Bartonella henselae infection in a dog with recalcitrant ineffective erythropoiesis

Ineffective erythropoiesis was diagnosed in an 8‐year‐old male castrated Labrador Retriever. Despite treatment with immunosuppressive therapy for suspected immune‐mediated erythrocyte maturation arrest, resolution of the nonregenerative anemia was not achieved. Following documentation of Bartonella henselae bacteremia by Bartonella alpha proteobacteria growth medium (BAPGM) enrichment blood culture, immunosuppressive therapy was discontinued, and the anemia resolved following prolonged antibiotic therapy. Bartonella immunofluorescent antibody testing was negative, whereas B henselae western blot was consistently positive. The contribution of B henselae bacteremia to ineffective erythropoiesis remains unknown; however, the potential role of B henselae in the pathophysiology of bone marrow dyscrasias warrants additional investigation.     

Autoantibodies and red blood cell membrane proteins in a case of canine autoimmune hemolytic anemia.

Autoantibodies and erythrocyte membrane proteins were analyzed in a case of a dog with autoimmune hemolytic anemia (AIHA). In crisis phase, antiglobulin test was positive. The eluate from the erythrocytes of the dog with AIHA gave agglutination against autologus erythrocytes. Immunoglobulin subclasses in the eluate were revealed to be IgG and IgA by the double diffusion test. Comparing the SDS polyacrylamide gel electrophoretic patterns of erythrocyte membrane proteins between the crisis and remission phases, there was a change in the protein on protein 4.1 region. However, there were no changes in blood group typings in two phases.     

Persistent reticulocytosis in a case of poodle macrocytosis

A healthy 14‐year‐old, male neutered, Miniature Poodle was found to have a persistent erythrocyte macrocytosis and reticulocytosis with a normal and stable HCT. The hematologic features of macrocytosis, increased Howell‐Jolly bodies, and metarubricytosis, in the absence of anemia or other cytopenias, combined with the cytologic evidence of bone marrow erythroid dysplasia, including megaloblastosis, binuclearity, increased mitotic activity, and nuclear fragmentation, are consistent with previous reports of congenital dyserythropoiesis termed poodle macrocytosis. We speculate that the additional presence of persistent reticulocytosis in the absence of an identifiable stimulus for accelerated erythropoiesis may represent a phenotypic variation of this inherited condition, and the morphologic abnormalities of the dyserythropoiesis are described.     

Application of a direct flow cytometric erythrocyte immunofluorescence assay in dogs with immune-mediated hemolytic anemia and comparison to the direct antiglobulin test.

A direct flow cytometric erythrocyte immunofluorescence assay (FC) was developed and compared with the direct antiglobulin test (DAT) for detection of erythrocyte-bound immunoglobulin (IgG and IgM) and complement (C3) in dogs with immune-mediated hemolytic anemia (IMHA). Tests were performed on erythrocytes from 13 healthy nonanemic dogs and from 13 anemic dogs with IMHA. The FC and DAT were negative for erythrocyte-bound immunoglobulin in all healthy dogs. The FC was negative for erythrocyte-bound C3 in 12 healthy dogs and positive in 1 healthy dog, and the DAT was negative for C3 in all healthy dogs. Of the 13 IMHA dogs tested for erythrocyte-bound IgG, 12 were positive using the FC and 7 were positive using the DAT. Sensitivity for the detection of erythrocyte-bound IgG in the 26 dogs was 92% for FC and 53% for DAT. Specificity for detection of erythrocyte bound IgG for FC and DAT was 100%. The addition of IgM and/ or C3 did not increase the sensitivity for FC or DAT. In this group of dogs, the FC provided a more rapid, cost-effective, sensitive, objective method to quantitate erythrocyte-bound immunoglobulin and/or complement compared with the currently used DAT.     

The anemia of chronic renal failure revisited

The mechanisms of the anemia of chronic renal failure are reviewed. Ineffective erythropoiesis is undoubtedly the major cause of this anemia. The contributions of inadequate erythropoietin secretion by the diseased kidneys, suppressive effects of uremic "toxins" on erythropoiesis and red cell survival, excess blood loss, and plasma concentrations of parathyroid hormone, calcium and phosphate are discussed.     

