative drugs, starvation, or change of housing.¹³ High-protein diets have increased resistance to the hemolytic crisis in sheep, but the role of extra molybdenum intake in these supplemented animals was unclear.^9,14 The mechanistic relationship between stressful events and hepatocyte release of copper is unknown.

Sources of copper that have been responsible for copper accumulation in animals include trace mineralized salt, inap­propriately formulated cereal grain mixtures, forages from pastures that have been fertilized by swine or chicken manure,¹⁵ orchard pastures that have been contaminated by copper- containing fungicides, rations containing more than 20% chicken litter, or diets that contain high concentrations of palm kernel oil. Other potential sources of copper include fencing and copper piping and overdoses of parenterally administered copper salts. Dairy goats have been poisoned by supplementation of trace minerals that were formulated for dairy cattle.

The toxic dose of copper for each species is variable and depends on duration of exposure, the animal's genetics, and the amount of molybdenum being fed. Diets with copper/ molybdenum ratios over 6 : 1 are more likely to result in copper poisoning than diets with lower ratios. The interaction between the two minerals may occur in the solid phase of rumen ingesta, where insoluble and nonabsorbable complexes among copper, sulfur, molybdenum, and large-molecular-mass protein com­plexes are formed.

The respective single toxic doses of copper for sheep and cattle range between 20 and 110 mg/kg and 220 to 880 mg/ kg of body weight. Most poisonings occur after long-term, low-dose ingestion of the element. Copper poisoning can occur in sheep after 2 months of daily dosing of 3.5 mg/kg of copper, feeding a ration that contains 20 ppm of copper for several months, or after a single SC injection of copper EDTA salt at a dose of 2 mg/kg.¹⁹ Sheep have been poisoned by subcutaneous injection of 3 to 4 mg/kg of copper-calcium EDTA but remained normal after 6 mg/kg of copper methion- ate.²⁰ One study that fed 3.7 mg/kg of copper daily for 84 days reported peak hepatic copper levels that ranged between 258 and 375 mg/kg wet weight by 26 days after the final copper feed.²¹ A single dose of 50 mg of copper caused acute hemolytic crisis in sheep when treated during midpregnancy. The largest incidence of hemolytic anemia occurred in Welsh sheep, and the lower cumulative incidence was in Cheviot sheep.²² Single doses of copper calcium ETDA at 25 mg/animal have killed lambs.²³ Cumulative doses of copper ranging between 12.8 and 22 g have produced hemolytic anemia in Suffolk lambs by 42 days.

Goats may show signs of copper poisoning by 144 days after daily feeding of a ration that contains 80 mg copper/kg. Placental transfer of copper occurs, but concentrations in the tissues of lambs are not toxic even during the hemolytic phase in the dam. Adult cattle may be poisoned by feeding 5 g of copper sulfate daily for as long as 4 months.

Clinical Signs

Animals exposed to high levels of copper are asymptomatic for weeks until the onset of hepatic necrosis.^25,26 Signs develop rapidly thereafter and are reflective of coexisting anemia, myopathy, and neurologic, renal, and hepatic disease. Clinical signs include inappetence, lethargy, weakness, recumbency, cool extremities, pallor, and grayish discoloration of the mucous membranes. Affected animals have a markedly increased pulse rate, poor pulse quality, tachypnea, hypotension, and hypother­mia. Urine is dark red from the presence of free hemoglobin products. Feces are dark or have yellowish discoloration but are normally formed and do not contain hemoglobin unless secondary abomasal ulceration has occurred. Animals may have marked petechiation of the conjunctival mucosa. Pregnant animals often abort secondary to hypoxemia. Recumbent patients usually expire without struggling.

Pathology

The tissues of hemolyzing animals are pale and icteric, and serous surfaces are covered by petechial and ecchymotic hemorrhages. The liver is often pale and yellow, and the lungs are firm. Kidneys turn black and have a metallic sheen due to entrapped hemoglobin. The urinary bladder of copper-poisoned animals is filled with serosanguineous urine. Microscopic changes include hemoglobinuric and tubular nephrosis and necrosis of the splenic follicles and hepatocytes. There is also biliary ductular proliferation and pericholangitis.^21,27 Hepatic necrosis may be detectable by microscopic examination for as long as 412 days post poisoning.

Diagnosis

Normal hepatic concentrations in sheep have been reported as 173 ± 130 (mean ± SD) and 129 ± 59 ppm (wet matter) for adults and lambs, respectively.²⁸ Wet matter hepatic concentra­tions of copper in poisoned animals were 429 ± 249 (mean ± SD) ppm. Dry weight concentrations may be greater than 3000 ppm. Hepatic concentrations are usually high before and after the hemolytic episode, but normal hepatic copper con­centrations in hemolyzing animals have been reported.¹⁹ The correlation between plasma and hepatic copper concentrations 29-30

in prehemolytic animals is poor.^29-30 Copper poisoning in animals with normal plasma concentrations and near-toxic liver levels have been reported.³¹ Normal plasma copper concentrations range from 13 to 20 μmolZL (0.8 to 1.2 μg/mL).²¹ Serum copper concentrations range between 0.60 and 1.50 μgZmL (0.6 to 1.5 ppm). Plasma copper concentrations that range between 2.4 and 20.0 μgZmL (2.4 to 20 ppm) are diagnostic for acute toxicosis.

Clinical Pathology

There are no consistent hematologic changes until 24 hours before the hemolytic crisis, when sudden concentrations of cytosolic hepatic enzymes rise coincidentally with a sharp rise in plasma copper concentration. Plasma copper concentrations fall rapidly following the hemolytic crisis and are often near normal by 4 days after hemolysis.²⁵ Copper concentrations within erythrocytes remain high.

