Diagnosis

Diagnosis of acute blood loss is based on clinical signs, evidence of recent hemorrhage, and anemia accompanied by hypoproteinemia. Hemoperitoneum and hemothorax may be suggested by ultrasound and by abdominocentesis and thoracocentesis, respectively.

Treatment

Treatment of acute blood loss should initially be aimed at stopping the hemorrhage. External hemorrhage may be managed by pressure wraps or appropriately placed ligatures; however, it may be inadvisable to attempt to control internal hemorrhage when the patient is a poor risk for general anesthesia and the source of hemorrhage may not be found. Hypovolemic shock should be treated by prompt intravenous (IV) administration of 40 to 80 mL/kg body weight of sodium-containing crystalloid solutions. Studies indicate that a small volume of hypertonic saline (4 to 6 mL/kg of 7.2% sodium chloride) may temporarily reverse the pathophysiologic sequelae of severe hemorrhagic shock.^1,2 The total volume of necessary crystalloid solution is usually much greater than the volume of blood lost because crystalloid solutions distribute throughout the extracellular space. The clinical response to fluid administration should be evaluated in light of ongoing losses to determine the necessary replacement volume.

If anemia becomes life threatening, whole-blood transfusion must be considered. A PCV less than 20% in an animal with acute blood loss suggests depletion of erythrocyte reserves, and persistent reduction of the PCV to 12% or less over 24 to 48 hours indicates the need for blood transfusion. A low but stable PCV (12% to 20%) does not necessitate transfusion because transfusion should be reserved for cases in which oxygen delivery to the tissues is inadequate to support life. Blood transfusion can only be viewed as a temporary therapeutic procedure because even crossmatch-compatible, allogeneic erythrocytes are removed from the circulation by the mononuclear phagocyte system within 2 to 4 days of transfusion.³ Horses and cattle display a high degree of blood type polymorphism, and minor antigenic incompatibilities are only delineated by blood typing.⁴ Serum antibodies against nonhost erythrocyte antigens (erythrocyte alloantibodies) probably mediate the short lifespan of transfused erythrocytes. Compatibility testing is used to avert life-threatening antigen-antibody reactions caused by major blood group mismatching.

The routine crossmatch involves incubating washed erythrocytes from donor (major) and recipient (minor) with serum from the other. Gross and microscopic examination for clumping demonstrates serum agglutinins in horses. Sensitized cattle erythrocytes do not become clumped in saline solution but do lyse in the presence of rabbit complement, so only a hemolytic crossmatch can be performed in this species. Not all equine erythrocyte alloantibodies act as agglutinins, and hemolysins must be detected by adding complement to the reaction mixture. Pooled rabbit serum must first be absorbed with equine erythrocytes to remove naturally occurring antibodies. The need for special handling and storage of rabbit serum makes hemolytic crossmatch procedures impractical for most veterinarians. These tests are best performed by veterinary hematology laboratories (e.g., Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis), which usually require serum and whole blood in sodium citrate or acid-citrate-dextrose (ACD) to crossmatch.

The first transfusion of whole blood to a horse or ruminant that has not been previously transfused or sensitized by immunization or pregnancy is usually well tolerated because natural alloantibodies are of low concentration and weak activity. After incompatible transfusions, alloantibodies develop rapidly, making subsequent transfusions more hazardous.

Blood for transfusion should be collected aseptically into sterile containers containing sodium citrate (2.5% to 4%) or ACD solution and used immediately. The necessary dosage can only be estimated, but in most cases, replacing 20% to 40% of the calculated blood loss is sufficient to maintain life until the bone marrow can respond. A drop in PCV from 36% to 12% in a 500-kg animal (8% body weight blood) represents a loss of 27 L of blood. In this case, 6 to 8 L of whole blood would be therapeutic and easily donated by another 500-kg individual. Blood warmed to 37° C (98.6° F) should be administered through an in-line filter to remove clots. After pretransfusion vital parameters have been recorded, 0.1 mL blood/kg body weight is given over 5 to 10 minutes and the evaluation repeated. If parameters and attitude are unchanged, the transfusion can be continued at a rate not to exceed 20 mL/kg/hr. The recipient should be continuously monitored so that the transfusion can be stopped if adverse reactions occur, such as tachypnea, dyspnea, restlessness, defecation, tachycardia, piloerection, muscle fasciculations, or sudden collapse. Although these signs may not indicate anaphylaxis, severe reactions should be treated with epinephrine (0.01 to 0.02 mL/kg, 1:1000). Mild signs may respond to a slowed transfusion rate or administration of corticosteroids or flunixin meglumine. Because it is often not possible to delineate the cause of transfusion reactions, the safest approach is to discontinue the blood and administer isotonic crystalloid solutions.

The prognosis is good for most cases of acute blood loss if hypovolemic shock is quickly treated and the bleeding stops. The normal bone marrow begins to replace lost cells within 5 days. Sequential analysis of the PCV will be necessary to determine whether blood loss is controlled. Examples of specific disorders follow.

Guttural Pouch Mycosis

See Chapter 31.

Hemoperitoneum in Horses

MONICA ALEMAN

Hemoperitoneum is the accumulation of blood in the abdominal cavity, which can be a life-threatening problem. Causes associated with hemoperitoneum in the horse include trauma, postoperative abdominal hemorrhage, neoplasia, complications from pregnancy and foaling (uteroovarian, middle uterine, and external iliac artery rupture), organ rupture, mesenteric injury, coagulopathies, ovarian hematoma, systemic amyloidosis, and idiopathic causes.^5-11 The underlying cause of hemoperitoneum may be identified in the majority of the cases (78%).¹¹ Trauma (mainly of spleen, as well as reproductive tract with associated vessels in mares) and neoplasia are the most common causes of hemoperitoneum.^7,11

A recent retrospective study of 67 horses with hemoperitoneum revealed that thoroughbreds and Arabians were overrepresented breeds.¹¹ In addition, middle-aged and older horses (>13 years old) were also overrepresented. Females may be overrepresented.^10,11 The most clinical signs include abdominal discomfort, lethargy, hypovolemic shock, pale mucous membranes, prolonged capillary refill time, tachycardia, and tachypnea.^10,11 Other clinical signs are anorexia, reluctance to move, weakness, trembling, cool extremities, and abdominal distention.¹⁰

Clinicopathologic abnormalities include anemia, neutrophilia, lymphopenia, thrombocytopenia, hypoproteinemia, hypocalcemia, and azotemia.¹⁰ Abnormalities in clotting parameters may be observed, depending on the cause. Hemorrhagic abdominal effusion is characterized by high red blood cell (RBC) count (>2.4 million RBCs/μL), PCV (≥18%), and total protein (≥3.2 g/dL), with a normal to high leukocyte count.¹⁰ Central venous pressure and blood lactate concentration appear to be early indicators of hypovolemia caused by acute blood loss.¹² Swirling fluid and the site of hemorrhage may be evident on abdominal ultrasound.

Primary goals of therapy consist of treating hypovolemic shock, restoring perfusion and oxygen delivery to tissues, correcting fluid deficits, stopping further blood loss, and preventing complications. Blood transfusion should be considered if anemia becomes life threatening. The use of antifibrinolytic and procoagulant agents have been reported in the literature, but controlled studies on its efficacy and safety in the horse with acute blood loss are lacking. Physical activity must be restricted in affected patients.

The short-term outcome is strongly associated with the underlying cause.¹¹ Horses with neoplasia, uterine artery rupture, mesenteric injury, or disseminated intravascular coagulation (DIC) are a greater risk of death. The survival rate has been reported to range from 51% to 74%.^10,11 Poor short-term outcome was significantly associated with high respiratory rate in one study.¹¹ Prepartum hemorrhage appears to be associated with a poorer prognosis than postpartum hemorrhage (100% vs. 20% mortality, respectively).¹¹

Hemothorax

Hemothorax may occur secondary to trauma (including lung biopsy), neoplasia, and strenuous exercise¹³ (see Chapter 31). Hemothorax in neonatal foals may be the result of lacerated lungs and vessels from fractured ribs¹⁴ (see Chapter 19).

Exercise-Induced Pulmonary Hemorrhage

Exercise-induced pulmonary hemorrhage (EIPH) has not been associated as a major cause of blood loss. EIPH is associated with high intensity exercise in horses. An estimated 14% to 75% of racehorses examined by endoscopy have EIPH.¹⁵ Based on bronchoalveolar lavage analysis, a study suggested that 100% of horses performing strenuous exercise have EIPH.¹⁶ EIPH has been reported in various breeds. A recent study reported that the frequency of EIPH is associated with race type, distance, gender, and age. In one study, epistaxis was more common in females, in older horses than in horses less than 2 years old, after steeplechase races than flat races, and with shorter distances (≤1600 m [1 mile] long).¹⁷ Recurrence rate in that study was reported to be 4.64%. The pathophysiology of EIPH is not completely known. For a complete description of this disorder, see Chapter 31.

Gastric Ulceration

MONICA ALEMAN

Gastric ulceration in horses is also known as equine gastric ulceration syndrome (EGUS). Although EGUS is not an important cause of blood loss, it has a high prevalence among adult horses. Thoroughbred racehorses have the greatest prevalence (82% to 93%), followed by endurance horses (67%), show horses (58%), hospitalized horses (49%), and geriatric patients with abdominal pain (18%).^18-22 Gastric ulcers were found in 66.6% of pregnant and 75.9% of nonpregnant mares in a thoroughbred breeding farm.²³ Clinical signs may include poor hair coat, decreased appetite, poor performance, nervous disposition, abdominal pain, teeth grinding, and salivation. Proposed causes of gastric ulceration include exercise, transportation, grazing deprivation, alternating periods of feeding and fasting, diets with high concentrate content, and confinement. For a complete description of gastric ulceration, see Chapter 32.

Right Dorsal Colitis

Right dorsal colitis (RDC) is an enteropathy associated with administration of NSAIDs, most often phenylbutazone.^24,25 Other proposed factors that predispose to RDC include infection, immune-mediated response, genetics, and stress. The pathogenesis of RDC is unknown. NSAIDs act by inhibiting cyclooxygenase (more inhibition of constitutively expressed COX-1 than inducible expressed COX-2 during states of inflammation), which will cause inhibition of the production of prostaglandin E resulting in hypoxic or ischemic GI mucosal damage and may delay mucosal healing.²⁵

Ponies and young performance horses appear to be predisposed to RDC. The clinical signs include inappetence, lethargy, intermittent or episodic colic, diarrhea, and weight loss.

Clinicopathologic abnormalities may include mild anemia, moderate to severe hypoproteinemia with hypoalbuminemia, hypocalcemia, and in some cases azotemia. Although mild anemia may be seen in most cases, horses occasionally present with severe anemia and hematochezia. RDC often develops over days or weeks. (See Chapter 32.)