Anemia and osteopetrosis in a dog

A 1-year-old, male Australian Shepherd Dog with consanguineous parents was discovered to have severe nonregenerative anemia associated with osteopetrosis. Diagnosis of the bone abnormality was established by skeletal radiography and microscopic examination of a rib biopsy specimen. The anemia was attributed to failure to develop normal marrow cavities combined with failure of extramedullary erythropoiesis. Although blood transfusions sustained the dog's life for 15 months, the dog died of a hemolytic transfusion reaction.     

Erythrocyte volume distribution analysis and hematologic changes in two horses with immune-mediated hemolytic anemia

Immune-mediated hemolytic anemia was diagnosed in two horses on the basis of regenerative anemia, increased erythrocyte fragility in hypotonic saline, autoagglutination, and a positive direct antiglobulin (Coomb's) test. During steroid therapy partial resolution of the anemia was indicated by rising packed cell volume, macrocytosis, and bone marrow erythroid hyperplasia. Using erythrocyte volume distribution histograms (erythrograms), the regenerative response was characterized by analysis of macrocytic and normocytic erythrocyte subpopulations. In both horses, a gradual net increase of about 2 X 10(6) macrocytes/microliter occurred over a four- to five-week period. Over the same interval there was a gradual decrease in the number of normocytes. We suggest that the macrocytes remained large through this period rather than contributing to normocyte population growth. Erythrograms may provide an additional means of evaluating erythrocyte regeneration in horses.     

Studies on erythrocyte metabolism following acute blood loss in the horse.

Anaemic horses do not provide reticulocytes during an erythropietic response. Acute blood loss was studied in 4 ponies as a model to monitor changes in erythrocyte metabolism during active erythropoiesis. Following phlebotomy, erythrocyte adenosine-5-triphosphate, adenosine-5-diphosphate, glucose-6-phosphate, fructose-6-phosphate, and phosphoenolpyruvate increased signifcantly. The remaining glycolytic intermediates did not change. It is suggested that the determination of erythrocyte adenosine-5-triphosphate may be used to evaluate erythrocyte response following acute blood loss in the horse.     

Anemia, splenomegaly, and increased osmotic fragility of erythrocytes in Abyssinian and Somali cats

OBJECTIVE: To determine clinical and clinicopathologic features of a chronic intermittent severe hemolytic anemia characterized by erythrocyte osmotic fragility in Abyssinian and Somali cats. DESIGN: Case series. ANIMALS: 13 Abyssinian and 5 Somali cats. PROCEDURES: History, pedigree information, and results of routine laboratory tests, special erythrocyte studies, and histologic evaluation of splenic and hepatic specimens were analyzed. RESULTS: Age at which clinical signs of anemia were first apparent ranged from 6 months to 5 years. Ten cats had splenomegaly. Most often, the PCV was between 15 and 25%, but it was as low as 5% at some times. The anemia was characterized by macrocytosis and mild to moderate reticulocytosis, but no poikilocytosis. Hyperglobulinemia, lymphocytosis, mild hyperbilirubinemia, and high hepatic enzyme activities were common findings. Results of Coombs tests and tests for infectious diseases were negative. The erythrocytic osmotic fragility was high in affected cats (mean osmotic fragility, 0.66 to 0.78%), compared with healthy cats (0.48 to 0.58). No specific membrane protein abnormality, erythrocyte enzyme deficiency, or hemoglobinopathy was identified. Histologic evaluation of splenic and hepatic specimens revealed extramedullary hematopoiesis and hemosiderosis. Four of the 5 Somali cats were closely related. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of results of pedigree analyses, the apparent breed predilection, and the exclusion of other known causes of anemia in cats, we believe that the hemolytic anemia in these cats was likely a result of a novel hereditary erythrocyte defect. A genetic predisposition to immune-mediated destruction of erythrocytes could not be ruled out.     

