Med. Weter. 2013, 69 (11) 674
Praca oryginalna Original paper
In cattle and other animal species, the production and differentiation of leukocytic cell lines takes place in the bone marrow. Pluripotent stem cells develop into colony-forming progenitor cells (CFU). Granulocytic and monocytic lines are produced from a shared progenitor cell (CFU-GM) which gives rise to cells that form granulocyte colonies (CFU-G), neutrophils, and macrophage colonies (CFU-M). Eosinophils and basophils evolve from a different type of progenitors (CFU-Eos and CFU-Baso). During hematopoiesis, leukocytes evolve through the following developmen-tal stages: neutrophilic, eosinophilic and basophilic granulopoiesis, monocytopoiesis, lymphopoiesis, and megakaryopoiesis. The results of hematological and biological tests performed prior to the study were within the reference values for the species. The aim of our study was to present the maturation and differ-entiation of white blood cells in the bone marrow of healthy Holstein-Friesian cattle.
Material and methods
The experimental material comprised 10 high-yielding, clinically healthy Holstein-Friesian cows aged 2-3 years. To rule out bone marrow diseases or dysfunctions, blood morphology and biochemical tests were performed by determining the enzymatic activity of aspartate transami-nase, alkaline phosphatase, lactate dehydrogetransami-nase, creatine kinease, as well as total protein and iron concentrations. Bone marrow aspirate smears were analyzed. Marrow samples were collected with a marrow biopsy needle with a length of 63 mm, 13G, from the medullary cavity of the
third and fourth ribs in the sternal region, and the material was subjected to a smear test (16). Prior to biopsy, hema-tological analyses were performed with the use of whole blood sampled from the caudal vein and stored in test-tubes with K2EDTA as the anticoagulant. Serum was separated by centrifuging whole blood samples. It was stored in test- -tubes with a coagulation activator and used in biochemical analyses. Marrow and peripheral blood smears were stained by the method proposed by May Grünwald-Giemsa (MGG). Marrow smears were stained for 80 seconds in line with the May-Grünwald protocol, and for 5 minutes according to the Giemsa protocol. Peripheral blood staining times were 3 and 12 minutes, respectively. The Giemsa stain was diluted with a phosphate buffer, pH 7.2, at a ratio of 1:10. The stained specimens were analyzed by immersion micro- scopy with 1000 × magnification. Peripheral blood smears were evaluated in a ADVIA 2120i hematology analyzer that determines standard morphological parameters (CBC) and reticulocyte parameters, as well as performs automatic smear analyses (6 DIFF). Biochemical tests were carried out in an Accent-200 biochemical analyzer. The objective of this study was to determine the activity and effective-ness of bone marrow hematopoiesis in healthy, adult cattle, and to compare hematopoietic activity levels in sick cattle.
Results and discussion
Granulocytic, monocyte-macrophage, and lymphoid development series have to be analyzed in evaluations of the leukocyte system. Examinations of bone mar-row samples collected from healthy HF cows revealed that the total counts of granulocytic series cells were
Assessment of leukocytes in the bone marrow
of healthy Holstein-Friesian cows
ANNA SNARSKA, WIOLETTA KRYSTKIEWICZ, WOJCIECH RĘKAWEK
Department of Internal Diseases, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego 14 St., 11-957 Olsztyn
Snarska A., Krystkiewicz W., Rękawek W.
Assessment of leukocytes in the bone marrow of healthy Holstein-Friesian cows Summary
Similarly to the production of red blood cells, the production and development of leukocytes takes place in the bone marrow in the process of hematopoiesis. The key stages in the development of leukocytic cell lines are: neutrophilic, eosinophilic and basophilic granulopoiesis, monocytopoiesis, lymphopoiesis and megakaryopoiesis. Leukocytes produced at various developmental stages are characterized by differently shaped nuclei, the presence or absence of nucleoli, and variations in cytoplasm staining. The experiment was carried out on 10 clinically healthy HF cows. Bone marrow aspirate smears stained by the MGG method were analyzed. The study found significant differences in the number, size, and staining intensity of individual myeloid cell lines.