Basic Physiology of Normal Hemostasis

Understanding the pathogenesis and manifestations of hemostatic disorders is based on knowledge of the normal physiologic mechanism of hemostasis. Hemostasis can be viewed as two interrelated components, coagulation and fibrinolysis (both with their respective inhibitors), which function to stop bleeding from a damaged blood vessel and maintain nutrient blood flow.

Coagulation is mediated by blood vessels, platelets, and blood procoagulant proteins. When a blood vessel is damaged, vasoconstriction occurs, followed by rapid adherence of platelets to subendothelial collagen. Platelet adhesion causes membrane conformational changes that trigger aggregation, contraction, and granule secretion (the basic platelet reaction). Platelet phospholipoprotein (platelet factor 3) provides the necessary surface to catalyze interactions among the activated coagulation proteins that result in thrombin formation. Coagulant proteins are localized to this hemostatic plug because the platelet surface protects them from plasma anticoagulants. Through an incompletely understood mechanism, platelets also prevent spontaneous hemorrhage into the skin and mucous membranes by maintaining “vascular integrity.”

Procoagulant proteins circulate in the blood as precursive forms (zymogens) that must be altered during coagulation to become active. Numerous communications exist between the traditional extrinsic and intrinsic pathways, although initiating mechanisms remain distinct.²⁶ The extrinsic system is initiated when lipoprotein tissue factor (TF) gains access to the bloodstream. TF is widely distributed in most tissues, including endothelial cells and monocytes, and may be increased or secreted in response to numerous pathologic stimuli, such as bacterial endotoxin. Intrinsic coagulation is initiated when blood is exposed to a negatively charged surface such as activated platelets. Because of reciprocal activation between factor XII and prekallikrein, the intrinsic coagulation pathway stimulates formation of numerous inflammatory mediators (e.g., kinins, complement). Both coagulation pathways culminate in the formation of activated factor X (Xa), by which thrombin is generated. In addition to catalyzing the conversion of fibrinogen to fibrin, thrombin promotes platelet aggregation, enhances cofactor activities of factors V and VIII, and activates factor XIII and protein C.²⁷ Mechanisms to localize coagulation to the site of vascular injury are critical to protect against generalized thrombosis.²⁸ Plasma anticoagulant proteins include the serpins, which inhibit activated coagulation factors, and the protein C system, which is directed against cofactors V and VIII.²⁹ Antithrombin III (AT III), the main physiologic inhibitor of thrombin and Xa, normally provides 70% of the anticoagulant activity of plasma. Although not absolutely needed, heparin accelerates AT III action by 2000-fold.³⁰ Activated protein C destroys factors V and VIII, ultimately limiting its own activation, which depends on thrombin and endothelial cofactor, thrombomodulin. Protein S enhances the anticoagulant ability of protein C.

The fibrinolytic system is activated simultaneously with coagulation and functions to prevent tissue ischemia by limiting the extent of fibrin clot formation. Plasmin, primarily responsible for degradation of fibrin, exists in the plasma as the zymogen plasminogen. Plasminogen has a high affinity for fibrin, as does tissue plasminogen activator (tPA); therefore, clots contain the necessary components to allow lysis from within, and systemic plasmin formation is avoided. Alpha-2-antiplasmin (α₂-AP), the main physiologic inhibitor of plasmin, competes with the binding of plasminogen to fibrin, and the clot contains equal amounts of both glycoproteins. Because of this molar balance between α₂-AP and plasminogen, a normal blood clot does not lyse spontaneously, despite fixation of tPA. Physiologic inhibitors of tPA (PAIs) are found in plasma, platelets, and endothelial cells, and platelet-derived PAI also protects a blood clot against premature lysis. Clot lysis is initiated if additional tPA is taken up from the surrounding tissues; stasis upstream from the occluded vessel is a potent stimulus for release of endothelial tPA. Conversion of plasminogen to plasmin allows partial digestion of fibrin and exposure of additional plasminogen-binding sites. When this additional plasminogen is converted to plasmin, the inhibitory effect of α₂-AP is overcome, and clot lysis is accelerated. Plasmin hydrolyzes fibrinogen and fibrin with equal affinity, as well as numerous other procoagulants, and it can activate complement and kininogen. The physiologic actions of plasmin are limited to the fibrin clot by the affinity between the latter and plasminogen and the presence of α₂-AP in blood. Because of multiple interactions between the coagulation and fibrinolytic systems, the most important factor that determines the rate of fibrinolysis is the rate of fibrin formation.³¹

Inherited Coagulation Disorders

Inherited deficiencies of factors VIII,³² IX, XI,³³ and prekallikrein³⁴ have been described in horses. Holstein cattle may have inherent factor XI deficiency.³⁵ Congenital factor VIII deficiency (hemophilia A) is sex linked and recessive, occurring only in males. Factor XI deficiency in cattle is transmitted as an autosomal recessive trait.³⁵ The inheritance pattern of the other deficiencies has not been proved.

Clinical signs of clotting factor deficiency reflect the tendency for abnormal hemorrhage from larger vessels (e.g., subcutaneous hematomas; hemarthroses; epistaxis; melena; hematuria; prolonged bleeding after trauma, diagnostic procedures, or surgery). Petechiae are a feature of vascular or platelet disorders and are not caused by clotting factor deficiency. Clinical signs do not always result from inherited clotting factor deficiencies. Cattle deficient for factor XI and horses with prekallikrein deficiency have complete in vivo coagulation competency. Prekallikrein appears to perform an accelerating rather than required role in activation of factor XII.²⁶ Activated factor XII activates factor XI, which then catalyzes the remainder of the clotting sequence. Factor XII is also capable of activating factor VII in the extrinsic pathway,²⁶ which may explain why factor XI deficiency does not cause a hemorrhagic diathesis. Values for the coagulant part of factor VIII (VIII:C) or factor IX must be reduced to less than 5% of normal before spontaneous hemorrhage occurs. Less severe deficiencies may result in excessive hemorrhage only after trauma.

The major differential diagnoses for heritable factor deficiencies include the acquired coagulation factor—deficient states, DIC, warfarin toxicosis (horses), moldy sweet clover toxicosis, and acute hepatic disease. The heritable clotting factor deficiencies involve proteins in the intrinsic pathway; thus a prolonged activated partial thromboplastin time (aPTT) is the only hemostatic abnormality. Acquired coagulation factor deficiencies involve proteins in the extrinsic or common pathways as well, causing a trend toward prolongation of both the prothrombin time (PT) and aPTT. The definitive diagnosis of heritable clotting factor deficiency must be based on specific quantitative assays of intrinsic clotting factors. The only possible treatment for heritable clotting factor deficiency is replacement of clotting factors through the administration of fresh plasma. Specific clotting factor concentrates are not commercially available for large animals, and the rarity of specific factor-deficiency states that produce clinical signs makes the development of these products unlikely. Because of the expense of therapy and the potential for complications, the long-term prognosis for horses with hemophilia A or multiple congenital coagulation factor defects is poor. Cattle with factor XI deficiency apparently live a normal life but may be more susceptible to secondary diseases.³⁵

Thrombasthenia in Horses

MONICA ALEMAN

Acquired Hemostatic Disorders*

MONICA ALEMAN (MA)

DEBRA DEEM MORRIS (DDM)

JOHN E. MADIGAN (JEM)

Acquired defects of hemostasis may be divided into those involving blood vessels, platelets, and coagulation factors, although some diseases affect more than one component.

Epidemiology

Since the disease was first reported in California,⁶⁴ cases have been diagnosed in Colorado, Connecticut,⁶⁵ Florida, Illinois, Minnesota, New Jersey, New York, Oregon, Washington, Wisconsin, Canada, Brazil, northern Europe, and Israel. Equine cases occur during late fall, winter, and spring. There is no apparent gender or age predilection. However, EGE appears to be less severe in younger horses. Persistent, chronic, or latent infections and carrier status have not been demonstrated and are unlikely to occur because the presence of A. phagocytophila is limited to the acute phase. Therefore, it is also unlikely that infected horses could serve as reservoirs. The disease is not contagious but could be readily transmitted through the administration of infected blood. The vectors of granulocytic ehrlichiosis are Ixodes pacificus in California, Ixodes scapularis in the East and Midwest (U.S.), and Ixodes ricinus in Europe.^66-69 Potential or proposed reservoirs are white-footed mice, chipmunks, white-tailed deer, dusky-footed wood rats, cervids, lizards, and birds.⁶⁹

Pathogenesis

The pathogenesis of EGE is unknown. Entry of the organism occurs after inoculation from a biting tick and presumed spread by blood and lymphatics. The organism has cell tropism toward neutrophils and eosinophils, where it replicates within vacuoles, forming characteristic morulae. Presumed cytolysis, induction of inflammation, and cell sequestration, consumption, or destruction result in the observed clinical signs and pancytopenia.⁷⁰ Cell-mediated and humoral-mediated immune responses develop in affected animals. Antibody titers peak at 19 to 81 days after the onset of clinical signs, and immunity may persist for a long time (2 years).⁷¹

Clinical Signs

Signs include reluctance to move, fever ranging from 39.4° to 41.3° C (102.9° to 106.3° F), mild to moderate tachycardia (50 to 60 beats/min), lethargy, decreased appetite, limb edema, petechiation, icterus, weakness, ataxia, and recumbency (reported in one case). Secondary trauma may result from falling in severely ataxic horses. These clinical signs appear to be less profound in younger horses. The presumptive incubation period after natural infection is believed to be less than 14 days. The prepatent period after experimental exposure to infected ticks or inoculation with infected blood is 8 to 12 days and 3 to 10 days, respectively. The disease is self-limiting and nonfatal provided no complications develop. However, affected horses may be predisposed to secondary bacterial, fungal, and viral infections.⁶¹ Abortions and laminitis have not been reported in affected horses.

Clinical Pathology

Laboratory alterations include anemia, leukopenia characterized by granulocytopenia and lymphopenia, and thrombocytopenia. Morulae may be observed within the cytoplasm of neutrophils and eosinophils during the acute phase of the infection.

Diagnosis

The definitive diagnosis is based on the presence of characteristic morulae (minimum of three) within the cytoplasm of neutrophils and eosinophils or positive PCR assay for A. phagocytophila in peripheral blood (buffy coat).⁷² Morulae may be observed in less than 1% of cells in the initial stages of the infection, up to 20% to 50% of the cells few days later. A-fourfold or greater increase in IFA titer of paired samples confirms recent exposure.⁷³

Pathologic Findings

Petechiae and ecchymosis of subcutaneous tissues and edema of the ventral abdomen, limbs, and prepuce are characteristic in infected animals. Proliferative and necrotizing vasculitis, thromboses, and perivascular cuffing in subcutaneous tissue, fascia, kidneys, heart, brain, lungs, ovaries, and testes have been reported.^61,70

Treatment, Prognosis, and Prevention

The treatment of choice is IV oxytetracycline, 7 mg/kg once daily for 5 to 7 days. Prompt response to treatment is seen within the first 24 hours. Supportive therapy may be necessary in some cases. The disease can be self-limiting in 2 to 3 weeks if untreated. The prognosis of the disease is excellent provided secondary complications are prevented. At present, prevention is limited to tick control.

