J Vet Diagn Invest 4:31-37 (1992)
Congenital dyserythropoiesis and progressive alopecia in Polled Hereford calves: hematologic, biochemical, bone marrow cytologic, electrophoretic, and flow cytometric findings D. J. Steffen, G. S. Elliott, H. W. Leipold, J. E. Smith Abstract. Congenital dyserythropoiesis with dyskeratosis is a slow, progressive, and often fatal disease in Polled Hereford calves. Affected calves have a macrocytic normochromic anemia with a mild reticulocytosis. Studies indicate that calves are hyperferremic with increased saturation of serum total iron binding capacity, which rules out iron deficiency as a cause. Other secondary causes of dyserythropoiesis, including cobalamin and folate deficiencies, are unlikely because serum cobalamin and folate levels of affected calves were normal. Virus isolation was negative, and failure to identify bovine retroviral antigens or antibodies from several calves suggested that viral agents were not involved. Bone marrow cytologic findings were similar to those in congenital hereditary dyserythropoiesis in humans and included occasional multinucleate cells, internuclear chromatin bridging between nuclei of partially divided cells, and, more frequently, irregular nuclear shapes and chromatin patterns. DNA content and cell cycle distribution of erythroid cells appeared normal, and no electrophoretic abnormalities were detected in erythrocyte membrane proteins. The Polled Hereford syndrome is similar in many ways to type I congenital dyserythropoiesis in humans and may be an appropriate biomedical model for studying erythroid proliferation during dyserythropoiesis. phorusb and Sorbitol dehydrogenase were determined.c Serum levels of cobalamin and folate were bioassayed.d The serum iron and total iron binding capacity were determined, and percent saturation of serum iron binding capacity was calculated.21 Thyroid assays were performed by a commercial laboratory” and included total thyroxine (T4), free T4, total triiodothyronine (T3), and free T3. Thyroid response to thyroid stimulating hormone (TSH) was evaluated by comparing differences in levels of thyroxine pre-TSH and 6 hr post-TSH administration. Each calf received 2.5 IU of Dermathycinf intravenously as a source of TSH for the response test. Bone marrow aspirates and biopsies were taken on the same days as blood samples for hematology. Samples were collected via a 6-in. 12-gauge Jamshidi needle.g Samples were collected from sternebrae with the calves in lateral recumbency, after local anesthesia and surgical scrub of the overlying skin. Marrow was aspirated by 8-10 cc negative pressure with a 12-ml plastic syringe. The pressure was released slowly after approximately 0.5 ml of marrow was collected to prevent excessive dilution by peripheral blood. Samples were mixed with 1 drop of ethylenediaminetetraacetic acid in isotonic saline to prevent coagulation. Marrow particles were placed on glass slides, and smears were made. Biopsies were obtained by redirecting and advancing the Jamshidi needle without the stylet after aspirates were collected. Biopsy cores were placed immediately in chilled Karnovsky’s fixative and fixed overnight.10 Biopsies were decalcified overnight in formic acid-sodium citrate solution then processed routinely, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin, Giemsa, and Pearl’s
A congenital syndrome of anemia and alopecia in Polled Hereford calves characterized by nonregenerative anemia with ineffective erythropoiesis and dermatitis has been described recently.22 Cutaneous lesions are progressive, and hyperkeratotic dermatitis with dyskeratosis of individual cells occurs Within the stratum spinosum and follicular infundibuli. Affected calves are unproductive and often die or must be disposed of prematurely. This paper further describes hematologic, biochemical, and bone marrow findings of affected calves. Materials and methods Ten Polled Hereford calves of similar ages, 5 normal and 5 previously diagnosed with congenital anemia and progressive alopecia, were compared. Calves were housed in barns or dry lots and fed alfalfa hay, grass hay, grain, and a protein supplement. Hemograms, including erythrocyte, leukocyte, reticulocyte, and differential blood counts,a were performed 3 times at monthly intervals. Serum sodium, potassium, chloride, glucose, urea nitrogen, creatinine, alkaline phosphatase, gamma glutamyl transferase, aspartate amino-transferase, total bilirubin, direct bilirubin, total protein, albumin, globulin, calcium, phoso-
From the Departments of Pathology (Steffen, Leipold, Smith) and Laboratory Medicine (Elliott), College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506. Received for publication April 15, 1991. 31
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Steffen et al. Table 1. Hematologic findings from dyserythropoietic and normal Polled Hereford calves.