Med. Weter. 2013, 69 (11) 675
consistent with the norms for the species. A significant increase in cell counts was noted in every developmen-tal stage of the eosinophilic line. Eosinophilic cells are much larger, and their granules are larger and less intensively stained than in other species. Monocyte- -macrophage series counts were within the norm given by Jain (11). Monocyte cytoplasm is denser and less foamy than in other species, for example in dogs, and therefore, it is more intensively stained. The nucleus is large, variously shaped, and it accounts for ⅓ of cytoplasm volume. The lymphocyte system is char-acterized by a much higher number of cells at every developmental stage, as demonstrated by the results of peripheral blood morphology tests.
Neutrophilic granulopoiesis. The myeloblast is the earliest, morphologically identifiable stage in the granulocytary series (12) with a relatively irregular size and shape. Our previous research indicates that myoblasts are larger in cattle than in goats, cats or dogs.
An oval-shaped nucleus generally occupies the most part of the cell, it is adjacent to the cell membrane, and it is stained purple. The cytoplasm is stained blue. The following developmental stage in neutrophilic granu-lopoiesis is the promyelocyte. It is the largest cell in the granulocytary series in both cattle and other animal species (Fig. 1). The nucleus is round or oval shaped with clearly visible nucleoli. Basophilic cytoplasm contains red-stained granules. As the cells develop, the cytoplasm becomes eosinophilic, and a transparent zone appears around the nucleus. The transparent zone becomes visible already in the myelocyte. Myelocytes have large, oblong nuclei with clear incisures and weakly accented nucleoli. Those cells are transformed into metamyelocytes, which resemble myelocytes in shape and size, but their nuclei do not contain nucleoli, they are not surrounded by a transparent zone, and few granules are observed in the cytoplasm (Fig. 2).
Tab. 1. Distribution of granulocytic series cells in the bone marrow (%) No of
animal
MBL PML MYE MET BAND SEG EMYE EMET EBAND EOS BMYE BMET BBAND BAS
% % % % % % % % % % % % % % 1 0.2 0.9 2.8 2.9 4.6 20.2 1.9 2.3 2.9 6.7 0 0 0 0 2 0.1 1.3 2.9 3.6 7.6 20.1 3.7 4.2 4.3 6.6 0.0 0.1 0.5 0.7 3 0.3 1.5 3.3 3.2 7.6 18.7 1.6 2.0 3.8 3.9 0.1 0.2 0.3 0.4 4 0.2 0.8 3.1 4.8 8.2 21.1 2.6 3.2 3.7 4.5 0.0 0.2 0.4 0.2 5 0.1 1.0 3.5 3.9 9.2 19.2 1.5 3.1 3.4 4.1 0.0 0.1 0.2 0.2 6 0.4 1.5 3.6 2.9 8.3 18.3 2.1 2.5 3.0 3.9 0.0 0.0 0.0 0.0 7 0.1 0.7 2.2 3.3 5.0 22.5 1.4 1.9 3.1 3.8 0.0 0.0 0.0 0.0 8 0.3 1.3 2.1 3.3 7.7 19.2 2.5 4.1 5.2 9.0 0.0 0.0 0.0 0.0 9 0.2 0.8 2.9 3.4 7.1 20.5 1.6 2.8 3.6 4.1 0.1 0.1 0.2 0.4 10 0.8 1.8 5.2 6.8 11.2 25.6 2.5 2.6 2.5 3.2 0.0 0.0 0.3 0.2
Explanations: MBL – myeloblast, PML – promyelocyte, MYE – neutrophilic myelocyte, MET – neutrophilic metamyelocyte, BAND – band neutrophil, SEG – neutrophilic granulocyte, EMYE – eosinophilic myelocyte, EMET – eosinophilic metamyelocyte, EBAND – band eosinophil, EOS – eosinophilic granulocyte, BMYE – basophilic myelocyte, BMET – basophilic metamyelocyte, BBAND – band basophile, BAS – basophilic granulocyte
Fig. 1. Lymphocytes, segments and bands, erythroblast, eosinophilic metamyelocyte. May-Grünwald, Giemsa stain.
Med. Weter. 2013, 69 (11) 676
Non-segmented (band) and segmented neutrophilic granulocytes appear at the last stage of neutrophilic granulopoiesis. Band granulocytes have characteristic S-shaped or U-shaped nuclei without constrictions, and they are stained dark blue. The cytoplasm takes on a clear pink hue. This developmental series ends with the segmented neutrophilic granulocyte. In cattle, the nucleus generally contains 3-4 segments, which form various shapes and configurations without becom-ing structurally disintegrated. In pathological states, such as inflammations, the number of segments may increase, and the nucleus may disintegrate. The above is also noted in glucocorticosteroid treatment (15, 19). Similarly to younger cell forms, the nucleus of a segmented neutrophilic granulocyte is stained dark blue. The cytoplasm is eosinophilic, and it takes on a pink or red color. Red-stained granules may appear in the cytoplasm of a neutrophilic granulocyte with a segmented nucleus in healthy animals (25).
