Platelets from bleeding Simmental cattle have a long delay in both ADP-activated expression of GpIIB-IIIA receptors and fibrinogen-dependent platelet aggregation.

We have previously reported that platelets from bleeding Simmental cattle do not aggregate in vitro in response to ADP, collagen and calcium ionophore A23187, though calcium mobilization and myosin light chain phosphorylation do occur. The aggregation abnormality, measured by aggregometry, was ascribed to abnormal cytoskeletal expression, with the maximal numbers of activated GpIIb-IIIa receptors per platelet no different from that seen in normal bovine platelets activated with ADP. We have therefore sought to compare the kinetics of micro-aggregation with the rate of expression of GpIIb-IIIa receptors required for mediating fibrinogen (Fg)-dependent platelet aggregation, to provide a more direct molecular explanation for the aggregation abnormality. We compared aggregation kinetics of ADP-activated platelets using both aggregometry and particle counting to monitor micro-aggregation. Fibrinogen receptor expression was monitored with FITC-labelled human Fg and with the reporting antibody for activated GpIIb-IIIa, FITC-PAC1, using flow cytometry. The affected platelets show a marked delay in onset of microaggregation for ADP-activated platelets stirred with human Fg, paralleded by an unusual delay in activated GpIIb-IIIa receptor expression (DARE) for otherwise competent Fg binding. The on-rates for Fg binding to platelets maximally pre-activated with PMA are identical for normal and affected platelets, whether comparing the binding of human or bovine Fg. The unique DARE syndrome explains the observed delay in aggregation of platelets from affected Simmental cattle and predicts the bleeding problems due to delayed binding of adhesive proteins.

Platelets from bleeding Simmental cattle mobilize calcium, phosphorylate myosin light chain and bind normal numbers of fibrinogen molecules but have abnormal cytoskeletal assembly and aggregation in response to ADP.

We have evaluated platelet function in normal Simmental cattle and in those with a congenital, inherited bleeding disorder previously attributed to impaired platelet aggregation. Affected platelets failed to aggregate and secrete in response to ADP and the ionophore A23187, and showed impaired aggregation responses to collagen and ionomycin. Aggregation and secretion of normal and affected platelets was similar in response to thrombin and PMA. Resting cytosolic calcium levels and calcium mobilization in response to ADP and ionomycin were similar in control and four affected animals. Normal and affected bovine platelets phosphorylated myosin light chain and pleckstrin in response to ADP and A23187. Transmission electron microscopy of affected platelets following stimulation with ADP, showed shape change and some degree of centralization of the actomyosin gel. Affected platelets had comparable numbers of GPIIb/IIIa complexes and expressed comparable numbers of fibrinogen receptors as normal platelets in response to ADP. Cytoskeletal assembly in affected platelets was normal in response to PMA but incomplete in response to ADP and A23187. Failure of platelet aggregation in bleeding Simmental cattle is predicted to arise from abnormal cytoskeletal assembly following calcium mobilization and phosphorylation of myosin light chain in response to ADP.

Preliminary studies of a platelet function disorder in Simmental cattle.

A bleeding disorder due to abnormal platelet function occurs in Simmental cattle. Whole blood from these animals underwent good clot retraction. Platelet aggregation in response to adenosine diphosphate (ADP) and collagen in a whole blood aggregation system was markedly impaired. Normal bovine platelets in a whole blood aggregation system showed very little aggregation in response to epinephrine and arachidonic acid. Aggregation in platelet-rich plasma was negligible in response to ADP, collagen and thrombin. Dense granule release of radiolabelled serotonin from the platelets of one affected cow was similar to that of normal bovine platelets. Platelet membrane glycoprotein electrophoresis with the platelets of one affected cow revealed no quantitative abnormalities. These findings reveal similarities and differences in thrombopathic Simmental platelet function when compared to human Glanzmann's thrombasthenia and Basset Hound thrombopathia.

Immunohistochemical detection of intermediate filament proteins in formalin fixed normal and neoplastic canine tissues.

Normal and well differentiated neoplastic canine tissues were immunohistochemically stained for keratin, vimentin and desmin intermediate filament proteins using commercially available monoclonal antibodies. Keratin was detected in 56 of 57 carcinomas, vimentin in 59 of 62 sarcomas and desmin in three of four muscle cell tumors. Most normal and neoplastic tissues expressed only one type of intermediate filament; exceptions were one hemangiosarcoma and one pulmonary carcinoma in which there was coexpression of vimentin and keratin proteins. Since immunohistochemical detection of intermediate filaments has tissue-specific distribution in the majority of well differentiated canine neoplasms, these stains may be useful in the differential diagnosis of anaplastic canine tumors. However, the monoclonal antibodies to cytokeratin which were tested in this study failed to detect intermediate filaments in liver, pancreas and salivary glands which suggests that these antibodies may also be unable to detect epithelial tumors derived from these tissues. In addition, in nine neoplasms, the normal tissues adjacent to neoplastic cells failed to stain for the intermediate filament normally expressed. When this occurs, evaluation of intermediate filament expression is invalid for the determination of tissue of origin of the neoplastic cells.