Potential role of serum inhibitors of erythropoiesis in the anemia associated with infection, renal disease and malignancy in the dog

Serum from dogs with anemia secondary to infection, malignancy and chronic renal disease was tested for the capacity to inhibit in vitro erythropoiesis. Serum inhibition was found in seven of 10 dogs with infectious diseases, four of 10 dogs with malignant diseases, and two of 10 dogs with chronic renal failure. The results were reproducible with at least three different marrow donor dogs. Serum inhibition did not correlate with decreased packed cell volumes. However, serum inhibition occurred in the two dogs with greatest azotemia suggesting that inhibitors of erythropoiesis are present only in advanced stages of renal failure.     

Immunoglobulin G subclass [IgG and IgG(T)] interaction with the P26 group specific antigen of equine infectious anemia virus: immunodiffusion and complement-fixation reactions.

Isolated equine immunoglobulin (Ig)G(T) antibodies to equine infectious anemia virus P26 antigen did not precipitate with antigen when the ratio of antibody to antigen was high. However, at lower ratios of antibody to antigen precipitation occurred. In addition, complement-fixation by IgG and P26 antigen was inhibited by high concentrations of IgG(T). The unusual reaction pattern noted with IgG(T) antibodies was still detectable by the immunodiffusion test for equine infectious anemia virus. In situations of nonprecipitability by IgG(T), the adjacent positive control line was inhibited, and this was interpreted as a positive reaction.     

Erythrogram and red cell distribution width of Equidae with experimentally induced anemia

The erythrogram (erythrocyte histogram) and red cell distribution width (RDW) were evaluated in 5 purebred horses and 1 pony of mixed breeding with experimentally induced anemia. Four horses were studied for 6 weeks after 20% of their estimated blood volume was removed on each of 2 consecutive days (40% total blood loss; acute blood-loss group). Two horses were given acetylphenyl hydrazine IV daily, until acute Heinz body hemolytic anemia was induced; the 2 horses were then evaluated for 6 weeks. One horse and the pony had 20% of their estimated blood volume removed via phlebotomy once each week for 8 weeks to induce iron-deficiency anemia (chronic blood-loss group); the horse had been partially depleted of iron before the study began. Weekly blood samples were examined for changes in the erythrogram, RDW, mean cell volume (MCV), and erythrocyte glucose-6-phosphate dehydrogenase activity. Fourteen days after acute blood loss, mild increases were seen in the MCV, which persisted to day 42. The RDW was increased at day 14 and remained increased until day 42; however, the percentage increase was double that of the MCV at days 14, 21, and 28. Erythrograms had mild extensions of the right slope at days 14 to 28. Mean erythrocyte glucose-6-phosphate dehydrogenase activity increased in all 3 groups, but individual concentrations were erratic. In the 2 horses with acute hemolytic anemia, modest increases of similar magnitude were seen in RDW and MCV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hematologic features of iron deficiency anemia in llamas.

Iron deficiency anemia was identified and characterized in three 14 to 29-month-old male llamas (llama Nos. 1-3) from separate herds in Colorado. The identification of iron deficiency anemia was based on hypoferremia (serum iron = 20-60 micrograms/dl), erythrocytic features, and hematologic response to iron therapy. The anemia was moderate and nonregenerative and characterized by erythrocyte hypochromia, microcytosis (mean cell volume = 15-18 fl), and decreased mean corpuscular hemoglobin concentration (36.0-41.0 g/dl). Morphologic features unique to llamas with iron deficiency anemia included irregular distribution of hypochromia within erythrocytes and increased folded cells and dacryocytes. The cause of iron deficiency was not determined. The llamas were treated with various doses and schedules of parenteral iron dextran. Two of the llamas were monitored for up to 14 months after the start of iron therapy and experienced increases in hematocrit and mean cell volume values. In one llama, progressive replacement of microcytic cells with normal cells was visualized on sequential erythrocyte volume distribution histograms following iron therapy.     