Anaplasmosis

GUY PALMER

Definition and Etiology

In veterinary medicine, anaplasmosis traditionally refers to disease characterized by progressive anemia caused by intraerythrocytic infection with Anaplasma marginale in cattle and Anaplasma ovis in sheep and goats. In addition, A. marginale subspecies centrale (also known as Anaplasma centrale) causes mild disease in cattle and has been used as a live vaccine to induce partial protection against A. marginale.

In the recent taxonomic reclassification of the tick-borne bacteria in the genera Anaplasma and Ehrlichia, Ehrlichia equi (cause of equine granulocytic ehrlichiosis), Ehrlichia phagocytophila (the cause of tick-borne fever in sheep, not recognized as a significant disease problem in North America), and the agent of human granulocytic ehrlichiosis (HGE) have been reclassified and unified as a single species, Anaplasma phagocytophila.⁶² Correspondingly, the disease caused by A. phagocytophila in humans is designated as human granulocytic anaplasmosis (HGA) and in horses as equine granulocytic anaplasmosis. In addition, A. phagocytophila is an emerging disease of dogs.¹¹² Although this discussion primarily addresses bovine anaplasmosis and, to a lesser degree, ovine and caprine anaplasmosis, it is important to emphasize that any descriptions of human anaplasmosis are in reference to infection with A. phagocytophila. There is no evidence that A. marginale or A. ovis are capable of infecting humans or any nonruminant mammalian species.

Clinical Signs and Differential Diagnosis

Clinical signs are highly variable, from acute severe disease to subclinical infection, and reflect variation in virulence among pathogen strains and age- and breed-related differences in host susceptibility. Age at the time of initial infection is a primary determinant of host susceptibility. Disease is often mild in calves in the first 6 to 9 months of life and increasingly severe in older cattle. The typical incubation period ranges from 15 to 30 days. Infections in calves are often asymptomatic, but mild lethargy and anorexia may be seen for 24 to 48 hours. In contrast, the early stage of acute anaplasmosis in adult cattle is typified by fever, with rectal temperatures ranging from 39.5° C to 41° C (103° F to 106° F). Within 12 to 24 hours the fever subsides, and the temperature may drop to normal and become subnormal before the animal dies. Anorexia and, in dairy cows, a dramatic decrease in milk production can usually be observed soon after a fever is detected. Concurrently, there is suppression of rumination, dryness of the muzzle, and lethargy. Cattle may stagger or become aggressive as a result of cerebral hypoxia associated with anemia. Care must be taken not to stress severely anemic cattle because this may result in collapse and death. Early, the mucous membranes are pallid, but they may be icteric if an animal has survived for 2 to 3 days past the acute crisis. Constipation is a consistent sign, with the feces dark brown and covered with mucus, and pollakiuria is characterized by dark-yellow urine. Hemoglobinuria does not occur. Abortion may occur when infection occurs late in gestation. If an animal survives the acute crisis, the convalescent period is protracted and depends on the severity of the anemia. Icterus and weight loss are more frequently observed in the convalescent period, which may last for 3 to 4 weeks. Recovered animals remain persistently infected for life and, as described later, are epidemiologically important as a reservoir for ongoing transmission. However, persistent infection is not associated with any disease or decrease in production status; therefore, no basis exists for a diagnosis of “chronic anaplasmosis.” Although A. ovis infection in sheep and goats is often asymptomatic, anemia occasionally becomes severe enough to produce signs similar to those seen during A. marginale infection of cattle.

Definitive diagnosis of acute anaplasmosis requires identification of A. marginale— or A. ovis—infected erythrocytes by microscopic examination of blood smears, concomitant with a significant decrease in hematocrit (see next section). The differential diagnosis requires consideration of the diseases that can produce anemia or icterus, including babesiosis, bacillary hemoglobinuria, and leptospirosis. In pastured cattle, hepatotoxic plant poisonings (Senecio) and other causes of liver disease that produce icterus must also be considered. Copper poisoning is considered in sheep.

Clinical Pathology and Serology

Because acute bovine anaplasmosis is characterized by anemia, a falling hematocrit is an excellent criterion for prognostic purposes and for determining the severity of infection. The packed cell volume (PCV) drops below 30% when the first clinical signs are observed and may drop precipitously within 24 to 48 hours. Death can occur during this period despite a PCV above 20%. In other cases, PCV may decrease below 10% before death. During this acute phase, A. marginale can be detected within the erythrocytes by microscopic examination of blood smears stained with Wright’s, new methylene blue, or Giemsa stain. The inclusion is composed of a small morula of two to eight individual organisms, and over 5%, up to 20% to 70%, of the erythrocytes may be infected. Later, after several days of anemia, the percentage of infected erythrocytes decreases dramatically, and evidence of RBC regeneration can be detected. There is anisocytosis, basophilic stippling, poikilocytosis, polychromatophilia, and reticulocytosis.

After recovery from acute disease, cattle and sheep remain persistently infected, with 0.000001% to 0.1% of erythrocytes being infected. These extremely low levels cannot be reliably detected by microscopic examination, and persistently infected animals, which serve as reservoirs for transmission, need to be detected serologically. Serologic diagnosis is most often accomplished through competitive enzyme-linked immunosorbent assay (cELISA),* which provides both high specificity and, unlike the complement fixation test, high sensitivity in detection of persistently infected carrier cattle.^113,114 The cELISA, approved as an official test by the U.S. Department of Agriculture (USDA) and the Office of International Epizootics (OIE), is conducted by most, if not all, state diagnostic laboratories. Currently, negative status with the cELISA is required for importation of live cattle into Canada. Although cattle will seroconvert by cELISA during acute infection, serology generally is of minimal utility in diagnosis of acute anaplasmosis.

Pathophysiology

Anaplasma species are naturally transmitted by Ixodidae ticks, most often the genera Dermacentor in the mainland United States and Rhipicephalus (including tick species previously classified in Boophilus) in tropical and subtropical regions worldwide. Although there is strong epidemiologic evidence of transmission by hematophagous flies, under experimental conditions this is very inefficient,¹¹⁵ and thus the field conditions that allow transmission remain poorly understood. In addition, direct iatrogenic transfer of infected blood (contaminated needles; dehorning, castrating, hormone-implanting, or ear-tagging instruments) can result in transmission. After transmission, sequential rounds of bacterial invasion of mature erythrocytes, replication, and egress result in a progressively increasing, cell-associated bacteremia, with a doubling time of approximately 24 hours. Clinical signs appear when greater than 1% of erythrocytes are infected and the severity roughly correlates with the percentage of infected erythrocytes. Anemia is at least partly caused by splenic and hepatic macrophage-mediated phagocytosis of both infected and uninfected erythrocytes. This appears to reflect both induction of autoantibodies against the RBC surface and induction of acute-phase reactants, including complement activation, during high-level rickettsemia. The regenerative response to anemia can be vigorous and does not appear to be suppressed by the infection.

Protective immunity appears to require induction of both antibody against the outer membrane proteins and macrophage activation for enhanced phagocytosis and bacterial killing.¹¹⁶ Although the immune response controls the acute phase of infection, organisms are not completely cleared from the blood because of the emergence of antigenic variants.¹¹⁷ These variants are responsible for persistent infection, characterized by recurring waves of bacteremia that reflect sequential emergence and then immune control of antigenically variant organisms.

Epidemiology

A. marginale is the most prevalent of the tick-borne infections of cattle worldwide and remains a serious constraint to livestock production in tropical and subtropical regions. However, anaplasmosis is also a significant problem in temperate regions. In the United States, infection is endemic in much of the West and occurs episodically in many historically nonendemic regions; anaplasmosis has been detected in at least 40 states. Canada is considered to be free of endemic anaplasmosis. Endemic regions are maintained by the prevalence of both competent arthropod vectors and persistently infected carrier cattle. These carrier cattle, which are typically asymptomatic, are efficient reservoirs for tick-borne transmission.^115,118 Vector activity varies by region, but outbreaks of anaplasmosis generally occur most frequently in the late spring and summer, when arthropod activity is highest. However, it should be emphasized that the determinants of tick-borne and fly-borne transmission are not well understood, and transmission is often unpredictable. In contrast, iatrogenic transmission can occur at any time and can be controlled by avoiding blood contamination during veterinary medical procedures. Although wild ruminants (e.g., deer, elk, bison) rarely have clinical disease and generally are asymptomatic, persistently infected carriers, their overall importance in the epidemiology of infection is unclear. Currently, wild ruminants are thought to play at most only a minor role in natural transmission.

Pathology

At necropsy, there are no pathognomonic lesions for the diagnosis of anaplasmosis. In acute anaplasmosis the blood is thin and watery and fails to clot readily. Mucous membranes, subcutaneous tissues, and skeletal musculature are pale (anemic pallor). In later stages of acute disease, however, the same tissues exhibit varying degrees of icterus. Splenomegaly is a consistent finding; hepatomegaly and distention of the gallbladder are common but seen less often. Urine is deep yellow, but neither hemoglobinuria nor hematuria occurs. The absence of hemoglobinuria helps differentiate anaplasmosis from other hemolytic diseases (babesiosis, bacillary hemoglobinuria, leptospirosis, onion toxicity, copper poisoning in sheep). Occasionally, petechiae may be found in the subepicardium, subendocardium, and other serous membranes. Detection of A. marginale—infected erythrocytes within capillaries of Giemsa-stained histologic sections can be used to confirm a diagnosis of anaplasmosis.

Treatment

Tetracyclines are the antibiotic of choice for treating acute disease, and resistance has not been reported. In acute anaplasmosis, oxytetracycline at 11 mg/kg IV every 24 hours for 3 to 5 days is effective. One to two administrations of long-acting oxytetracycline at 20 mg/kg IM at 72-hour intervals is also an effective treatment. In addition to antibiotic therapy, supportive therapy is important. If the PCV is 12% or lower, whole-blood transfusion may be indicated to prevent death and shorten the convalescent period; 4 to 8 L of whole blood is usually administered to an adult animal. A PCV of 8% or lower indicates an unfavorable prognosis, and death often occurs despite appropriate antibiotic and supportive therapy. Importantly, the oxytetracycline regimen used to treat acute anaplasmosis is not effective in completely clearing the animal of the organism, and recovered animals become persistently infected carriers. Although prior studies showed that long-acting oxytetracycline at 20 mg/kg every 3 days for four successive treatments resulted in clearance, more recent studies using this regimen and variations of this regimen have indicated that clearance is not achieved.¹¹⁹ If required for exportation, clearance should be confirmed by conversion to seronegative status.