Determination of plasma fibrinogen concentration in the horse.

The microhematocrit heat-precipitation methods of Millar et al (1971) and Schalm et al (1975) were compared with the reference clottable protein method of Ratnoff and Menzie (1951) in the measurement of plasma fibrinogen concentration in horses. The millar et al method was more precise and accurate and showed better positive correlation with the reference method than did the Schalm et al method. There was no significant difference in the plasma fibrinogen concentration between healthy Thoroughbreds and healthy horses of other breeds. Horses with bacterial pneumonia and abscesses had significantly greater plasma fibrinogen values than did those with colic, fractures, and nonsuppurative wounds. The Millar et al method was recommended as a simple, relatively accurate procedure for the determination of plasma fibrinogen that might be used as an additional diagnostic and prognostic indicator in the laboratory investigation of diseases in the horse.

Hematologic abnormalities preceding apparent recovery from feline leukemia virus infection.

A 5-month-old domestic short-haired cat was referred to the Western College of Veterinary Medicine because of hemorrhage from the mouth. Neutropenia and bone marrow dyscrasia developed within 2 weeks, and results of a fluorescent antibody test for feline leukemia virus (FeLV) were positive. During the next 6 months, the feline oncornavirus cell membrane-associated antibody titer increased, the hematologic abnormalities regressed, and the cat became negative to the FeLV fluorescent antibody test (FeLV test). The results of 3 consecutive FeLV tests were negative, and the cat has been clinically normal for the past 18 months.

Cold-agglutinin hemolytic anemia and Haemobartonella canis infection in a dog.

Cold-agglutinin hemolytic anemia in a dog was manifested by weakness, progressive hemolytic anemia, hematuria, and hemoglobinuria. Corticosteroid therapy failed to alleviate the anemia. The condition became complicated by Haemobartonella canis infection berminally. Prominent postmortem findings included disseminated thrombosis and infarction, glomerulonephritis, and thickened alveolar membranes.

Chronic granulocytic leukemia in a horse.

A nine year old quarter horse exhibited progressive weight loss and inappetance over a 47 day period. There was clinical evidence of pleuritis and pneumonia substantiated by leukocytosis and elevated protein in pleural fluid. Over the entire period the horse was neutropenic and had circulating abnormal immature granulocytes and low numbers of blast cells. Anemia and thrombocytopenia progressively worsened. Bone marrow examination revealed very few mature granulocytes but large numbers of immature cells of the granulocytic series and marked megaloblastic transformation of erythroid cells. These findings were consistent with chronic granulocytic leukemia.

Clinical and laboratory findings of a bleeding disorder in Simmental cattle.

Eight Simmental cattle were presented to the Western College of Veterinary Medicine over the past decade with historic or current prolonged episodes of bleeding. Spontaneous epistaxis, superficial hematomas, and prolonged bleeding following injury or minor surgery (such as tattooing) were frequent observations. Animals presented because of prolonged hemorrhage were anemic and debilitated. Compensatory erythropoiesis was compromised in these animals by depletion of iron stores due to chronic blood loss. Screening tests to assess coagulation were normal as were numbers and morphology of platelets. In vitro platelet aggregation was abnormal in all affected cattle. Three animals were euthanized due to uncontrollable hemorrhage but the precise source of hemorrhage was undetectable. These findings are consistent with bleeding due to a platelet function disorder which has been described previously in Simmental cattle.

Bovine postparturient hemoglobinuria: a review of the literature.

Predisposing causes of bovine post-parturient hemoglobinuria are summarized along with suspected pathogeneses, clinical signs, laboratory findings, clinical management and early experimentation.

Moldy sweetclover poisoning in cattle.

Some selected clinical and laboratory aspects of moldy sweetclover poisoning in cattle are reviewed. The prothrombin time is preferred when the disease is suspected. The bleeding time and whole blood clotting time test are also used. Dicoumarol is not always detectable in the suspected feed which may be due to sampling technique or inaccuracy in the laboratory assay. The most effective treatment is a whole blood transfusion. Vitamin K(1) (naturally occurring vitamin K) is an effective antidote but too expensive. Vitamin K(3) (synthetic vitamin K) in both the injectable and oral forms are not as effective as K(1) but are used extensively for treatment and prevention. Feeding the suspected feed for two weeks followed by a one week withdrawal successively or by dilution with other feeds will help to reduce the incidence of disease if other feeds are not available. Suspected feed should not be fed to cattle for at least three weeks before surgery or parturition.