Prussian blue reaction (PPB)J Cellularity of marrow and iron stores and distribution of cells were assessed. Cytology smears were stained with Wright’s stain and by periodic acid-Schiff (PAS) and PPB reactions.2,5 Five hundred cell differential counts were made from the Wright’s=stained smears. Three-milliliter bone marrow samples from 3 affected and 3 control calves were also collected into 25 units of preservative-free heparin. Single-cell suspensions were obtained by forcing aspirates through a series of progressively smaller needles (21-27 gauge), and rubricytes were isolated by centrifugation in Ficoll-HYPaqueh at 1,000 x g and 4 C for 30 min. Erythroid cells were rinsed in phosphate-buffered saline (PBS) and resuspended at a concentration of 5 x 106 cells/ ml. Cytospin preparations were made from a small amount of this sample to verify the cell type present. Reference cells were prepared from bovine peripheral blood. Ten milliliters of peripheral blood was collected in 25 units of preservative-free heparin. Samples were mixed with ammonium chloride, centrifuged, and resuspended in PBS at a concentration of 1.5 x 106 cells/ml. Two hundred microliters of the marrow cell sample was combined with 100 µ1 of reference leukocytes and 1 ml of chilled propidium iodide and incubated on ice for a minimum of 10 min.i Analyses were performed with flow cytometry to determine ploidy. Cell cycle distribution analyses were done similarly, omitting addition of the reference cell
25
population . Red fluorescence (FL2-R) was used to quantify DNA, and an average of 17,857 events (cells) were counted per sample.j Computerized data analysis was done.k Erythrocyte membrane ghosts were prepared at 4 C from the peripheral blood erythrocytes of 3 affected calves, dams of 2 affected calves, and 6 unrelated cattle. Erythrocytes were lysed in sodium phosphate lysing buffer (10.0 mM, pH 7.4) after several 0.9% saline washes and buffy coat removal.6 Diisopropyl fluorophosphate (DIFP)1 was added to the lysing buffer to inhibit protease activity. After lysis, membranes were washed repeatedly in sodium phosphate buffer to remove hemoglobin and other cytoplasmic proteins. Polyacrylamide gel electrophoresis was performed in the presence of sodium dodecyl sulfate (SDS) and 7.5% acrylamide, and the gels were stained with Coomassie blue to demonstrate membrane proteins.8 Proteins also were electrophoresed as above then blotted onto Immobilin-P transfer membranes.m,23 Proteins on the Immobilin-P membranes were demonstrated using Ponceau-S solution and glycoproteins were demonstrated by using a commercial glycan detection kit for detecting glycoconjugates with oxidizable OH groups.n Virus isolation was attempted from buffy coat preparations from 5 calves. Preparations were passed blindly on embryonic bovine kidney cells 3 times, and observations were made for cytopathic effects. After 3 passes, cultures were examined by fluorescent antibody techniques with bovine viral diarrhea and Parainfluenza-3 (P13) conjugates. Serum samples from
Table 2. Serum chemistry and enzyme levels of dyserythropoietic and normal Polled Hereford calves.
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Table 3. Iron, cobalamin, and folate status of dyserythropoietic and normal Polled Hereford calves.
5 calves also were used to attempt detection of bovine retroviral infection.o Agar gel immunodiffusion tests were used for checking bovine leukemia virus and bovine syncytium virus antigens, and bovine immunodeficiency-like virus antigen detection was attempted by western blotting technique. Statistical analysis was done with a statistical software packagep using proc ANOVA for analysis of variance and animals nested within group (affected or control) as the error term when analyses were done several times at monthly intervals. Probabilities of 90% erythroid cells with little contamination by granulocytes. The DNA index was not different between affected and unaffected calves = 1.15). Cell cycle distribution was also similar between the 2 groups (Table 7). No electrophoretic abnormalities of erythrocyte membrane proteins (gels stained with Coomassie blue, Ponceau-S) or glycoproteins (membranes stained with glycan detection kit) were detected. Samples from all calves had 9 intensely staining protein bands detectable with Coomassie blue stains (Fig. 5) similar to previous reports of normal bovine erythrocytes.14 Several protein bands of lesser intensity were consistently present in affected and control calves. All proteins from unaffected and affected calves had identical migration patterns. Table 6.
Figure 1. Bone marrow cytologic features of dyserythropoietic Polled Hereford calf. Notice erythroid dominance in this preparation. Wright’s stain.
Glycan detection demonstrated an intense staining reaction in samples from several calves near the top of the gel that corresponded to the major glycoprotein previously described in cattle.14 Identical patterns were present in normal and affected calves. Intensity of staining in the band varied among individual calves, independent of the presence or absence of disease. No evidence of bovine retroviral infection was found. Virus isolation attempts were negative in all but 1 calf. In 1 instance, cell cultures were positive when stained with fluorescent antibody against PI3 viral antigen. Discussion The macrocytic, poorly regenerative anemia observed was similar to that previously described for affected calves. 22 Macrocytosis was significant, when cell size was compared with that of age-matched calves. An increase in erythrocyte size as calves age has been demonstrated,13,18 so caution is warranted when comparing data with normal laboratory values based on adults. The reticulocytosis, although statistically significant, was mild considering the severity of the anemia. Biologically significant differences have not been found for most serum analytes. Serum proteins, which were low in the previous report,22 were not significantly
Quantitative bone marrow cytologic findings in dyserythropoietic and normal Polled Hereford calves.