Clinicopathologic changes that occur during the acute hemolytic crisis include Heinz body formation, intravascular hemolysis, methemoglobinemia (as much as 5%), decreased PCV, and increased concentrations of plasma bilirubin, AST, GGT, alkaline phosphatase, total bilirubin, creatine kinase, creatinine, plasma urea nitrogen, and plasma ceruloplasmin. Urine is dark brown to black and contains high concentrations of protein, blood, and hemoglobin casts. Microscopic examina­tion of urine may detect erythrocytic casts and inflammatory cells.³² Sheep that survive the hemolytic episode develop reticulocytosis by 4 days after the onset of clinical signs.³³

Hepatic concentrations of 16 mmol copper/kg of dry matter are the threshold for development of hemolytic anemia. The half-life of copper in the liver of untreated poisoned sheep has been estimated to be 175 ± 91 days,²¹ and high copper concentrations in hepatic tissues can be found for as long as 100 days after initiation of the hemolytic episode.²⁹

Kidney concentrations of copper in animals with hemolytic crisis are 15 and 50 ppm for dry and wet weight volumes, respectively. Sheep may have increased plasma concentrations of γ-glutamyltransferase (GGT) and AST for 3 days before the onset of hemolysis, and glutamate dehydrogenase may be increased for as long as 700 days after cessation of copper ingestion.²¹ Fecal copper concentrations may exceed 10,000 ppm during the hemolytic episode.

Treatment

Animals with acute hemolytic syndrome should be insufflated with oxygen and treated with vitamin E (3 to 5 daily doses consisting of 3000 IU/dose). If PCV is below 8%, animals should be given packed washed homologous erythrocytes. Additional therapy should include D-penicillamine (Cuprimine [Merck Sharp & Dohme, Rahway, N.J.] at 52 mg/kg of body weight daily for 6 days), anhydrous sodium sulfate (1 g/sheep daily for 6 days), and ammonium molybdate (100 mg/sheep daily for 6 days). Treatment with D-penicillamine increases urinary copper excretion by 10- to 20-fold.³⁴ Single-dose therapy with D-penicillamine (28 mg/kg body weight) increases copper excretion, but the effect is transient and insignificant for reducing the total hepatic copper load.³⁵

Dietary supplementation with 7.7 ppm ammonium molyb­date also results in hepatic copper concentrations that are 40% lower than in unsupplemented controls. Addition of 7 or 15 mg molybdenum for 80 days to experimentally poisoned sheep has reduced hepatic copper concentration in sheep by 34% and 46%, respectively.³⁶ Copper-poisoned cattle have been successfully treated with oral sodium molybdate (3 g daily) and sodium thiosulfate (5 g daily). When introduced into the sulfur-rich rumen contents, molybdenum salts complex with high-molecular-weight proteins in the solid phase of the digesta.³⁷ The protein-bound thiomolybdate aggregates strongly chelate copper in insoluble and indigestible complexes. Molybdenum salts also enhance biliary copper excretion and selectively remove copper from hepatic metallothionein.^16,38,39 For poisoned cattle, top dressing food with 500 to 1000 mg ammonium molybdate daily for 18 days markedly reduced the amount of hepatic copper.⁴⁰

Parenterally administered ammonium tetrathiomolybdate has been recommended for treatment of acute hemolytic crises due to copper poisoning. IV administration of ammonium tetrathiomolybdate reduces both lysosomal and cytosolic copper in hepatocytes. The drug has been given as a 5% solution in sterile saline with an IV dosing schedule of either 50 to 100 mg twice weekly for up to 11 weeks, or as 3 doses consisting of 3.4 mg/kg of body weight on alternate days and beginning at the onset of hemolytic crisis.^41-45 Concomitant administration of 36 mg of xylazine IV increased copper excretion in bile by as much as 2.25-fold over controls that were treated with thiomolybdate only.⁴⁶ Parenteral administration of thiomolyb­date results in transient increases of copper concentrations in blood for as long as 24 hours. Because copper is acid insoluble, the effect is to protect copper-exposed animals from hemolytic crises and reduce tissue damage during a hemolytic event.^47,48 Biweekly injection of 100 mg molybdenum as tetrathiomo­lybdate increased daily hepatic biliary copper excretion by a factor that ranges between 150% and 300%.⁴⁹

Prevention

Owing to the sporadic nature of copper poisoning and lack of signs during the accumulative phase, it can be difficult to prevent. Clients should be counseled to purchase concen­trates with label claims for the species being supplemented. Copper-supplemented salts should be restricted, especially in sheep. Pastures heavily contaminated by copper can be top dressed with molybdenum phosphate (113 g molybdenum/ acre). Fencing and plumbing that contain copper sulfate or metallic copper should be removed from the environment. One study in sheep receiving 36 mg copper/kg daily for 18 months controlled the hemolytic crisis and associated mortality due to copper poisoning by SC injection of sterile ammonium tetrathiomolybdate (containing 3 to 4 mg molybdenum) on alternate days for three injections.⁴⁴ Addition of ammonium molybdate at 7.7 ppm and sodium sulfate at 4200 ppm reduced liver copper and decreased plasma ceruloplasmin in experi­mentally exposed lambs.³⁰

Feeding experimentally poisoned lambs sodium molybdate and sodium sulfate at respective daily dosages of 20 mg and 6 g reduced hepatic copper content after 90 days.⁵⁰ Addition of 3 mg/kg of food (dry matter) molybdenum in the form of tetrathiomolybdate reduced hepatic copper accumulation by as much as 33-fold.⁴³