Eosinophilic and basophilic granulopoiesis. The production and development of eosinophils and ba-sophils is determined by interleukin-5 (IL-5) (6). The first morphologically identifiable cell in eosinophilic granulopoiesis is the eosinophilic promyelocyte, which is characterized by an oval-shaped, slightly concave nucleus with nucleoli. Its cytoplasm is stained blue, and it has red granules. The next developmental stage is the eosinophilic myelocyte, which has a round shape and a nucleus with visible nucleoli (2). Basophilic granules are not found in the cytoplasm, but an abundance of eosinophilic granules is noted. The nuclei of eosino-philic metamyelocytes and granulocytes are much smaller, often S-shaped or C-shaped, and the cytoplasm contains numerous eosinophilic granules. The oldest cell in this developmental series is the segmented eosinophil. In cattle, the cell nucleus features up to 4 constrictions (26). The cytoplasm is stained inten-sive red with clear eosinophilic granules. Segmented eosinophils are released into the bloodstream, and
they participate in type 1 immune response and type 2 hypersensitivity response (16).
Monocytopoiesis and lymphopoiesis. Lymphocytes and monocytes develop from a shared progenitor cell (CFU-GM). The first morphologically identifiable cell in the monocytic series is the monoblast (8). The lymphoblast is the equivalent of monoblasts in lymphopoiesis. Monoblasts have dark blue nuclei and bluish cytoplasm. They are the largest cells to be released from the bone marrow into peripheral blood. Their nuclei are large, irregular in shape, pink colored, and surrounded by blue cytoplasm. Lymphocytes have small quantities of cytoplasm and large nuclei that fill nearly the entire cell. A physiologically high number of lymphocytes is reported in healthy cattle aged up to 15 months. This group of cells can be subdivided into three main lymphocyte categories: T lymphocytes that develop in the thymus, B lymphocytes that mature in the bone marrow, and NK cells that do not have the features of T or B lymphocytes.
Megakaryopoiesis. The production and maturation of thrombocytes involves a completely different pro-cess than that observed in the cells of other develop-mental series. Megakaryoblasts, promegakaryocytes, and megakaryocytes are produced as a result of DNA endoreplication and intracellular nuclear division. The resulting platelets are not typical blood morphotic ele-ments. They are formed by the splitting of cytoplasm fragments, and they are released from the bone marrow into peripheral blood (3).
The analysis of leukocytes in bone marrow aspirate smears combined with blood morphology tests sup-ports the diagnosis of the progression of inflamma-tions in cattle (20). Bovine inflammainflamma-tions are difficult to diagnose due to physiologically high lymphocyte counts. The above is also clearly demonstrated dur-ing evaluations of bone marrow aspirate smears in stained specimens. Every inflammation mobilizes the body’s immune system which consists of lymphocytes,
Tab. 2. Distribution of monocyte-macrophage series cells in the bone marrow (%)
No of animal MNBL PROMN MON
1 0.1 0.1 1.9 2 0.0 0.0 1.7 3 0.2 0.0 2.0 4 0.4 0.2 1.0 5 0.1 0.1 2.0 6 0.2 0.1 1.8 7 0.1 0.1 1.6 8 0.1 0.1 1.9 9 0.2 0.3 2.3 10 0.3 0.1 1.9
Explanations: MNBL – monoblast, PROMN – promonocyte, MON – monocyte
Tab. 3. Distribution of lymphoid series cells in the bone marrow (%)
No of animal LBL PLYM LYM PLBL PLAZ LC
1 1.0 2.9 8.4 0.0 0.3 0.3 2 2.1 2.2 9.0 0.4 7.1 0.1 3 0.9 2.8 10.8 0.0 3.7 0.1 4 1.5 2.8 11.2 0.0 0.6 0.2 5 2.3 3.0 10.5 0.0 0.2 0.1 6 1.6 1.9 12.5 0.1 0.9 0.1 7 2.2 3.1 11.2 0.1 1.1 0.1 8 2.1 2.9 11.3 0.0 3.4 0.2 9 1.9 2.8 10.6 0.1 1.8 0.1 10 2.3 2.7 109 0.1 2.5 0.1
Explanations: LBL – lymphoblast, PLYM – prolymphocyte, LYM – lymphocyte, PLBL – plasmocytoblast, LC – lymphoid cells
Med. Weter. 2013, 69 (11) 677