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Congenital dyserythropoiesis and alopecia in calves
Figure 2. Bone marrow cytologic features of dyserythropoietic Polled Hereford calf. Note multinucleate rubricyte. Wright’s stain.
Figure 4. Higher magnification of cellular features of Fig. 3. Note internuclear chromatin bridging (arrow). Wright’s stain.
lower in these calves. Serum iron was significantly increased in affected calves, increasing the saturation of serum iron binding capacity, which remained norma1.20 The macrocytic, normochromic nature of the anemia and the elevated serum iron suggest that deficiencies in iron uptake and utilization are not occurring. Hyperferremia is common in children affected with congenital dyserythropoiesis and often leads to hemochromatosis .3,7 Increased iron is absorbed during anemic states and is not adequately excreted, even though serum iron may be elevated.20 The significance of decreased serum thyroxine in affected calves remains unclear. However, results of
TSH response indicated that primary thyroid function remained intact, so the mild hypothyroidism must be secondary to other processes. Two calves were treated with thyroid hormone replacementq starting at 10 mg/ kg daily and adjusted to maintain pretreatment thyroxine levels near normal. No improvement was noted in the severity of anemia or skin condition after 12 weeks, and therapy was discontinued. Bone marrow evaluation demonstrated marked erythroid predominance with numerous mitotic figures. That pattern is consistent with ineffective erythropoiesis. Significant morphologic abnormalities justify classifying the anemia as dyserythropoietic. Many abnormalities (nuclear shape changes, multinuclearity, nuclear cytoplasmic asynchrony, maturation arrest, and internuclear chromatin bridging) resemble those of congenital dyserythropoietic anemia type I (CDA I) in humans.4,11,12,16,17 Unsuccessful attempts to identify several secondary causes of dyserythropoiesis, including iron deficiency, B vitamin deficiency, and viral infection, suggest that it may be a primary congenital dyserythropoiesis. Few DNA ploidy and cell cycle distribution studies of CDA erythroblasts in humans have been done. Those completed demonstrate marked abTable 7. Cell cycle distribution (%) of erythroid cells from dyserythropoietic and normal Polled Hereford calves.
Figure 3. Bone marrow cytology from dyserythropoietic Polled Hereford calf. Note mitotic figures (arrowheads), irregular chromatin (small arrows), and internuclear chromatin bridging (large arrows). Wright’s stain.
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Steffen et al.
Figure 5. Coomassie blue stained erythrocyte membrane proteins in a polyacrylamide gel. Lane 1-high molecular weight marker; lane Z-proteins from unrelated heifer of different breed; lane 3proteins from dyserythropoietic Polled Hereford calf; lane 4-proteins from dam of affected Polled Hereford calf; lane 5-proteins from second affected Polled Hereford calf; lane 6-proteins from unrelated Polled Hereford cow, lane 7-low molecular weight marker.
normalities in the DNA content of erythroblasts in type III CDA and less dramatic changes in erythroblasts with CDA types I and II. 24,26-28 Changes usually have been associated with binucleate and multinucleate cells. The absence of significant changes in rubricyte DNA content in our calves might have been related to the lower number of binucleate and multinucleate cells in the samples and the probable loss of many binucleate cells when we attempted to gate off doublets during flow cytometric analysis. In only 2 cases of CDA type I in humans has cell cycle analysis been reported, both in the same study, and DNA content of a combined total of only 233 cells was reported.28 Methods used in human cases allowed correlation of DNA quantity and cell morphology and included all binucleate or multinucleate cells in the analysis. More studies in humans and calves using comparable techniques of cell cycle analysis are needed before a final evaluation of dyserythropoiesis in calves as a model for CDA type I in humans can be made. The normal cellular DNA content and cell cycle distribution suggest that nuclear morphologic abnormalities are occurring after cytoplasmic division and may represent abnormal completion of cytokinesis and inhibited postmitotic nuclear membrane fusion. Electrophoresis of peripheral blood erythrocyte membrane proteins failed to demonstrate differences
in protein migration or glycosylation between affected and unaffected calves. This result suggests that major defects in erythrocyte membrane protein structure or glycosylation are not present, a finding similar to most cases of CDA I of humans and distinct from CDA II erythrocytes, in which defects of membrane glycosylation have been reported.1,9 Although no mechanism to explain the dyserythropoiesis could be determined, morphologic evidence suggests rapid cell proliferation with an extended maturation time for affected erythroid cells. Nuclear shape and chromatin abnormalities and internuclear chromatin bridging suggest impaired cytokinesis during terminal cell divisions within the erythroid series and prolonged duration of cells within the marrow. We conclude that additional studies of dyserythropoiesis in Polled Hereford calves are needed to further clarify this recently described syndrome. Initial hematologic studies demonstrated many similarities to human CDA type I, making dyserythropoiesis in calves a possible biomedical model for studying other dyserythropoietic anemias. Acknowledgement This research was part of the Regional Project NC-2. It is contribution no. 91-451-J from the Kansas Agriculture Experiment Station and was supported by grant no. HLO 1877 from the National Institutes of Health.