Prevention and Control

Control measures vary depending on the geographic region and type of livestock production system. In endemic regions with high transmission rates, such as those in tropical countries, beef cattle are often allowed to become naturally infected at a young age and remain asymptomatic carriers with minimal risk of later acute disease. In regions with lower transmission rates, live blood-based vaccines may be used to ensure infection of cattle at a young age. This is exemplified by the use of trivalent (A. marginale, Babesia bovis, Babesia bigemina) live vaccine in Australia. Similarly, live vaccines based on A. centrale or weakly virulent strains of A. marginale are typically used in Africa, Asia, and Central and South America. However, these are not licensed for use in the United States, largely because of the risk of transmitting known or newly emergent pathogens contaminating the blood-based vaccine. The exception to this is the licensing of a live vaccine* for use in California. Importantly, these live vaccines should only be used in young animals and are contraindicated for use in older and especially pregnant animals. Killed vaccines are less efficacious and require multiple immunizations, but these can induce at least partial protection against severe morbidity and mortality. Unfortunately, none of the federally licensed killed vaccines previously marketed in the United States are currently available. However, an experimental killed vaccine* has been licensed for use in 14 states and Puerto Rico.

In the absence of immunoprophylaxis, anaplasmosis is usually controlled by preventing transmission. Although it is difficult to prevent completely the contact of ticks and biting flies with cattle grazing on open ranges or farm pastures, strategic use of acaricides and insecticides can reduce transmission during periods of high vector activity. Periodic spraying for tick control and the use of insecticide-impregnated ear tags or insecticide dust bags for biting-fly control are cost-effective, not only for control of anaplasmosis but also for control of pinkeye and for directly reducing irritation and increasing weight gain. Because blood-contaminated instruments and needles can mechanically transmit infection, appropriate sanitary measures should be implemented when injections or surgical procedures are performed.

Maintenance of an A. marginale–free herd in nonendemic areas can be accomplished by quarantine and serologic screening of all additions using the USDA-approved cELISA. Within endemic regions, however, this requires extreme vigilance in screening and prevention of both direct contact and sharing pasture with other domestic and wild ruminants, which may result in vector-borne transmission. The risk of maintaining a fully susceptible herd within an endemic region should not be taken lightly.

BABESIOSIS IN THE BOVINE

JERRY L. ZAUGG

BABESIOSIS IN THE HORSE

EPERYTHROZOONOSIS IN CATTLE

The causal agent is Eperythrozoon wenyoni (Mycoplasma wenyoni). Infection is usually latent, producing no clinical signs in normal cattle, but it may become apparent in animals that have been severely stressed by some other systemic disease. The disease can be produced experimentally if infected blood is administered to splenectomized calves. Even under experimental circumstances, clinical signs consist of mild depression, fever, and modest anemia. The disease in cattle is of little clinical consequence, except for the potential for confusion should the organism be seen on stained blood films. Occasionally, cattle may have swollen and tender teats and legs.¹⁴⁸

EPERYTHROZOONOSIS IN SHEEP AND GOATS

The causal organism in sheep and goats is Mycoplasma ovis (Eperythrozoon ovis), which appears to be very similar morphologically and serologically to the species found in cattle. The disease can produce more prominent clinical signs in sheep, with profound depression, anemia, and significant death losses in young lambs.¹⁴⁹ Erythrocyte destruction is thought to be caused by intravascular hemolysis and erythrophagocytosis.

Diagnosis

Diagnosis of leptospirosis can be challenging because of variable serologic results. Microscopic agglutination titer (MAT) in serum and fluorescent antibody titer (FAT) in infected tissues have been routinely used for diagnosis. The sensitivity and specificity of FAT in tissues of aborted fetuses are almost 100%.¹⁵⁵ In cases of uveitis, high antibody titers in aqueous humor may be found despite variable serum titers, suggesting local antigenic stimulation.¹⁵⁶ Serum titers of 1:6400 are considered significant and consistent with infection.¹⁵⁷ Serum titers of 1:3200 that are stable for 2 to 3 weeks are not considered significant. Rising titers after 2 to 3 weeks are considered significant, and the patient should be isolated and treated. Other diagnostic modalities are PCR, histology, and bacterial isolation. Recently, Divers¹⁵⁵ isolated genes of two surface proteins of L. pomona (immunoglobulin-like proteins A and B) that are expressed only during active infection. These proteins may improve the diagnosis and development of a protective vaccine for leptospirosis.

Treatment

Treatment for leptospirosis has been advocated with ampicillin, amoxicillin, procaine penicillin (22,000 IU/kg IM every 12 hours), oxytetracycline (5 to 10 mg/kg IV every 24 hours, slow in fluids not containing calcium), or doxycycline (7 to 10 mg/kg PO every 12 hours) for 7 days. The inflammatory response in horses with ERU and immune-mediated keratitis has been managed with corticosteroids and cyclosporine, but this may provide only temporary relief. Affected horses tend to develop ocular complications (e.g., cataracts, glaucoma, blindness, intractable pain) that may result in enucleation. The inability to cure many cases of ERU may be caused by the lack of treatment for the possibly active infection. Therefore, treatment should be directed toward controlling the ocular inflammatory response and ongoing infection, if indicated. Systemic antimicrobial therapy may not be successful in ERU cases resulting from an intact blood ocular barrier despite being inflamed.¹⁵⁵

Prevention

Prevention must be aimed at controlling exposure to shedding hosts, infected animals, and contaminated fomites. Thorough cleaning and disinfection of contaminated areas and proper disposal of infected material are essential measures to minimize exposure. Pregnant mares should be isolated from other animals because bacterial shedding may last several weeks. If abortions occur in endemic areas, it is recommended to test horses for leptospiral antibodies and isolate those horses with a serum titer of 1:6400 or higher.¹⁵⁷ Also, horses with a negative or low titer should be retested in 2 to 3 weeks. Horses with rising titers should be isolated. Attempts to decrease shedding in horses with oxytetracycline, penicillin G, and streptomycin have not been effective. Vaccination is sometimes performed on farms with endemic abortions or high rates of uveitis.¹⁵⁵

Serosurveillance and Detection

EIA is a USDA-regulated disease. In 2003 the USDA estimated that serosurveillance of almost 2 million samples cost U.S. horse owners more than $48 million. USDA-accredited laboratories use a testing scheme that detects the presence of serum antibody to the virus. The Coggins’ test, developed by Dr. Leroy Coggins in 1970, provided the first efficient serologic test for detection of EIAV-infected animals; it remains a USDA-accepted test.^160,161 In addition, four ELISAs are licensed tests, one of which is a competitive ELISA (cELISA). These ELISAs detect antibody directed at the transmembrane glycoprotein (gp45) and the p26 antigen.¹⁶² All areas in the United States, Canada, and Mexico accept either the Coggins’ test or one of the licensed ELISAs when testing for EIA is required for entry. Equids that cross state lines or that attend many equestrian events are required to be seronegative for EIA; however, the required period of seronegativity between tests varies according to the state. No tests are currently approved by the USDA for diagnosis of EIA based on detection of viral nucleic acid.

Serosurveillance can lead to the detection of horses that are infected with EIAV but that otherwise appear healthy. Indeed, most infected horses are inapparent carriers. However, blood transferred from an inapparent carrier to another horse, through instruments (e.g., needles, nasogastric tubes, dental equipment), blood products, or insect vectors (primarily tabanids), can lead to infection with a fatal outcome.

Clinical Findings

The clinical form of EIA is characterized by three defined, temporal stages of disease: acute, chronic, and inapparent. The acute stage occurs with the initial burst of viremia and is characterized by high fever, thrombocytopenia, and nonspecific signs of malaise, including lethargy and inappetence. Ecchymoses and petechiae may also be detected on the mucous membranes. During the chronic stage, similar clinical features occur during recurrent episodes of viremia, interspersed with periods of clinical quiescence and low viremia. Throughout the chronic stage the clinical episodes tend to diminish gradually in severity and duration. The inapparent stage occurs once levels of viremia are immunologically contained and no clinical signs are detected. Horses experiencing clinical disease at any point may develop DIC and die. In addition, episodes of stress, including transportation, racing, and extreme temperatures, may precipitate clinical disease. Furthermore, corticosteroids have been used experimentally to induce recrudescence of clinical disease.¹⁶³

Other chronic manifestations of EIA include the development of dependent edema, weight loss, anemia, and other ill-thrift signs. Rare manifestations that may develop include leukoencephalitis and enterocolitis.¹⁶⁴ The expression of clinical disease, particularly during acute infection, likely involves multiple factors, including inoculating dose, virulence factors of the inoculating viral strain, immune status and immunogenetics of the host, age, and stress. Indeed, clinical disease occurs concomitantly with high-titer viremia. Horses with high-titer viremia pose the greatest risk of transmission to uninfected horses.

Lentiviruses have a DNA-dependent RNA polymerase (reverse transcriptase) that misincorporates nucleotides, resulting in changes in the viral genome.¹⁶⁵ This mechanism allows for generation of viral variants that differ genetically from preexisting ones. As a result, genetic and antigenic variation of epitopes for neutralizing antibody and cytotoxic T lymphocytes allows viral escape from adaptive immune responses.^166,167 Antigenic variation plays a central role in recurrence of clinical disease throughout the chronic stage of infection and thwarts attempts to develop an effective vaccine.¹⁶⁵

The EIA virus can replicate effectively in monocytes, dendritic cells, and tissue macrophages.^168-170 In addition, EIAV has been shown to replicate in endothelial cells.¹⁷¹ EIAV replication in endothelial cells may play a role in the development of DIC by viral damage to endothelial cells, with subsequent exposure of subendothelium.¹⁷¹ This in turn could lead to platelet aggregation and formation of thrombi.¹⁷² In addition, damaged endothelium may result in development of dependent tissue edema by transudation of fluid through compromised small vessels.¹⁷¹

Necropsy Findings and Clinical Pathology

Necropsy examination of horses that die of EIA may demonstrate splenomegaly, lymphadenopathy, hepatomegaly, pronounced hepatic lobular architecture, ecchymoses of the mucosa and viscera, dependent subcutaneous edema, and thrombotic disease of small vessels. Histologic findings may include a mononuclear cell infiltrate of periportal regions of the liver, adrenals, spleen, lymph nodes, meninges, and lungs. Hemosiderophages are frequently detected in the spleen, lymph nodes, liver, and bone marrow. Glomerulonephritis from immune complex deposition is often observed.