Erythrocyte macrocytosis in feline leukemia virus associated anemia

Using erythrocyte volume distribution histograms (erythrograms), erythrocyte macrocytosis and anisocytosis were quantitated in 139 cats tested for feline leukemia virus group-specific antigen. Feline leukemia virus-negative cats with non-regenerative anemia or normal packed cell volumes had normal mean corpuscular volume values. Uninfected cats with regenerative anemia had prominent significantly increased macrocytosis and anisocytosis (p less than 0.01). Ninety percent of 62 feline leukemia virus-positive cats had altered erythrograms. Thirty-three feline leukemia virus-positive cats with non-regenerative anemia had marked macrocytosis. Their mean corpuscular volume values (mean 60 fl +/- 2 fl standard error, reference range of 37-49 fl) were significantly greater than those of feline leukemia virus-negative cats except for those with regenerative anemia. Feline leukemia virus-positive, non-anemic cats had significantly increased mean corpuscular volume values of intermediate magnitude. Nine adult cats experimentally infected with feline leukemia virus developed non-regenerative anemia with significant increases in mean corpuscular volume and anisocytosis. However, the macrocytosis observed in these cats was considerably less than in naturally occurring feline leukemia virus-positive cats with non-regenerative anemia. These observations indicate there are events in the pathogenesis of feline leukemia virus-associated anemia other than simple erythroid hypoplasia. We suggest that hemolysis and erythrocyte regeneration occur before erythroid hypoplasia and may partially account for macrocytosis observed in the face of non-regenerative anemia.     

Determination of erythrocyte pyruvate kinase deficiency in Basenjis with chronic hemolytic anemia

Erythrocyte pyruvate kinase (PK) deficiency was first described in Basenjis 20 years ago. Although the approach to diagnosis had not been well established, a screening program to detect heterozygotes was thought to have eliminated PK deficiency from the Basenjis of the United States. Four not closely related Basenjis with severe chronic hemolytic anemia, from various parts of the United States, were studied. Their PCV ranged from 16 to 25% and their reticulocyte count was always above 15%. A progressive osteosclerosis was seen in all of the Basenjis and hepatic failure developed in 2 of them. The erythrocyte intermediary metabolite patterns indicated a glycolytic defect at the PK step. Erythrocyte PK activities were markedly increased in the anemic Basenjis, compared with those of a control group, but the enzyme in these Basenjis had abnormal kinetic properties and was thermolabile. An antibody against R-type PK, the regular erythrocyte PK form, did not neutralize the PK activity of affected Basenjis, and results of electrophoretic studies suggested the expression of M2-type PK, a leukocyte and fetal erythroid PK-type. Clinically healthy heterozygous Basenjis had half-normal R-type PK activity and did not express the M2-type in their erythrocytes. We conclude that severe chronic hemolytic anemia, caused by erythrocyte PK deficiency, and associated osteosclerosis still develop in Basenjis. A definitive diagnosis cannot be reached by simply measuring erythrocyte PK activity; rather, diagnosis requires measurement of glycolytic substrate accumulation and enzyme stability and immunologic or electrophoretic studies of erythrocyte PK.     

The kinetics of hematopoiesis in the light horse III. The hematological response to hemolytic anemia.

The hematological response to acetylphenylhydrazine hemolytic anemia was studied in three standardbred horses. The lifespan of erythrocytes produced during the most severe phase of the anemia were measured with 75-selenomethionine and found to be 144 days as compared to the 139 day lifespan in response to hemorrhagic anemia or 155 days in normal standardbred horses measured previously using the same technique. The erythrocyte counts returned to initial values in 42 days (37, 34 and 54 days) a mean erythrocyte production of 6.4 times 10-12 erythrocytes/day. The mean hemoglobin production was 0.31 gm/kg body weight/day as compared to 0.11 gm Hb/kg/day previously observed in response to hemorrhagic anemia. The mean increase in erythrocyte mean cell volume was 12 mu-3 during the acute response phase to hemolytic anemia in contrast to the absence of a significant increase in the mean cell volume as previously observed during response to hemorrhagic anemia. Free Heinz bodies separated from erythrocytes during the acute phase could not be differentiated from platelets on the hemocytometer counting chamber with standard techniques.     

Acquired methemoglobinemia in anemic cattle infected with Theileria sergenti.