Sources and manufacturers a. Coulter sPlus IV, Coulter Electronics, Hialeah, FL. b. Dacos, Coulter Corporate Communications, Hialeah, FL. c. Abbot VP Biochromatic Analyzer, Abbot Laboratory, Dallas, TX. d. Vitamin Diagnostics, Driftwood Beach, CA. e. Endocrine Laboratory, Michigan State University, East Lansing, MI. f. Coopers Animal Health, Kansas City, MO. g. Baxter Pharmaceuticals, St. Louis, MO. h. Pharmacia, Uppsala, Sweden. i. DNA IC particles kit, Benton Dickenson, Mountain View, CA. j. FAC Scan Analyzer, Benton Dickenson, Mountain View, CA. k. Cell Fit version 1.2, Benton Dickenson, Mountain View, CA. 1. Sigma Chemical Co., St. Louis, MO. m. Millipore Corp., Bedford, MA. n. Boehringer Mannheim Biochemicals, Indianapolis, IN. o. National Animal Disease Center, Ames, IA. p. SAS Institute, Car-y, NC. q. Thyro-L, Vet-A-Mix, Shenandoah, IA.
References 1. Anselstetter V, Horstmann HJ, Heimpal H: 1977, Congenital dyserythropoietic anemia, types I and II: abberant pattern of erythrocyte membrane proteins in CDA II, as revealed by twodimensional polyacrylamide gel electrophoresis. Br J Haematol 35:209-215. 2. Beutler E: 1990, Blood, marrow, and urine iron stains. In: Hematology, ed. Williams WJ, Beulter E, Erslev A, Lichtman MA, 4th ed., pp. 1701-1702. McGraw-Hill, New York, NY.
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Congenital dyserythropoiesis and alopecia in calves 3. Bird AR, Jacobs P, Moores P: 1987, Congenital dyserythropoietic anaemia (type II) presenting with haemosiderosis. Acta Haematol (Basel) 78:33-36. 4. Conde E, Mazo E, Baro J, et al.: 1983, Transmission and scanning electron microscopy study on congenital dyserythropoietic anemia type I. Acta Haematol (Basel) 70:243-249. 5. Davey FR, Nelson DA: 1990, Periodic acid Schiff (PAS) stain. In: Hematology, ed. Williams WJ, Beutler E, Erslev A, Lichtman MA, 4th ed., pp. 1748-1750. McGraw-Hill, New York, NY. 6. Dodge JT, Mitchell C, Hanahan DJ: 1963, The preparation and chemical characteristics of hemoglobin free ghosts of human erythrocytes. Arch Biochem Biophys 100:119-130. 7. Facon T, Mannessier L, Lepelley P, et al.: 1990, Congenital dyserythropoietic anemia type I report on monozygotic twins with associated hemochromatosis and short stature. Blut 61: 248-250. 8. Fairbanks G, Steck TL, Wallach DFH: 1971, Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606-2617. 9. Fukuda MN, Masri KA, Dell A, et al.: 1989, Defective glycosylation of erythrocyte membrane glycoconjugates in a variant of congenital dyserythropoietic anemia type II: association of a low level of membrane-bound form of galactosyltransferase. Blood 73:1331-1339. 10. Glauert AM: 1975, Fixation, dehydration, and embedding of biological specimens, pp. 47-48. North Holland, New York, NY. 11. Heimpal H, Forteza-Vila J, Queisser W, Spiertz E: 1971, Electron and light microscopic study of erythropoiesis of patients with congenital dyserythropoietic anemia. Blood 27:299-310. 12. Hiraoka A, Kanayama Y, Yonezawa T, et al.: 1983, Congenital dyserythropoietic anemia type I: a freeze fracture and thin section electron microscopic study. Blut 46:329-338. 13. Holman HH: 1956, Changes associated with age in the blood picture of calves. Br Vet J 112:91-104. 14. Kobylka D, Khettry A, Shin BC, Carraway KL: 1972, Proteins and glycoproteins of the erythrocyte membrane. Arch Biochem Biophys 148:475-487. 15. Luna LG: 1968, Manual of histologic staining methods of the Armed Forces Institute of Pathology, 3rd ed., pp. 6-9. McGrawHill, New York, NY. 16. Maeda K. Saeed S, Rebuck J, Raymond W: 1980, Type I dys-
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erythropoietic anemia: a 30-year follow-up. Am J Clin Pathology 73:433-438. 17. Mori PG, Fauareto F, Schenone A, et al.: 1986, Congenital dyserythropoietic anemia type I: report in a pair of siblings. Acta Haematol (Basel) 75:219-223. 18. Schalm OW: 1972, Differential diagnosis of anemia in cattle. J Am Vet Med Assoc 161:1269-1275. 19. Schultz WJ: 1989, A comparison of commercial kit methods for assay of vitamin B12 in ruminant blood. Vet Clin Pathol 16: 102-107. 20. Smith JE: 1989, Iron metabolism and its diseases. In: Clinical biochemistry of domestic animals, ed. Kaneko JJ, 4th ed., pp. 256-273. Academic Press, San Diego, CA. 2 1. Smith JE, Moore K, Schoneweis D: 1981, Coulometric technique for iron determination. Am J Vet Res 42:1084-1085. 22. Steffen DJ, Leipold HW, Gibb J, Smith JE: 1991, Congenital anemia, dyskeratosis and progressive alopecia in Polled Hereford calves. Vet Pathol 28:234-240. 23. Towbin H, Staehelin T, Gordon J: 1979, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc Nat1 Acad Sci USA: 76:4350-4354. 