Clinicopathologic abnormalities vary depending on the stage and severity of infection. During clinical disease, a frequently profound thrombocytopenia develops concurrently with each successive episode of fever. Platelet function of EIAV-infected horses has been demonstrated to be hypofunctional, possibly exacerbating bleeding tendencies and the progression to DIC in clinically affected equids.¹⁷³ Anemia from intravascular hemolysis, extravascular hemolysis, and depression of bone marrow erythropoiesis may progressively develop during the chronic stage of infection.¹⁷⁴ During the inapparent stage, horses typically have increased plasma total solids and globulin concentrations, mild anemia, and decreased albumin concentration.¹⁷⁵ Polyclonal B-cell activation is also evident, suggesting chronic antigenic stimulation despite clinical quiescence.¹⁷⁵ Concomitant fever and thrombocytopenia should prompt immediate consideration of EIA. A history of recurring fever is also suggestive of EIA, particularly when other causes of fever have been ruled out.

Prevention, Control, and Regulatory Considerations

It is well recognized that EIAV seroprevalence varies with the region of the United States.¹⁷⁴ States considered at a high risk include Texas, Oklahoma, Louisiana, and Arkansas, whereas the mid-Atlantic area, New England, Alaska, and Hawaii are considered low-risk regions. The remaining states are classified as intermediate-risk areas. Despite regional differences in risk to noninfected horses, sporadic outbreaks do occur. Veterinarians should therefore encourage all horse owners to test for EIAV annually, test new arrivals, and maintain good fly control. In addition, the veterinary community should encourage organizers of equestrian events to require proof of seronegativity of attending horses.

Once a seroreactor is identified, all contact horses are quarantined and undergo testing until two negative test results at 30- to 60-day intervals are obtained for all individuals in the quarantined herd.¹⁶² The required interval is based on the need to allow recently infected horses to seroconvert. Seroreactors may be either quarantined or euthanized, depending on state regulations. Quarantined horses must be at least 200 yards (180 m) from seronegative animals, the distance shown to be sufficient to obviate viral spread by insect vectors. In addition, seroreactors remaining in permanent quarantine need to be identified. A USDA code number followed by the letter A is assigned to the quarantined horse and permanently applied to the horse as a brand or lip tattoo.

Because an eradication program has never been implemented in the United States for EIA, persistence of reservoirs of horses infected with EIAV must be expected. Indeed, the vast majority of horses in the United States are not tested on an annual basis. To encourage horse owners voluntarily to maintain vigorous surveillance testing, the veterinary community needs to educate owners about the consequences of infection by EIAV. Horse owners are often unprepared to deal with the financial and emotional losses associated with detection of a horse seropositive for EIAV.

Autoimmune Hemolytic Anemia

GARY P. CARLSON

Neonatal Isoerythrolysis in Horse and Mule Foals

MONICA ALEMAN

Neonatal isoerythrolysis (NI) is the most common alloimmune disease in neonatal foals 7.5 hours to 12 days old (median. 2.5 days).¹⁹⁰ NI is characterized by hemolytic anemia, icterus, and hemoglobinuria. Clinicopathologic abnormalities include anemia, high serum indirect and direct bilirubin concentrations, and sorbitol dehydrogenase activity. NI is caused by a blood group incompatibility between the foal and dam. The foal inherits from the sire and expresses an erythrocyte antigen (alloantigen) that is not present in the mare. The hemolytic syndrome is mediated by maternal antibodies against foal erythrocytes (alloantibodies) absorbed from colostrum. Blood factors associated with NI in horse foals are Qa, Qb, Qc, Aa, Pa, and Dg, and donkey factor in mule foals.¹⁹⁰ The most common antigens are Qa and Aa. Foals with NI may also present with alloimmune thrombocytopenia. (See Chapters 19 and 53.)

Definition and Etiology

An acute hemolytic anemia can develop after exposure to a variety of oxidizing agents. These include drugs such as phenothiazine, methylene blue, and acetylphenylhydrazine and plants such as wild or domestic onions, members of the Brassica family (rape or kale), and wilted or dried leaves of the red maple (Acer rubrum).^191-197 Red maple toxicosis (RMT) has been reported in horses, zebras, and alpacas.^195,198-200 Heinz body hemolytic anemia also occurs in sheep on specially formulated diets that are low in molybdenum, which results in chronic copper toxicity, as herd problems in cattle grazing rye grass (Secale cereale)²⁰¹ or selenium-deficient pastures in Florida,²⁰² and with selenium deficiency as a contributing factor in postparturient hemoglobinuria of cattle in New Zealand.²⁰³ These agents produce or allow oxidative denaturation of hemoglobin and resultant aggregation of the protein globin, which appears as Heinz body inclusions within the RBCs. Heinz body anemia has been seen in association with lymphoma in a horse, possibly from failure of the reticuloendothelial system (RES) to remove the Heinz bodies, as reported in horses with EIA.²⁰⁴

Clinical Signs and Differential Diagnosis

Clinical signs vary with the species involved, specific toxin or toxic metabolites, amount of toxin ingested, time course of the disease process, and occurrence of complicating secondary factors, such as hemoglobin nephrosis and acute renal failure. Weakness, lethargy, anorexia, and exercise intolerance are the usual presenting complaints, and death losses can occur. Mucous membranes are generally pale with variable to marked icterus. The heart and respiratory rates are generally elevated, but rectal temperature is usually within normal limits. Horses with RMT may be subclinical or may present with lethargy, muddy or cyanotic mucous membranes, tachycardia, inappetence, weakness, colic, icterus, brown discoloration of the blood, and pigmenturia. Rarely, sudden death occurs.²⁰⁰ Pyrexia and hypothermia have also been documented. Most horses develop clinical RMT during summer and fall. The high mortality rate in horses with RMT may relate to the combination of a rapidly progressive hemolytic anemia and the formation of methemoglobin. Urine output may be reduced, and the urine may be dark because of the presence of hemoglobin, methemoglobin, or bilirubin.

It is not possible to differentiate Heinz body hemolytic anemia from other potential causes of hemolytic anemia without laboratory evaluation. The absence of fever may help to differentiate these anemias from infectious causes of hemolytic anemia. History of exposure to potential oxidizing agents and the fact that these toxic plants may produce death losses or clinical signs in multiple animals at the same time should help to differentiate these cases from autoimmune hemolytic anemia.

Clinical Pathology

Poisoning or intoxication resulting in Heinz body formation usually causes acute and profound anemia. In the early stages a very high percentage of erythrocytes may have Heinz body inclusions. Later, as these cells are removed from the circulation and replaced by young cells from the bone marrow, the relative number of affected cells may decrease greatly. Heinz bodies are round, oval to serrated, refractile granules usually located near the cell margin or protruding from the cell and best visualized with vital stains such as crystal violet or new methylene blue applied to unfixed blood smears. Heinz bodies appear as bluish green inclusions with the new methylene blue stain. Fixing of blood smears with methanol in preparation for staining with the classic Wright’s stain interferes with stain uptake, and Heinz bodies appear as a pale area within or projecting from the cell margin and can easily be missed. After the first 3 or 4 days, the anemia is usually associated with hematologic evidence of an active erythrogenic response in all species except the horse. The total plasma proteins usually remain within normal limits, and the Coombs’ test is negative. Red maple poisoning also results in depleted RBCs, reduced glutathione, methemoglobinemia, increased osmotic fragility, and modest elevations of liver-derived serum enzyme activities.

The rapid and profound erythrocyte destruction may lead to hemoglobinemia and hemoglobinuria. The development of renal failure secondary to hemoglobin nephrosis is a definite risk in these animals and is reflected by modest to marked increases in the blood urea nitrogen (BUN) and creatinine, as well as changes in the urinalysis.^196,197 These parameters should be monitored in severely affected animals. As with other causes of hemolytic anemia, serum bilirubin, particularly the indirect reacting bilirubin, is elevated.

The clinicopathologic abnormalities of horses with RMT include anemia (PCV as low as 7.5%), eccentrocytes, Heinz bodies, and elevated plasma methemoglobin concentration.²⁰⁰ Other significant findings consist of inflammatory leukogram and renal insufficiency (75% and 40% of cases, respectively).²⁰⁰ Mild elevation of serum total bilirubin is observed in most cases, and hemoglobinuria is seen in all affected patients.

Pathophysiology

Heinz bodies are formed by the precipitation of oxidatively denatured hemoglobin. The hemoglobin contained within the RBC is constantly undergoing mild oxidative stress associated with oxygen transport, as well as generation of superoxide radicals and hydrogen peroxide within the cell. A number of reducing mechanisms within the RBC counteract these oxidative processes through production of NADPH and reduced glutathione. The occurrence of Heinz body hemolytic anemia could be viewed as a consequence of exposure to oxidative stresses that simply overwhelmed the cells’ reductive capacity. Selenium deficiency results in a decrease in glutathione peroxidase, a selenium-containing enzyme; in special circumstances, selenium deficiency may contribute to Heinz body formation by impeding the ability of the cells to respond to oxidative stress. Substantial species variation in the rate of Heinz body formation relates to the chemical structure of hemoglobin and the efficacy of erythrocyte-reducing mechanisms in the face of oxidative stress.¹⁹¹ RBCs with Heinz bodies are less deformable than normal cells and are rapidly removed from the circulation by the RES in the spleen, where they are phagocytized and broken down. Old or senescent erythrocytes are thought to be more prone to develop Heinz bodies. Splenectomy or corticosteroid therapy may alter Heinz body clearance mechanisms, allowing significant numbers of affected RBCs to remain in the circulation of otherwise normal animals.