A study of the inheritance of a bleeding disorder in Simmental cattle.

A study was designed to determine the inheritance pattern of a blood platelet aggregation disorder in Simmental cattle utilizing embryo transfer technology. A Simmental donor cow that had previously produced a calf with the platelet aggregation disorder was superovulated and mated to a bull that had also produced affected offspring. Twenty-seven calves were produced from the 63 (42.9%) embryos transferred. This somewhat lower than expected pregnancy rate is suggestive of an increased rate of embryo loss. Twenty-three of 25 (92%) calves had normal platelet aggregation patterns and 2 failed to show any evidence of platelet aggregation. Data are suggestive that inheritance is not simple Mendelian recessive. A more likely scenario is that the defect is the result of the inheritance of at least 2 genes, which is also consistent with the sporadic incidence reported in the population at large.

A comparison of parameters used to assess liver damage in sheep treated with carbon tetrachloride.

Changes in serum enzyme levels, liver histology and liver function tests have been correlated to determine the usefulness of these tests in assessing liver status. The effects of carbon tetrachloride administration on these parameters has been determined in a group of 20 sheep. Normal levels, elevated levels after injury and the effect of elapsed time after injury are reported for serum glutamic dehydrogenase, sorbitol dehydrogenase, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, lactate dehydrogenase, fructose-1-phosphate adlolase, alkaline phosphatase, cholesterol and proteins. Variation in the time of elevation of enzyme activities may be useful in determining the elapsed time between acute injury and serum sampling. In comparison to sheep fed an adequate diet, a diet with a restricted protein intake was associated with increased severity of histological lesions and decreased liver function.

Serum iron, total iron binding capacity, plasma copper and hemoglobin types in anemic and poikilocytic calves.

Ninety-eight calves were studied to determine if anemia and poikilocytosis were related to iron or copper status or hemoglobin type. No significant differences were found in serum iron, total iron binding capacity, marrow iron, plasma copper or hemoglobin type between affected and normal calves. Poikilocytes were strongly inversely correlated (-0.9177) with age. Calves less than six weeks of age had more poikilocytes, lower serum iron, higher total iron binding capacity, less adult hemoglobin and more neonatal and fetal hemoglobin than calves greater than six weeks of age.

The effect of oral phenylbutazone on whole blood platelet aggregation in the dog.

Platelet aggregation to collagen, arachidonic acid and adenosine diphosphate was evaluated in six dogs using a whole blood electronic aggregometer. The six dogs were then given phenylbutazone orally according to four different dosage levels and durations of treatment. Aggregation responses were measured at established intervals of time following phenylbutazone administration. Data on untreated dogs indicated that arachidonic acid, at a final concentration of 50 micrograms/mL and collagen, at a final concentration of 5 micrograms/mL, were useful agents for studying whole blood platelet aggregation in the dog, but adenosine diphosphate, at a final concentration of 30 microM was not. The high single dose (900 mg) of phenylbutazone significantly inhibited platelet aggregation to arachidonic acid at 1.5,4,7 and 12 hours following administration. The results indicated that the whole blood electronic aggregometer was of limited value in detecting subtle changes in platelet aggregation. It was concluded, however, that the instrument is potentially useful as a rapid screening aid for detecting canine patients at high risk of platelet-related bleeding problems.

The diagnostic significance of a positive direct antiglobulin test in anemic cats.

The preparation of a feline Coombs serum (rabbit antifeline gamma globulin) is described. The direct antiglobulin test using this serum was performed on 20 anemic and 20 healthy control cats. Red cell membrane antibodies were detected in cats with feline leukemia virus infection and in others with inflammatory and neoplastic diseases. A low titre of cold agglutinating antibody was present in a high proportion of the control cats. Positive direct antiglobulin tests were noted in cats without overt hemolytic disease. It was concluded that the direct antiglobulin test in anemic cats has certain diagnostic limitations. A positive reaction should be interpreted cautiously especially when there is no clinical or laboratory evidence to support a diagnosis of autoimmune hemolytic anemia.

Congenital hemolytic anemia in the Basenji dog due to erythrocyte pyruvate kinase deficiency.

Congenital hemolytic anemia in the Basenji dog resembles pyruvate kinase (PK) deficiency in man as it is characterized by an abbreviated erythrocyte life span, an intense reticulocytosis, type II autohemolysis and splenomegaly. Glucose utilization and lactate production were inadequate with respect to the immature cell population. Analysis of enzymes involved in erythrocyte glycolysis revealed a deficiency of pyruvate kinase.