To investigate the mechanism of anemia accompanying Japanese bovine theileriosis, we examined whether production of methemoglobin (MetHB), an indicator of erythrocyte oxidation, was associated with anemia in cattle experimentally infected with Theileria sergenti. The percentage of MetHB, which is an oxidized form of hemoglobin, increased according to the onset of anemia. During severe anemia, high levels of acquired methemoglobinemia were observed in all infected cattle. A significant correlation (r=-0.649; P<0.01) between an increase in MetHB concentration and a decrease in packed cell volume (PCV) was observed. It was considered that hemoglobin oxidation may be one of the aggravating factors of anemia in T. sergenti infection.     

Immune-mediated hemolytic anemia induced by penicillin in horses

Three horses developed severe, immune-mediated hemolytic anemia after treatment with penicillin. The horses had positive direct antiglobulin (Coombs) tests and high titers of IgG antibody that agglutinated penicillin-coated equine red cells. Two of the horses were tested for antibodies to autologous red cell antigens; autoantibodies were not present. Titers of antipenicillin antibody decreased after penicillin was discontinued but IgG antibody was detectable months after recovery. One of the horses was challenged with penicillin; antibody titer increased slightly, but anemia did not develop. Antipenicillin antibody of the IgM class was present in low titer in 23 (77%) of 30 non-anemic horses tested. Apparently, the horse is similar to man in that penicillin-induced anemia is rare but the percentage of individuals with antipenicillin antibody is high.     

Corpuscular oxidation in newborn crossbred calves naturally infected with Theileria annulata

Erythrocytic lipid peroxidation has been implicated as a cause of anemia in Theileria annulata infection in cattle. The present study aimed to evaluate oxidative damage of membrane lipids and proteins in addition to hemoglobin (Hb) as three criterions of erythrocyte oxidation and their relation to erythrocyte deformability and anemia of newborn crossbred calves (Friesian × Egyptian Balady breed) naturally infected with T. annulata. Twenty-five T. annulata-infected calves (aged 20-30 days) along with 15 age matched healthy controls were used. Percentage of parasitemia varied from 12% to 63% (34.76 ± 3.05%). In comparison to controls, infected calves showed increased levels (P<0.001) of lipid peroxidation (malondialdehyde; MDA, 52%) and protein oxidation (protein carbonyls; PCs, 132%) in erythrocyte membrane as well as increased values of Hb oxidation (methemoglobin; MetHb, 186%), corpuscular osmotic fragility (15.1%) and hemolysis (free Hb; 195.5%). Parasitemia was positively correlated with MDA (r=0.41, P=0.039), PCs (r=0.45, P=0.023) and MetHb (r=0.40, P=0.042). Also, percent of erythrocytic deformability (echinocytosis) was positively correlated with MDA (r=0.49, P=0.013) and PCs (r=0.63, P<0.001). On the other hand, erythrocytic packed cell volume was negatively correlated with MDA (r=-0.44, P=0.028), PCs (r=-0.72, P<0.001) and MetHb (r=-0.42, P=0.037). In conclusion, T. annulata infection is associated with a parasitic burden-dependant oxidative damage to the erythrocyte membrane protein and lipid contents in addition to Hb. This oxidative damage is linked to the morphological changes of the erythrocyte and may act as mechanisms contribute to pathogenesis of anemia in T. annulata infection in newborn calves.     

Decrease in erythrocyte survival in Theileria sergenti-infected calves determined by non-radioactive chromium labelling method.

Pathogenesis of anemia in the calves infected with Theileria sergenti was investigated from the viewpoint of erythrocyte survival decrease in the circulating blood. For investigation of erythrocyte survival a method of erythrocyte labelling with non-radioactive chromium (50Cr) was utilized. It was found that (1) the erythrocyte survival decreased markedly in the T. sergenti-infected calves compared with that in the uninfected calves; the survival rate of 25.7% for infected calves and 86.0% for uninfected ones on the fourth day after re-introduction of the labelled erythrocytes into the original donors, and that (2) the survival of non-parasitized erythrocytes in the infected calves was also decreased, which indicates no obvious relationship between parasitism and decrease in survival of erythrocytes.