24. Ucci G, Riccardi A, Dormer P, et al.: 1985, Proliferation kinetics of bone marrow cells in congenital dyserythropoietic anemia type II. Blut 50:219-224. 25. Vindelov LL, Christensen IJ: 1983, Limits of detection of nuclear DNA abnormalities by flow cytometric DNA analysis: results obtained by set methods of sample storage, staining, and internal standardization. Cytometry 3:332-339. 26. Wickramasinghe SN, Goudsmit R: 1979, Some aspects of the biology of multinucleate and giant erythroblast in a patient with CDA, type III. Br J Haematol 41:484-495. 27. Wickramasinghe SN, Parry TE, Williams C, et al.: 1982, A new case of congenital dyserythropoietic anaemia, type III: studies of the cell cycle distribution and ultrastructure of erythroblasts and of nucleic acid synthesis in marrow cells. J Clin Pathol 35: 1103-1109. 28. Wickramasinghe SN, Pippard MJ: 1986, Studies of erythroblast function in congenital dyserythropoietic anaemia, type I: evidence of impaired DNA, RNA, and protein synthesis and unbalanced globin chain synthesis in ultrastructurally abnormal cells. J Clin Pathol 39:881-890.
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Congenital dyserythropoiesis and progressive alopecia in Polled Hereford calves: hematologic, biochemical, bone marrow cytologic, electrophoretic, and flow cytometric findings D. J. Steffen, G. S. Elliott, H. W. Leipold, J. E. Smith Abstract. Congenital dyserythropoiesis with dyskeratosis is a slow, progressive, and often fatal disease in Polled Hereford calves. Affected calves have a macrocytic normochromic anemia with a mild reticulocytosis. Studies indicate that calves are hyperferremic with increased saturation of serum total iron binding capacity, which rules out iron deficiency as a cause. Other secondary causes of dyserythropoiesis, including cobalamin and folate deficiencies, are unlikely because serum cobalamin and folate levels of affected calves were normal. Virus isolation was negative, and failure to identify bovine retroviral antigens or antibodies from several calves suggested that viral agents were not involved. Bone marrow cytologic findings were similar to those in congenital hereditary dyserythropoiesis in humans and included occasional multinucleate cells, internuclear chromatin bridging between nuclei of partially divided cells, and, more frequently, irregular nuclear shapes and chromatin patterns. DNA content and cell cycle distribution of erythroid cells appeared normal, and no electrophoretic abnormalities were detected in erythrocyte membrane proteins. The Polled Hereford syndrome is similar in many ways to type I congenital dyserythropoiesis in humans and may be an appropriate biomedical model for studying erythroid proliferation during dyserythropoiesis. phorusb and Sorbitol dehydrogenase were determined.c Serum levels of cobalamin and folate were bioassayed.d The serum iron and total iron binding capacity were determined, and percent saturation of serum iron binding capacity was calculated.21 Thyroid assays were performed by a commercial laboratory” and included total thyroxine (T4), free T4, total triiodothyronine (T3), and free T3. Thyroid response to thyroid stimulating hormone (TSH) was evaluated by comparing differences in levels of thyroxine pre-TSH and 6 hr post-TSH administration. Each calf received 2.5 IU of Dermathycinf intravenously as a source of TSH for the response test. Bone marrow aspirates and biopsies were taken on the same days as blood samples for hematology. Samples were collected via a 6-in. 12-gauge Jamshidi needle.g Samples were collected from sternebrae with the calves in lateral recumbency, after local anesthesia and surgical scrub of the overlying skin. Marrow was aspirated by 8-10 cc negative pressure with a 12-ml plastic syringe. The pressure was released slowly after approximately 0.5 ml of marrow was collected to prevent excessive dilution by peripheral blood. Samples were mixed with 1 drop of ethylenediaminetetraacetic acid in isotonic saline to prevent coagulation. Marrow particles were placed on glass slides, and smears were made. Biopsies were obtained by redirecting and advancing the Jamshidi needle without the stylet after aspirates were collected. Biopsy cores were placed immediately in chilled Karnovsky’s fixative and fixed overnight.10 Biopsies were decalcified overnight in formic acid-sodium citrate solution then processed routinely, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin, Giemsa, and Pearl’s