Gallic acid is a strong oxidant present in red maple leaves. Gallic acid has been implicated in the oxidation of hemoglobin, methemoglobin formation, and Heinz body anemia.^195,196,205 Methemoglobin is the result of oxidative change of hemoglobin iron to the nonfunctional ferric state (see discussion of nitrate poisoning, Chapter 54). This is normally prevented by glutathione reductase, ascorbic acid, and reduced glutathione. Methemoglobin cannot load or transport oxygen and, when present in sufficient quantities, results in a brown color of peripheral blood and mucous membranes. An estimation of methemoglobin concentration in a blood sample can be made by comparing the hemoglobin concentration measured by the cyanmethemoglobin method, which measures all forms of hemoglobin, with that measured by the oxyhemoglobin method, which only measures oxyhemoglobin, not other forms such as methemoglobin. Methemoglobinemia and hemolytic anemia have been reported in a mare and her dam in association with decreased levels of RBC glutathione and glutathione reductase, presumably as a result of an inherited enzymatic defect.²⁰⁶

Treatment

Treatment primarily involves removal from the source of toxicity and provision of supportive care. Blood transfusion can be very beneficial in severely anemic patients, particularly when there is insufficient evidence of active erythropoietic response. Iron-containing hematinics are of little benefit. Intravenous fluid therapy is indicated in animals with hemoglobinuria or azotemia to reduce the potential for further renal damage. High doses of vitamin C (ascorbic acid, 50 to 100 g IV daily), together with fluids and transfusion, were thought to aid recovery in two horses with red maple poisoning.²⁰⁷ One report suggests that vitamin C therapy may have little impact on survival of affected horses, and when methylene blue was used to treat the associated methemoglobinemia in two horses, both died.²⁰⁸ The goal of therapy should be to improve tissue oxygenation and perfusion and control inflammation and pain (use of NSAIDs). Horses treated with corticosteroids have an increased likelihood of death.²⁰⁰

Prognosis

Prognosis in animals with modest anemia and evidence of response is good if the inciting factor can be controlled or eliminated. The mortality rate of horses with experimental or naturally occurring RMT is reported to be 60% to 65%.²⁰⁸ In animals with rapidly progressive and profound anemia, prognosis is poor unless blood transfusion is undertaken. Complications are associated with hypoxia, hypoperfusion, and inflammation. Horses may develop acute renal failure, colic, pyrexia, and laminitis. A recent retrospective study of 32 horses reported a fatality rate of 59%.²⁰⁰ It has been proposed that the severity of anemia is associated with mortality, but the previous study showed no such association.²⁰⁰

Intravascular Hemolysis Following Cutaneous Burns

MONICA ALEMAN

Intravascular hemolysis has been reported in horses after severe cutaneous burns affecting more than 25% of their body surface. Plasma on admission showed hemolysis along with abnormal RBC morphology, increased osmotic fragility, hemoglobinuria, azotemia, and hemoglobin pigment nephropathy.²⁰⁹ The pathophysiology of hemolysis is unclear but suspected to be associated with the production of hydroxyl radicals by complement-activated neutrophils. Abnormal findings reported in humans include intravascular hemolysis, increased RBC osmotic fragility, decreased membrane deformability, RBC crenation, eccentrocytosis, spherocytosis, bud formation, fragmentation, and vesiculation.²¹⁰ Presence of these abnormalities depends on the severity of burn trauma.²¹⁰ Pseudothrombocytosis has been reported, presumably caused by the incorrect recognition by automatic counters of RBC microvesicles as platelets.²¹⁰ Early fluid therapy and free-radical scavengers have been beneficial for the treatment of burns in humans, along with supportive and wound care, control of pain and inflammation, and prophylaxis of sepsis. A recent murine study suggested that the level of plasma free hemoglobin is related to the size and depth of burn injury, which may be difficult to determine in some cases.²¹¹

L-Tryptophan—Indole Intoxication

GARY P. CARLSON

Experimental studies in ponies have demonstrated an acute hemolytic process associated with orally administered L-tryptophan, which is converted to indole in the GI tract.²¹² Intoxication was associated with an acute onset of restlessness, tachypnea, intravascular hemolysis, and hemoglobinuria. At necropsy there was evidence of hemoglobinuric nephrosis and bronchiolar degeneration in some ponies. Similar clinical signs were noted after oral administration of tryptophan at 0.35 to 0.60 g/kg and indole at 0.1 to 0.2 g/kg.^212,213 Intravascular hemolysis was associated with increased osmotic fragility and with Heinz body formation in a few of the experimental ponies.

Water Intoxication

GARY P. CARLSON

Massive water intake may produce marked hypotonicity of the body fluids, with subsequent intravascular hemolysis of erythrocytes.²¹⁴ This problem has been described as a naturally occurring entity in milk-reared calves when first given access to unlimited quantities of water.^215,216 Severe neurologic signs may be seen, including depression, convulsions, and coma; respiratory distress, hemoglobinuria, and death losses occur in some cases. Clinicopathologic features include hemolytic anemia, hypoproteinemia, hyponatremia, hypochloremia, hyposmolality, hemoglobinuria, and hyposthenuria. A sudden decrease in serum osmolality is believed to result in osmotic lysis of erythrocytes.²¹⁷ Fragility of the erythrocytes to osmotic shock is greatest in calves between 4 and 5 months of age. Treatment is primarily a matter of temporarily restricting water and providing supportive care. Calves with marked hyponatremia (sodium 110 mmol/L) that are manifesting neurologic signs may benefit from hypertonic saline, mannitol, and corticosteroids. The goal of treatment is the restoration of serum sodium to 120 to 125 mmol/L without overcorrection. Death losses can occur in as soon as 2 hours, but most calves recover without long-term adverse effects.

Postparturient Hemoglobinuria

GARY P. CARLSON

A syndrome of intravascular hemolysis, hemoglobinuria, and anemia has been recognized in postparturient dairy cattle worldwide.²¹⁸ The disease occurs sporadically, and the incidence is relatively low. Affected animals are most often high-producing multiparous cows that develop clinical signs during the first month after calving.²¹⁹ Depression, decreased feed consumption, and decreased milk production are associated with hemoglobinuria, anemia, and icterus. The anemia is often marked and after 4 or 5 days is associated with evidence of a strong erythropoietic response. The precise mechanism causing the intravascular hemolysis has not been fully defined. The condition has been related to the marked hypophosphatemia often found in affected cows and the moderately low phosphate levels in unaffected herdmates. Hypophosphatemia arises from inadequate dietary phosphorus intake in animals grazing phosphorus-deficient soils or fed fodder grown on such soils. Low intracellular phosphate concentration may interfere with energy metabolism, thus affecting cell viability and the ability of RBCs to deal with potential hemolysins, such as saponins from sugar beets or alfalfa. A postparturient hemolytic problem has been described as a herd problem in New Zealand associated with copper deficiency and Heinz body formation²⁰³ (see previous section).

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

Copper Toxicosis

LISLE W. GEORGE

Copper is an essential nutrient for domestic animals, but excessive supplementation can result in toxicity. Copper poisoning is a common intoxication in ruminants. The toxicodynamics and clinical syndromes have been reviewed.^220-222 Lambs are most susceptible to the effects of copper, but poisonings have occurred in adult sheep, goats, and cattle. Merino sheep are more resistant than British breeds to the toxic effects of copper.²²³ Cattle have been poisoned by ingesting diets containing 37 mg of copper/kg of feed for 2 years.²²⁴ Cattle fed 12 mg or more of copper/kg have been reported to develop subclinical hepatic disease.²²⁵

Pathogenesis

Elemental copper is an essential trace mineral that has a narrow therapeutic window. Dietary requirements for growing sheep range between 4 and 6 parts per million (ppm), but toxicity can occur whenever sheep are exposed to diets containing as little as 10 to 20 ppm.^226-228 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.²²⁹ Ionized copper is approximately 20% of the total plasma copper. Between 70% and 90% of the 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.²²⁹ Accumulation of copper occurs because daily hepatic biliary copper excretion amounts to less than 1% of ingested copper in ruminants.²³⁰ 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.^223,231

Free inorganic copper is an oxidant and can participate in Fenton’s reaction. Cellular damage is related to the production of oxidative hydroxides and peroxides, but not to copper oxidation. These reactive intermediates are thought to initiate lipid peroxidation and oxidative denaturation of proteins within erythrocytes. Oxidation of heme protein produces Heinz bodies, and oxidation of the hemin produces methemoglobinemia. Oxidized erythrocytes spontaneously lyse intravascularly, or they are sequestered in the spleen and degraded to constituent amino acids. Vitamin E is also denatured. This latter reaction removes a potent antioxidative protective factor and enhances the cellular susceptibility to additional oxidants. Differences in genetic susceptibility to copper poisoning are unknown.

The hemolytic phase of copper poisoning is often initiated by noxious stimuli that could include shipping, hierarchic change, administration of oxidative drugs, starvation, or change of housing.²³² Feeding of high-protein diets has increased the resistance in sheep to the hemolytic crisis; however, the role of extra molybdenum intake in these supplemented animals was unclear.^228,233 The mechanistic relationship between the stressful events and the hepatocyte release of copper is unknown.

Etiology

Sources of copper that have been responsible for copper accumulation in animals include trace-mineralized salt, inappropriately formulated cereal grain mixtures, forages from pastures fertilized by swine or chicken manure,²³⁴ orchard pastures contaminated by copper-containing fungicides, rations containing more than 20% chicken litter, and diets that contain high concentrations of palm kernel oil. Other potential sources of copper accumulation include fencing and copper piping and overdoses of parenterally administered copper salts. In one recent case at the University of California, Davis, dairy goats were 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 the duration of exposure, the animal’s genetics, and the amount of molybdenum being fed. Diets that contain copper/molybdenum ratios greater than 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, when insoluble and nonabsorbable complexes of copper, sulfur, molybdenum, and large—molecular-mass protein are formed. The highly complexed copper is unabsorbed and thus not toxic. High dietary intake of zinc and iron also inhibits absorption of copper, and although the precise mechanism of antagonism is unknown, the elements share a competitive affinity for metallothioneine.^235-237 Competition for this protein could alter the storage capacity for copper. Soil sulfates reduce molybdenum absorption by plants, thereby increasing the potential for pathologic accumulation of copper in herbivores.

The respective single toxic doses of copper for sheep and cattle range from 20 to 110 mg/kg and 220 to 880 mg/kg body weight. Most poisonings occur after a 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 copper, feeding a ration that contains 20 ppm copper for several months, or after a single subcutaneous injection of copper—ethylenediaminetetraacetic acid (EDTA) salt at 2.0 mg/kg.²³⁸ Sheep have been poisoned by subcutaneous injection of 3 to 4 mg/kg copper-calcium EDTA, but remained normal after 6 mg/kg copper methionate.²³⁹ Sheep have died after single injections of 2 mg/kg diethylamine copper oxyquinoline sulfonate.²³⁹ One study that fed 3.7 mg copper/kg daily for 84 days reported peak hepatic copper levels between 258 and 375 mg/kg wet weight by 26 days after the final copper feed.²⁴⁰ A single dose of 50 mg copper caused acute hemolytic crisis in sheep when treated during midpregnancy. The highest incidence of hemolytic anemia occurred in Welsh sheep, with a lower cumulative incidence in Cheviot sheep.²⁴¹ Single doses of copper-calcium ETDA at 25 mg/animal has killed lambs.²⁴² 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 copper sulfate daily for as long as 4 months. Calves may be poisoned after 6 to 8 weeks of feeding milk replacer containing 115 ppm copper.^229,243 Horses are resistant to high dietary concentrations of copper and may remain clinically normal after prolonged feeding of diets containing levels as high as 791 ppm. In these horses, hepatic concentrations of copper at the end of the feeding period reached 4000 ppm. Adult ponies treated once with oral copper at 40 mg/kg did not develop hemolytic anemia.