monocytes, and granulocytes (4). This results from increased activity levels in the bone marrow and an enhanced release of immature cells, in particular white blood cells, into peripheral blood (17). Whole blood smears reveal mostly neutrophilic granulocytes with non-segmented nuclei, and an increase in the counts of neutrophilic granulocytes with segmented nuclei (7). When the bone marrow is mobilized, immature cells of the granulocytic series, such as myelocytes and meta-myelocytes, are released into peripheral blood. In in-flammations, higher promyelocyte, metamyelocyte and myelocyte counts are observed in bone marrow aspirate smears. The number of lymphocytes and lymphoblasts increases, and elevated promonocyte and monocyte levels are noted in the monocyte-macrophage system. Viral diseases, including BVD/MD, accompanied by a drop in peripheral leukocyte counts, are characterized by a decrease in the number of neutrophilic metamye- locytes (MYE), eosinophilic myelocytes (EMYE) and band eosinophils (EBAND) (14). The above changes lead to peripheral leukopenia and neutropenia (1, 5). Bone marrow analysis pointed to the activation of the entire white blood cell system with a particularly high increase in granulocyte precursor cell counts. Mastitis caused by Streptococcus agalactiae was accompanied by a drop in neutrophil counts in peripheral blood and bone marrow with a simultaneous increase in neutro-phil levels in milk from the infected quarters (9, 13). The M:E ratio (ratio of cells belonging to the line of the myeloid cells to nucleated erythroid), which was below 1.0 in healthy animals, significantly exceeded 1.0 in infected cows. The above suggests that the trans-fer of neutrophils from peripheral blood to infected tissue constitutes a highly effective line of defense against necrotic mastitis. Bone marrow analyses sup-port determinations of pathogenic microorganisms of the genera Leishmania, Histoplasma, Trypanosoma and Mycoplasma (18, 21, 24). Similarly to bacterial infections, the presence of the above pathogens in-creases the number of band neutrophils with basophilic cytoplasm and toxic granulation. The results of bone marrow evaluation reveal the advancement of inflam-mation or bacterial invasion caused by the presence of pathogens, such as Streptococcus agalactiae, in the bone marrow. The leukocyte system is also activated by external pathogens such as Psoroptes ovis. As a re-sult of long-term pathogenic invasions, myelocyte and metamyelocyte counts increase, the M:E ratio exceeds 1.0, and a higher number of neutrophils and eosino-phils is noted. To conclude, the examination of bone marrow leukocytes at different developmental stages supports the diagnosis and reveals the progression of some pathological states in animals (10, 22, 23, 25).
References
1. Ammann V. J., Fecteau G., Helie P., Desnoyers M., Hebert P., Babkine M.: Pancytopenia associated with bone marrow aplasia in a Holstein heifer. Can. Vet. J. 1996, 37, 493-495.
2. Babior B. M., Golde D. W.: Production, distribution and fate of neutrophils. Williams Hematology, New York 1995, 773-779.
3. Burstein A., Breton-Gorius J.: Megakaryopoesis and platelet formation. Williams Hematology, New York 1995, 1149-1161.
4. Burvenich C., Paape M. J., Hill A. W., Guidry A. J., Miller R. H., Heyneman R.,
Kremer W. D. J.: Role of neutrofil leucocyte in the local and systemic reactions
during experimentally induced E. coli mastitis in cows immediately after calving. Vet. Quart. 1994, 16, 45-50.
5. Cullor J. S., Smith W., Zinkl J. G., Dellinger J. D., Boone T.: Hematologic and bone marrow changes after short- and long-term administration of two recombinant bovine granulocyte colony-stimulating factors. Vet. Pathol. 1992, 29, 521-527.
6. Galli S. J., Dvorak A. M.: Production, biochemistry and function of basophils and mast cell. Williams Hematology, New York 1995, 805-810.
7. Guidry A. J., Paape M. J.: Effects of bovine neutrophil maturity on phago- cytosis. Am. J. Vet. Res. 1976, 37, 703-705.