A congenital syndrome of anemia and alopecia in Polled Hereford calves characterized by nonregenerative anemia with ineffective erythropoiesis and dermatitis has been described recently.22 Cutaneous lesions are progressive, and hyperkeratotic dermatitis with dyskeratosis of individual cells occurs Within the stratum spinosum and follicular infundibuli. Affected calves are unproductive and often die or must be disposed of prematurely. This paper further describes hematologic, biochemical, and bone marrow findings of affected calves. Materials and methods Ten Polled Hereford calves of similar ages, 5 normal and 5 previously diagnosed with congenital anemia and progressive alopecia, were compared. Calves were housed in barns or dry lots and fed alfalfa hay, grass hay, grain, and a protein supplement. Hemograms, including erythrocyte, leukocyte, reticulocyte, and differential blood counts,a were performed 3 times at monthly intervals. Serum sodium, potassium, chloride, glucose, urea nitrogen, creatinine, alkaline phosphatase, gamma glutamyl transferase, aspartate amino-transferase, total bilirubin, direct bilirubin, total protein, albumin, globulin, calcium, phoso-
From the Departments of Pathology (Steffen, Leipold, Smith) and Laboratory Medicine (Elliott), College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506. Received for publication April 15, 1991. 31
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Steffen et al. Table 1. Hematologic findings from dyserythropoietic and normal Polled Hereford calves.
Prussian blue reaction (PPB)J Cellularity of marrow and iron stores and distribution of cells were assessed. Cytology smears were stained with Wright’s stain and by periodic acid-Schiff (PAS) and PPB reactions.2,5 Five hundred cell differential counts were made from the Wright’s=stained smears. Three-milliliter bone marrow samples from 3 affected and 3 control calves were also collected into 25 units of preservative-free heparin. Single-cell suspensions were obtained by forcing aspirates through a series of progressively smaller needles (21-27 gauge), and rubricytes were isolated by centrifugation in Ficoll-HYPaqueh at 1,000 x g and 4 C for 30 min. Erythroid cells were rinsed in phosphate-buffered saline (PBS) and resuspended at a concentration of 5 x 106 cells/ ml. Cytospin preparations were made from a small amount of this sample to verify the cell type present. Reference cells were prepared from bovine peripheral blood. Ten milliliters of peripheral blood was collected in 25 units of preservative-free heparin. Samples were mixed with ammonium chloride, centrifuged, and resuspended in PBS at a concentration of 1.5 x 106 cells/ml. Two hundred microliters of the marrow cell sample was combined with 100 µ1 of reference leukocytes and 1 ml of chilled propidium iodide and incubated on ice for a minimum of 10 min.i Analyses were performed with flow cytometry to determine ploidy. Cell cycle distribution analyses were done similarly, omitting addition of the reference cell
25
population . Red fluorescence (FL2-R) was used to quantify DNA, and an average of 17,857 events (cells) were counted per sample.j Computerized data analysis was done.k Erythrocyte membrane ghosts were prepared at 4 C from the peripheral blood erythrocytes of 3 affected calves, dams of 2 affected calves, and 6 unrelated cattle. Erythrocytes were lysed in sodium phosphate lysing buffer (10.0 mM, pH 7.4) after several 0.9% saline washes and buffy coat removal.6 Diisopropyl fluorophosphate (DIFP)1 was added to the lysing buffer to inhibit protease activity. After lysis, membranes were washed repeatedly in sodium phosphate buffer to remove hemoglobin and other cytoplasmic proteins. Polyacrylamide gel electrophoresis was performed in the presence of sodium dodecyl sulfate (SDS) and 7.5% acrylamide, and the gels were stained with Coomassie blue to demonstrate membrane proteins.8 Proteins also were electrophoresed as above then blotted onto Immobilin-P transfer membranes.m,23 Proteins on the Immobilin-P membranes were demonstrated using Ponceau-S solution and glycoproteins were demonstrated by using a commercial glycan detection kit for detecting glycoconjugates with oxidizable OH groups.n Virus isolation was attempted from buffy coat preparations from 5 calves. Preparations were passed blindly on embryonic bovine kidney cells 3 times, and observations were made for cytopathic effects. After 3 passes, cultures were examined by fluorescent antibody techniques with bovine viral diarrhea and Parainfluenza-3 (P13) conjugates. Serum samples from
Table 2. Serum chemistry and enzyme levels of dyserythropoietic and normal Polled Hereford calves.