Clinical Signs

Animals that have been exposed to high levels of copper are asymptomatic for weeks until the onset of hepatic necrosis.^244,245 Signs develop rapidly after the onset of hepatic necrosis and reflect coexisting anemia, myopathy, and neurologic, renal, or hepatic disease. Clinical signs include inappetence, lethargy, weakness, recumbency, cool extremities, pallor, and grayish discoloration of the mucous membranes. Affected animals have a greatly increased pulse rate, tachypnea, hypotension, and hypothermia. The pulse amplitude of the linguofacial artery is decreased. Urine is dark red due to the presence of free hemoglobin products. The animals may have marked petechiation of the conjunctival mucosa. Pregnant animals often abort because of hypoxemia. Feces are dark or have yellowish discoloration, but are normally formed, and do not contain hemoglobin unless secondary abomasal ulceration has occurred. Recumbent patients usually expire without struggling. Exposed ewes that survive copper poisoning tend to have dystocia and dilation failure in subsequent lambing periods.

Pathology

The tissues of hemolyzing animals are pale and icteric. The serous surfaces are covered by petechial and ecchymotic hemorrhages. The liver is often pale and yellow, and the lungs are firm. The kidneys are black and have a metallic sheen because of entrapped hemoglobin. The urinary bladder of copper-poisoned animals is filled with serosanguineous urine. Microscopic changes include hemoglobinuric and tubular nephrosis, as well as necrosis of the splenic follicles and hepatocytes. There is also biliary ductular proliferation and pericholangitis.^240,246 Hepatic necrosis may be detectable by microscopic examination for as long as 412 days after poisoning. Microscopic changes in the brain of poisoned animals include spongy degeneration of the pons and brainstem.

Diagnosis

Normal hepatic copper concentrations in sheep have been reported as 173 ± 130 ppm (mean ± SD) and 129 ± 59 ppm (wet matter) for adults and lambs, respectively.²⁴⁷ Wet matter hepatic concentrations of copper in poisoned animals were 429 ± 249 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.²³⁸ The correlation between plasma and hepatic copper concentrations in prehemolytic animals is poor.^248,249 Copper poisoning in animals with normal plasma concentrations and near-toxic liver levels has been reported.²⁵⁰ Normal plasma copper concentrations range from 13 to 20 μmol/L (0.8 to 1.2 μg/mL).²⁴⁰ Serum copper concentrations range between 0.60 and 1.50 μg/mL (0.6 to 1.5 ppm). Plasma copper concentrations that range between 2.4 and 20.0 μg/mL (2.4 to 20 ppm) are diagnostic of acute toxicosis.

Clinical Pathology

There are no consistent hematologic changes until 24 hours before the hemolytic crisis, when sudden concentrations of cytosolic hepatic enzymes increase coincidentally with a sharp rise in plasma copper concentration. Plasma copper concentrations fall rapidly after the hemolytic crisis and are often near normal by 4 days posthemolysis.²⁴⁴ The concentrations of copper within erythrocytes remain high.

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 nontreated poisoned sheep has been estimated at 175 ± 91 days,²⁴⁰ and high copper concentrations in hepatic tissues can be found for as long as 100 days after the initiation of the hemolytic episode.²⁴⁸

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 aspartate aminotransferase (AST) for 3 days before onset of hemolysis, and glutamate dehydrogenase may be increased for as long as 700 days after cessation of copper ingestion.^240,251 Fecal copper concentrations may exceed 10,000 ppm during the hemolytic episode.

Clinicopathologic changes that occur during the acute hemolytic crisis include Heinz body formation; intravascular hemolysis; methemoglobinemia (as much as 5%); decreased PCV; increased concentrations of plasma bilirubin, AST, GGT, alkaline phosphatase, total bilirubin, creatine kinase, creatinine, and plasma urea nitrogen; and increased 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.²⁵² Sheep that survive the hemolytic episode develop reticulocytosis by 4 days after onset of clinical signs.²⁵³

Treatment

Animals with acute hemolytic syndrome should be treated with insufflated oxygen and vitamin E (three to five daily doses of 3000 IU/dose). If PCV is less than 8%, animals should be given packed, washed homologous erythrocytes. Additional therapy should include D-penicillamine (Cuprimine* at 52 mg/kg daily for 6 days), anhydrous sodium sulphate (1 g/sheep daily for 6 days), and ammonium molybdate (100 mg/sheep daily for 6 days). D-Penicillamine therapy increases urinary copper excretion by 10- to 20-fold.²⁵⁴ Single-dose therapy with D-penicillamine (28 mg/kg) increases copper excretion, but the effect is transient and insignificant for reducing the total hepatic copper load.²⁵⁵

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.²⁵⁶ Copper-poisoned cattle have been successfully treated with oral sodium molybdate (3 g daily) and sodium thiosulphate (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.²⁵⁷ The protein-bound thiomolybdate aggregates strongly chelate copper in insoluble and indigestible complexes. Molybdenum salts also enhance biliary copper excretion and remove copper selectively from hepatic metallothioneine.^235,258,259 For poisoned cattle, top dressing of food with 500 to 1000 mg ammonium molybdate daily for 18 days greatly reduced the amount of hepatic copper.²⁶⁰

Parenterally administered ammonium tetrathiomolybdate has been recommended for treatment of acute hemolytic crises caused by copper poisoning. Intravenous administration of ammonium tetrathiomolybdate reduces both lysosomal and cytosolic copper in hepatocytes. The drug has been given at 0.2 mg/kg molybdenum (thiomolybdate), as IV injections of 50 to 100 mg twice weekly for up to 11 weeks, or as three doses of 3.4 mg/kg on alternate days and beginning at the onset of hemolytic crisis.^261-265 Concomitant administration of 36 mg xylazine IV increased copper excretion in bile by as much as 2.25-fold over controls that were treated with thiomolybdate only.²⁶⁶ The parenteral administration of the 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 the copper-exposed animals from hemolytic crises and reduce the tissue damage during an hemolytic event.^267,268 Biweekly injection of 100 mg molybdenum as tetrathiomolybdate increased daily hepatic biliary copper excretion by a factor ranging between 150% and 300%.²⁶⁹

Prevention

Because of the sporadic nature of the poisoning and lack of signs during the accumulative phase, copper poisoning can be difficult to prevent. Clients should be counseled to purchase concentrates with label claims for the species that are being supplemented. Copper-supplemented salts should be restricted, especially in sheep. Heavily copper-contaminated pastures can be top-dressed with molybdenum phosphate at 113 g per acre. Fencing and plumbing that contain copper sulfate or metallic copper should be removed from the environment. One study controlled the hemolytic crisis and associated mortality from copper poisoning in sheep receiving 36 mg copper/kg daily for 18 months, by subcutaneous (SC) injection of sterile ammonium tetrathiomolybdate containing 3 to 4 mg molybdenum on alternate days for three injections.²⁶⁴ The sheep in this study were given either IV or SC molybdenum at dosages that amounted to 1.7 and 3.4 mg/kg ammonium tetrathiomolybdate. 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.²⁴⁹

Feeding of sodium molybdate and sodium sulfate at respective daily dosages of 20 mg and 6 g to experimentally poisoned lambs reduced hepatic copper content after 90 days.²⁷⁰

Molybdenum and sulfur protect sheep by converting ingested copper to nonabsorbable thiomolybdate complexes in the rumen. The antagonistic effects are greatest at times of rapid hepatic accumulation of copper. Addition of 3 mg/kg of food (dry matter) molybdenum as tetrathiomolybdate reduced hepatic copper accumulation by as much as 33-fold.²⁶³ Lambs that were fed diets containing 7.7 ppm ammonium molybdate after weaning until 14 weeks of age had reduced hepatic copper and decreased ceruloplasmin concentrations.²⁴⁹ Cattle can tolerate indefinite periods of feeding copper at dosages less than 0.6 mg/kg daily.²⁵⁰ Addition of 220 to 420 ppm zinc may be protective against copper poisoning in sheep, but this is not used in clinical situations.²⁵⁷

Hemolytic Syndrome in Horses with Liver Failure

GARY P. CARLSON

A fulminant, intravascular hemolytic syndrome has been reported as a near-terminal event in horses with either acute or chronic liver failure.²⁷¹ Marked hemoglobinemia and hemoglobinuria are associated with intense icterus. The sclera and conjunctiva often take on a distinctive, deep—reddish orange color. The onset of intravascular hemolysis is sudden and rapidly progressive. The prognosis is highly unfavorable because almost all horses that develop this syndrome die or must be euthanized because of clinical deterioration.²⁷² Intravenous fluids, corticosteroids, and supportive care do not appear to alter the clinical course or the unfavorable outcome.

The cause of this syndrome has not been determined, but apparently it is not related to a release of hepatic copper stores. The hemolysis appears to be associated with increased erythrocyte osmotic fragility. Human patients with liver cirrhosis develop a hemolytic syndrome associated with alterations in the exchangeable lipoproteins of the RBC membrane. It is not known if a similar mechanism is responsible in horses, but morphologic alterations in the RBCs of these horses resemble the “burr cells” described in human patients.²⁷³ Bile acids and their salts are greatly increased in liver failure and could play a contributing role in the hemolytic process. At necropsy, widespread hemorrhagic lesions that resemble those described for DIC are often present.²⁷³ DIC and the activation of various mediators may play a role in the terminal stages of this almost invariably fatal process.

“Pink Tooth,” Congenital Erythropoietic Porphyria, Toxic Porphyria

GARY P. CARLSON

A rare congenital disorder of hemoglobin production inherited as an autosomal recessive trait has been recognized primarily in Holstein cattle, but it has also been reported in Shorthorns and Jamaican cattle.^274,275 This disorder is commonly called “pink tooth” and is characterized by slow growth rates in calves, photosensitization and exfoliation of nonpigmented skin when exposed to sunlight, reddish brown teeth, and modest anemia. The teeth and bones exhibit a pink fluorescence under ultraviolet (UV) light, and the urine is brownish red because of uroporphyrin. The condition is present at birth, and the metabolic defect in these cattle is a hereditary deficiency of the enzyme uroporphyrinogen III cosynthetase, which catalyzes an essential step in the synthesis of the porphyrin structure of hemoglobin.²⁷⁵ This leads to the accumulation of uroporphyrin and coproporphyrin, which deposit in the teeth, where porphyrin is concentrated in the dentine, bones, and other tissues. Several factors contribute to the variable anemia seen in these cattle. A reduced intravascular RBC lifespan is related to the high concentration of uroporphyrin and coproporphyrin within the cells. Porphyrins may induce hemolysis and also delay maturation of the RBC series in the bone marrow, although there is often evidence of active erythropoietic response in the peripheral blood.²⁷⁵

There is no treatment for this inherited disorder, but genetic counseling is advisable. Substantial efforts have been made to reduce the incidence of pink tooth in the Holstein breed. It may be possible to detect carrier animals. Affected cattle have much lower levels of uroporphyrinogen III cosynthetase than normal cattle, and carrier animals have intermediate levels of this enzyme. Despite their rather serious problems, these cattle do reasonably well if housed indoors out of direct sunlight. The principal differential consideration is chronic fluorosis, which also produces brown discoloration of the teeth. However, the teeth of cattle with chronic fluorosis do not fluoresce under UV light.