8. Hammer R. F., Weber A. F.: Ultrastructure of agranular leukocytes in peripherial blood of normal cow. Am. J. Vet. Res. 1974, 35, 527-536. 9. Heynemann R., Burvenich C., Vecauteren R.: Interaction between the
respiratory burst activity of neutrophil leukocytes and experimentally induced Escherichia coli mastitic in cows. J. Dairy Sci. 1990, 73, 985-994.
10. Hoedemaker M., Lund L. A., Wagner W. C.: Influence of arachidonic-acid metabolites and steroids on function of bovine polymorphonuclear neutrophils. Vet. Res. 1992, 53, 1534-1539.
11. Jain N. C.: Essentials of Veterinary Hematology. Lea&Febrider, Philadelphia 1993.
12. Jain N. C.: Schalms Veterinary Hematology. Lea&Febiger, Philadelphia 1986. 13. Jolly R. D., Walkley U.: Lysosomal storage diseases of animals: an essay in
comparative pathology. Vet. Pathol. 1998, 34, 527-548.
14. Makoschey B., Lieber-Tenorio E. M., Goovaerts Y. M., Biermann D., Pohlenz
J. F.: Leukopenia and thrombocytopenia in pigs after infections with bovine
viral diarrhoea virus-2 (BVDV-2). Dt. Tierärztl. Wschr. 2002, 109, 225-230. 15. Maryuama, Minagawa M., Shimizu T., Oya H., Yamamoto, Musha N., Abo W.,
Weerasinghe A., Hatakeyama K., Abo T.: Administration of glucocortico-
steroids markedly increases the numbers of granulocytes and extrathymic T cells in the bone marrow. Cell Imunol. 1999, 194, 38-35.
16. Merris V. Van, Meyer E., Dosogne H., Burvenich C.: Separation of bovine bone marrow into maturation-related cell fractions. Vet. Immunol. Immunopathol. 2001, 83, 11-17.
17. Nagahata H., Kehrli M. E., Murata H., Okada H., Noda H., Kociba G. J.: Neutrophil function and pathologic findings in Holstein calves with leukocyte adhesion deficiency. Am. J. Vet. Res. 1994, 55, 40-48.
18. Neimark H., Kocan K. M.: The cell-wall rickettsia. Eperythrozoon wenyonii is a Mycoplasma. FEMS Microbiol. Let. 1997, 156, 287-291.
19. Roth J. A., Kaeberle M. L., Appell L. A., Nachreiner R. F.: Association of increased 17-estradiol and progesterone blood values with altered bovine polymorphonuclear leukocyte function. Am. J. Vet. Res. 1983, 44, 247-253. 20. Schalm O. W., Lasmanis J.: Cytologic features of bone marrow in normal and
mastitic cows. Am. J. Vet. Res. 1976, 37, 359-363.
21. Smith J. A., Thrall M. A., Smith J. L., Salman M. D., Ching V., Collins J. K.: Eperythrozoon wenyonii infection in dairy cattle. J. Am. Vet. Med. Assoc. 1990, 196, 1244-1250.
22. Steffen D. J., Elliott G., Leipold H. W., Smith J. E.: Congenital dyserythtropoesis and progressive alopecia in Polled Hereford calves: hematologic, biochemical, bone marrow cytologic, electrophoretic, and flow cytometric findings. J. Vet. Diagn. Invest. 1992, 4, 31-37.
23. Stockham L., Kjemtrup A. M., Conrad P. A.: Theileriosis in a Missouri beef herd caused by Theileria buffeli: case report, herd investigation, ultrastructure, phylogenetic analysis and experimental transmission. Vet. Pathol. 2000, 37, 11-21.
24. Swenson C., Jacobs R.: Spherocytosis associated with anaplasmosis in two cows. J. Am. Vet. Med. Assoc. 1986, 188, 1061-1063.
25. Waage: Influence of a new infection with bovine virus diarrhoea virus on udder health in Norwegian dairy cows. Prev. Vet. Med. 2000, 43, 123-133. 26. Wardlaw A. J., Kay A. B.: Eosinophils: production, biochemistry and function.
Williams Hematology, New York 1995, 798-805.
Corresponding author: Anna Snarska Ph.D., Department of Internal Diseases, Oczapowskiego 14 St., 11-957 Olsztyn, Poland