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Table 3. Iron, cobalamin, and folate status of dyserythropoietic and normal Polled Hereford calves.
5 calves also were used to attempt detection of bovine retroviral infection.o Agar gel immunodiffusion tests were used for checking bovine leukemia virus and bovine syncytium virus antigens, and bovine immunodeficiency-like virus antigen detection was attempted by western blotting technique. Statistical analysis was done with a statistical software packagep using proc ANOVA for analysis of variance and animals nested within group (affected or control) as the error term when analyses were done several times at monthly intervals. Probabilities of 90% erythroid cells with little contamination by granulocytes. The DNA index was not different between affected and unaffected calves = 1.15). Cell cycle distribution was also similar between the 2 groups (Table 7). No electrophoretic abnormalities of erythrocyte membrane proteins (gels stained with Coomassie blue, Ponceau-S) or glycoproteins (membranes stained with glycan detection kit) were detected. Samples from all calves had 9 intensely staining protein bands detectable with Coomassie blue stains (Fig. 5) similar to previous reports of normal bovine erythrocytes.14 Several protein bands of lesser intensity were consistently present in affected and control calves. All proteins from unaffected and affected calves had identical migration patterns. Table 6.
Figure 1. Bone marrow cytologic features of dyserythropoietic Polled Hereford calf. Notice erythroid dominance in this preparation. Wright’s stain.
Glycan detection demonstrated an intense staining reaction in samples from several calves near the top of the gel that corresponded to the major glycoprotein previously described in cattle.14 Identical patterns were present in normal and affected calves. Intensity of staining in the band varied among individual calves, independent of the presence or absence of disease. No evidence of bovine retroviral infection was found. Virus isolation attempts were negative in all but 1 calf. In 1 instance, cell cultures were positive when stained with fluorescent antibody against PI3 viral antigen. Discussion The macrocytic, poorly regenerative anemia observed was similar to that previously described for affected calves. 22 Macrocytosis was significant, when cell size was compared with that of age-matched calves. An increase in erythrocyte size as calves age has been demonstrated,13,18 so caution is warranted when comparing data with normal laboratory values based on adults. The reticulocytosis, although statistically significant, was mild considering the severity of the anemia. Biologically significant differences have not been found for most serum analytes. Serum proteins, which were low in the previous report,22 were not significantly
Quantitative bone marrow cytologic findings in dyserythropoietic and normal Polled Hereford calves.
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Congenital dyserythropoiesis and alopecia in calves
Figure 2. Bone marrow cytologic features of dyserythropoietic Polled Hereford calf. Note multinucleate rubricyte. Wright’s stain.
Figure 4. Higher magnification of cellular features of Fig. 3. Note internuclear chromatin bridging (arrow). Wright’s stain.
lower in these calves. Serum iron was significantly increased in affected calves, increasing the saturation of serum iron binding capacity, which remained norma1.20 The macrocytic, normochromic nature of the anemia and the elevated serum iron suggest that deficiencies in iron uptake and utilization are not occurring. Hyperferremia is common in children affected with congenital dyserythropoiesis and often leads to hemochromatosis .3,7 Increased iron is absorbed during anemic states and is not adequately excreted, even though serum iron may be elevated.20 The significance of decreased serum thyroxine in affected calves remains unclear. However, results of
TSH response indicated that primary thyroid function remained intact, so the mild hypothyroidism must be secondary to other processes. Two calves were treated with thyroid hormone replacementq starting at 10 mg/ kg daily and adjusted to maintain pretreatment thyroxine levels near normal. No improvement was noted in the severity of anemia or skin condition after 12 weeks, and therapy was discontinued. Bone marrow evaluation demonstrated marked erythroid predominance with numerous mitotic figures. That pattern is consistent with ineffective erythropoiesis. Significant morphologic abnormalities justify classifying the anemia as dyserythropoietic. Many abnormalities (nuclear shape changes, multinuclearity, nuclear cytoplasmic asynchrony, maturation arrest, and internuclear chromatin bridging) resemble those of congenital dyserythropoietic anemia type I (CDA I) in humans.4,11,12,16,17 Unsuccessful attempts to identify several secondary causes of dyserythropoiesis, including iron deficiency, B vitamin deficiency, and viral infection, suggest that it may be a primary congenital dyserythropoiesis. Few DNA ploidy and cell cycle distribution studies of CDA erythroblasts in humans have been done. Those completed demonstrate marked abTable 7. Cell cycle distribution (%) of erythroid cells from dyserythropoietic and normal Polled Hereford calves.