An additional form of altered porphyrin metabolism, congenital erythropoietic porphyria, has been described in humans and cattle.²⁷⁵ In humans the mode of inheritance is autosomal dominant, whereas in cattle the disorder appears to have a recessive pattern of inheritance and may be sex linked because it is only seen in females. The disease does not produce anemia, porphyrinuria, or discoloration of the teeth. This disorder is caused by a deficiency of ferrochelase (heme synthase), which leads to high concentrations of erythrocyte and fecal protoporphyrins. Porphyria has also been reported in swine, with a dominant pattern of inheritance.²⁷⁵ There is little effect on the health of the pigs and no photosensitivity, but the teeth have reddish brown discoloration. Although similarities exist with bovine congenital erythropoietic porphyria, the precise defect in swine has not been found.

Finally, animals may develop acquired toxic porphyrias. This can occur with heavy metal poisonings, principally lead, but has also been produced experimentally with hexachlorobenzene and other chemicals. Lead inhibits several key enzymes of heme synthesis. Inhibition of aminolevulinate dehydrase leads to an accumulation of aminolevulinic acid, and decreased aminolevulinate dehydrase activity is a sensitive indicator of lead poisoning. Lead also inhibits ferrochelase, leading to marked elevation of erythrocyte zinc protoporphyn IX, the measurement of which provides a means of monitoring lead exposure.²⁷⁵

Primary Erythrocytosis

Polycythemia vera, an idiopathic myeloproliferative disorder characterized by excessive proliferation of erythroid, myeloid, and megakaryocytic elements, has not been reported in large animals.

Secondary Erythrocytosis

Sporadic Lymphoma

Adult Lymphoma (Bovine Leukemia Virus)

The adult or enzootic form of lymphoma is the most common neoplastic disease of cattle and is associated with BLV infection. Rates of lymphoma in cattle vary considerably, probably reflecting variation in BLV infection rates. In 1978 the U.S. condemnation of carcasses because of lymphoma was reported to be 170 per 100,000 head slaughtered.³³² A high condemnation rate, sometimes exceeding 1%, was reported for slaughtered California dairy cows,³³³ and about 1.73% of BLV-infected cattle had lymphoma.³³⁴ The rate of cattle condemnations resulting from lymphoma appears to have increased over time.³³³

Epidemiology

The epidemiology of adult lymphoma is not completely understood. Herd size might be positively correlated with a high rate of lymphoma, which may reflect higher rates of BLV infection in large herds or a preponderance of susceptible pedigrees in some of the herds studied.^334,335 Susceptibility to BLV infection is associated with BoLA type,^336,337 which may explain why higher rates of lymphoma are found in certain families of cattle. The progression of disease to lymphocytosis in some BLV-infected cows may also be influenced by BoLA type, suggesting a possible genetic predisposition to development of lymphoma through the antigens of the major histocompatibility complex (MHC) encoded at closely linked loci. Other potentially contributing factors, including nutrition, concurrent infections, and environmental/meteorologic stressors, have not been explored sufficiently. There is no evidence for a seasonally associated pattern to appearance of clinical lymphoma.³³³ Antigenic and molecular evidence for the presence of BLV in mammary glandular epithelium has been demonstrated.³³⁸ The effect of persistent mammary BLV infection on the mammary gland is not completely understood. Although one study identified increased average milk production in BLV-infected versus uninfected cattle,³³⁹ others report negative associations between milk production and BLV status.^340,341

Economics

The economic importance of adult lymphoma varies according to production type.^341,342 Producers incur losses from lymphoma associated with death of cattle (particularly genetically valuable livestock), loss of milk production, costs of treatment and diagnosis, and premature replacement costs for cattle dying or culled as a result of lymphoma. The latter may partly explain an observed increased rate of culling found for BLV-infected dairy cattle.^339,343 In addition to premature culling and replacement, losses are incurred because of failure to retain salvage value of cows condemned for lymphoma at slaughter.³³³ A hidden cost of lymphoma also may be in the perpetuation of BLV-infected cattle through in utero infection of calves born to cows with lymphoma.^344,345

Diagnosis

Cattle presented with the adult or enzootic form of lymphoma usually are older than 4 years but may be as young as 2 years. Cattle are often presented with a history of loss in condition, an abrupt drop in milk production (over a few days), enlarged peripheral nodes, exophthalmos, or partial to complete anorexia, particularly with regard to grain or concentrates. Because dry cows are not observed as closely as lactating cows and are not monitored for milk yield, lymphoma is less likely to be recognized until they freshen. Subclinical lymphoma may be diagnosed in cows submitted for routine reproduction examinations; other signs may include diarrhea, ataxia, paresis, ketosis, and infertility.

Physical examination often reveals an organ system failure resulting from tumor involvement. Thoracic auscultation may reveal cardiac dysrhythmia, tachycardia, tachypnea, and hyperpnea. Common sites of lymphoid tumor predilection in adult lymphoma include right atrium, uterus, retrobulbar space, abomasum, and spinal cord (epidural space); involvement of rumen, colon, and kidney is also seen. Dependent, pitting edema is common when cervical or supramammary lymph nodes are involved. Involvement of the intestinal lymphatics is often associated with generalized dependent edema anterior to the udder. Peripheral nodes typically found to be enlarged are the prescapular, femoral, and supramammary nodes. Feces may be scant, pasty, or watery and probably reflects the presence of lesions within the GI tract. Melena may be present in animals with ulceration of the abomasum and lymphoid cell infiltration in the abomasal wall. Rectal palpation can be a useful in cases lacking peripheral node enlargement or exophthalmos. Tumor masses palpated in the abdomen typically are multiple and range in size from only slightly enlarged lymph nodes to massive lesions half a meter in diameter. The internal iliac nodes are involved in most cattle with abdominal tumors.

Tumors tend to be firm but not hard and may feel slightly lobulated. Differentiation by palpation of lymphoma tumors from other masses is highly subjective. Carcinomas tend to be of similar consistency but are seldom larger than 15 cm in diameter and are usually associated with intestinal tissue. Lymph nodes of cows with a carcinoma are not usually enlarged unless a secondary infection is present. Melanomas are generally less than 15 cm in diameter and hard, sometimes with protrusions 1 to 2 cm high along the surface. They usually are not found associated with lymph nodes. Masses of fat necrosis tend to be firm and are usually associated with omental tissue. Internal abscesses are often single and associated with the uterus, as a consequence of uterine tear and infection during parturition, or with gluteal muscles of the pelvic ceiling, as a consequence of injection-related infections. Exploratory laparotomy may be indicated as an additional diagnostic tool in difficult cases, particularly in valuable cattle.

The hemogram of cattle with lymphoma often is generally unremarkable. Anemia, characterized as microcytic and hypochromic, may be present in cattle with GI hemorrhage. Fibrinogen levels have been inconsistent in lymphoma, and therefore its measurement may be helpful only in differentiating an abscess. Approximately 30% of cattle with BLV infection develop persistent lifelong lymphocytosis, composed mostly of B cells.^346,347

Cytology of aspirates of tumors or tumorous nodes may not always be a reliable diagnostic tool, although it can be helpful. Discrimination cannot be made between a normal node responding to an infectious agent and a node involved with neoplastic lymphocytes; in both situations, cells resembling young, poorly differentiated lymphocytes may be present. Examination of nontumorous active lymph nodes may reveal an elevated proportion of large, young lymphocytes that could resemble those of lymphosarcoma. Histopathologic examination of biopsied tumors or nodes may be more useful than aspirates in making a diagnosis. However, cytology and culture of aspirates could be helpful in differentiating a tumor from an abscess. Failed attempts at collection of cerebrospinal (lumbosacral) fluid in cattle with recumbency or hindlimb weakness may be associated with the presence of lymphoid tumors in the vertebral canal; cytologic examination of cells from the collection needle in such cases may reveal abnormal lymphoid cells.

Standard serologic testing for BLV involves identification of antibody against the 51-kilodalton (kD) envelope glycoprotein (gp51). Although BLV diagnosis historically involved the use of the agar gel immunodiffusion (AGID) test, that method has been shown to be less sensitive than the enzyme-linked immunosorbent assay (ELISA).³⁴⁸ ELISA test kits are available commercially that detect antibodies to gp51 in serum and milk.* Diagnosis is also possible by use of PCR to detect BLV nucleic acid,^349-351 as well as by a combined PCR-ELISA that is more sensitive than staining of PCR amplicons with ethidium bromide.³⁵² ELISA-positive cattle that were not detected by PCR testing have been reported, most likely related to a low number of infected circulating lymphocytes at sample collection.³⁵³ The PCR by itself is considered unreliable for routine detection of BLV in herds with a high prevalence of disease.³⁵⁴ The ability of PCR to detect a BLV infected animal is improved by means of a nested PCR, which was reported to be effective in diagnosing infection when seroconversion had not yet occurred.³⁵⁵ The presence of antibodies to gp51 is generally considered to be a prerequisite to a diagnosis of lymphoma, except for cows during the periparturient period, when circulating antibodies may fall below the level detectable by a serologic test.³⁵⁶ The mere presence of BLV antibodies does not necessarily mean that an animal has lymphoma, which is found in only about 1.7% of BLV-infected cattle.³⁵⁷ However, serology can be helpful in predicting the chance of lymphoma in cattle seropositive to the gp51 antigen. Sera from cattle with lymphoma generally have high titers to the gp51 antigen (score of 3 or 4 on AGID), compared with those of infected cattle without lymphoma (score of 1 or 2). In addition, cattle that do not have lymphoma tend not to have antibodies to the p24 antigen of BLV, as detected by AGID. The percentage of cattle with histologically confirmed lymphoma but in which lymphoma was not diagnosed by a gp51-positive and p24-negative test result (false-negative rate of diagnostic test) has been found to be only 0.21%, using the AGID test.³³⁴ Unfortunately, most diagnostic laboratories do not have the capability to perform the test using the p24 antigen.

At necropsy, tumors are found enclosed in a capsular-like tissue that, when sectioned with a knife, results in an eversion of tumor stroma. Cut tumor tissue is cream colored and friable, with little binding structure. Centers of tumors may appear necrotic and mushy, whereas peripheral regions are firmer and pink to white. Histopathology provides the only definitive diagnosis. Tissue should be biopsied by surgical removal of as much of the node (or mass) as possible.