Figure 3. Bone marrow cytology from dyserythropoietic Polled Hereford calf. Note mitotic figures (arrowheads), irregular chromatin (small arrows), and internuclear chromatin bridging (large arrows). Wright’s stain.
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Steffen et al.
Figure 5. Coomassie blue stained erythrocyte membrane proteins in a polyacrylamide gel. Lane 1-high molecular weight marker; lane Z-proteins from unrelated heifer of different breed; lane 3proteins from dyserythropoietic Polled Hereford calf; lane 4-proteins from dam of affected Polled Hereford calf; lane 5-proteins from second affected Polled Hereford calf; lane 6-proteins from unrelated Polled Hereford cow, lane 7-low molecular weight marker.
normalities in the DNA content of erythroblasts in type III CDA and less dramatic changes in erythroblasts with CDA types I and II. 24,26-28 Changes usually have been associated with binucleate and multinucleate cells. The absence of significant changes in rubricyte DNA content in our calves might have been related to the lower number of binucleate and multinucleate cells in the samples and the probable loss of many binucleate cells when we attempted to gate off doublets during flow cytometric analysis. In only 2 cases of CDA type I in humans has cell cycle analysis been reported, both in the same study, and DNA content of a combined total of only 233 cells was reported.28 Methods used in human cases allowed correlation of DNA quantity and cell morphology and included all binucleate or multinucleate cells in the analysis. More studies in humans and calves using comparable techniques of cell cycle analysis are needed before a final evaluation of dyserythropoiesis in calves as a model for CDA type I in humans can be made. The normal cellular DNA content and cell cycle distribution suggest that nuclear morphologic abnormalities are occurring after cytoplasmic division and may represent abnormal completion of cytokinesis and inhibited postmitotic nuclear membrane fusion. Electrophoresis of peripheral blood erythrocyte membrane proteins failed to demonstrate differences
in protein migration or glycosylation between affected and unaffected calves. This result suggests that major defects in erythrocyte membrane protein structure or glycosylation are not present, a finding similar to most cases of CDA I of humans and distinct from CDA II erythrocytes, in which defects of membrane glycosylation have been reported.1,9 Although no mechanism to explain the dyserythropoiesis could be determined, morphologic evidence suggests rapid cell proliferation with an extended maturation time for affected erythroid cells. Nuclear shape and chromatin abnormalities and internuclear chromatin bridging suggest impaired cytokinesis during terminal cell divisions within the erythroid series and prolonged duration of cells within the marrow. We conclude that additional studies of dyserythropoiesis in Polled Hereford calves are needed to further clarify this recently described syndrome. Initial hematologic studies demonstrated many similarities to human CDA type I, making dyserythropoiesis in calves a possible biomedical model for studying other dyserythropoietic anemias. Acknowledgement This research was part of the Regional Project NC-2. It is contribution no. 91-451-J from the Kansas Agriculture Experiment Station and was supported by grant no. HLO 1877 from the National Institutes of Health.
Sources and manufacturers a. Coulter sPlus IV, Coulter Electronics, Hialeah, FL. b. Dacos, Coulter Corporate Communications, Hialeah, FL. c. Abbot VP Biochromatic Analyzer, Abbot Laboratory, Dallas, TX. d. Vitamin Diagnostics, Driftwood Beach, CA. e. Endocrine Laboratory, Michigan State University, East Lansing, MI. f. Coopers Animal Health, Kansas City, MO. g. Baxter Pharmaceuticals, St. Louis, MO. h. Pharmacia, Uppsala, Sweden. i. DNA IC particles kit, Benton Dickenson, Mountain View, CA. j. FAC Scan Analyzer, Benton Dickenson, Mountain View, CA. k. Cell Fit version 1.2, Benton Dickenson, Mountain View, CA. 1. Sigma Chemical Co., St. Louis, MO. m. Millipore Corp., Bedford, MA. n. Boehringer Mannheim Biochemicals, Indianapolis, IN. o. National Animal Disease Center, Ames, IA. p. SAS Institute, Car-y, NC. q. Thyro-L, Vet-A-Mix, Shenandoah, IA.
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