Ultrastructural characteristics of the blood cells of chickens commonly reared under backyard poultry farming in Mizoram,India

The present study was undertaken to characterize the ultrastructural morphology of the blood cells of commonly reared chickens in the state of Mizoram, India under backyard poultry farming. For this study, 2 ml of whole blood was aseptically collected from the wings veins of 12 chickens of three different breeds namely the Zoar, Aseel and Rhode Island Red and processed for ultrastructural imaging under standard protocols. Under scanning electron microscopy (SEM) the matured erythrocytes of Zoar, Aseel and Rhode Island Red appeared elliptical in shape while the leukocytes and thrombocytes appeared round in shape with variable surface modifications. Under transmission electron microscopy (TEM) the granules of the heterophils of Zoar, Aseel and Rhode Island Red appeared predominantly fusiform in shape, the granules of the eosinophils appeared round in shape and that of the basophils appeared pleomorphic in shape. The cytoplasm of the monocytes, medium-to-large lymphocytes and thrombocytes of Zoar, Aseel and Rhode Island Red under TEM appeared to be vacuolated and granular while that of the small lymphocytes appeared to be granular but non-vacuolated. The study concluded that the ultrastructural characteristics of the blood cells of the three breeds of chicken studied were almost similar to the blood cells of other birds reported earlier.     

Morphology of blood cells in carp (Cyprinus carpio L.)

The morphology of blood cells in the carp was investigated by light and electron microscopy. Erythrocytes, thrombocytes, lymphocytes, granulocytes and monocytes were identified as the peripheral blood cells. Thrombocytes were round to long oval, each containing vesicular and microtubular structures and an oval nucleus with abundant heterochromatins. Lymphocytes were divided into three types in size, small, medium and large. Some of the small and medium lymphocytes were alpha-naphthyl-acetate esterase (ANAE) positive, while large lymphocytes were pyroninophilic. Granulocytes were distinguished into three types (type I, type II and type III) according to the morphology of the nucleus and granules. Type I granulocytes possessed lobulated nuclei and a large number of cytoplasmic granules, each of which was oval and contained electron-dense materials and a crystalloid. Type II granulocytes had small eccentric nuclei and were subdivided into IIa and IIb granulocytes by electron microscopic analysis. Granules of type IIa granulocytes were furnished with an electron-dense rim. Granules of type IIb granulocytes were larger than those of type IIa, containing randomly distributed electron-dense and electron-lucent materials. Type III granulocytes possessed round nuclei and a few large granules. The granules were filled with regularly arranged fibriform materials and some needle-like structures. Monocytes were morphologically similar to those of mammals.     

Ultrastructural characteristics of blood cells of juvenile loggerhead sea turtles (Caretta caretta)

Ultrastructural characteristics of erythrocytes, heterophils, eosinophils, lymphocytes, monocytes and thrombocytes of the loggerhead sea turtle (Caretta caretta) were evaluated, using blood samples from 15 healthy juvenile animals. Except for the eosinophils, the rest of the white blood cells from loggerhead turtles had similar ultrastructural characteristics compared with blood cells from other sea turtle species. Eosinophils from loggerhead turtles were homogeneous in size, and no crystalline structures were observed within the granules. This paper provides an ultrastructural characterization of blood cells of loggerhead sea turtles, as a reference for future haematological studies of this species.     

Cytochemical staining characteristics of chicken heterophils and eosinophils

Cytochemistry was used to differentiate chicken heterophils and eosinophils in blood smears and cytospin preparations of isolated leukocytes. Cytochemical staining also was performed to evaluate any alteration of cell staining characteristics after heterophils had been isolated using discontinuous Ficoll-Hypaque gradients. Cytochemical staining reactions of heterophils and eosinophils were the same before and after discontinuous gradient cell separation. Characteristic positive reactions did not occur when chicken heterophils were stained with alkaline phosphatase, peroxidase, Sudan black B, acid phosphatase, alpha-naphthyl acetate esterase, naphthol AS-D chloroacetate esterase, periodic acid-Schiff, or Luna's eosinophil granule techniques. Chicken eosinophils stained positively when peroxidase, Sudan black B, acid phosphatase, and Luna methods were performed. In highly cellular cytospin preparations, peroxidase reactions readily distinguished the few contaminating eosinophils (< 1%) from the heterophils.     

Heterophilic leucocytes are the predominant granulocytes in the ovary and ultimobranchial glands of the adult fowl

Pieces of ovary and ultimobranchial gland from adult laying hens were examined by an indirect peroxidase technique which differentiated tissue eosinophil and heterophil leucocytes. The results showed that in both organs, brown-staining peroxidase-positive eosinophils were greatly out-numbered by pink- to red-staining heterophils, thus demonstrating that eosinophils are not the dominant granulocyte present, as has been widely reported. It is suggested that the heterophils may be required in a phagocytic role.     

A new method for the identification and enumeration of chicken heterophils and eosinophils

A method for the direct counting of chicken heterophils and eosinophils using Phloxine B stain and a hemacytometer is described. A solution of 0.1% Phloxine B in 50% propylene glycol stained heterophils and eosinophils obtained from peripheral blood. The cytoplasm of heterophils became completely stained, whereas that of the eosinophils stained incompletely, thereby enabling these cells to be distinguished easily from each other. Identification of cell type was confirmed by comparing cell counts prepared from blood smears stained with Wright's stain with wet mounts (hemacytometer preparations) stained with Phloxine B.     

鸡骨髓内嗜酸性粒细胞发生的形态特点

对雏鸡骨髓内嗜酸性粒细胞发生的形态结构进行了光镜和电镜观察。嗜酸性颗粒从早幼阶段开始出现，随着细胞的不断发育，颗粒的数量逐渐增多。嗜酸性颗粒呈圆形，大小相近，着桔红色，内部结构均匀，电子密度中等，胞核轮廓清楚，异染色质较少。     

Separation of turkey heterophils from blood using two-step Ficoll-Hypaque discontinuous gradients

A method is presented to separate turkey heterophils from anticoagulated whole blood using two-step Ficoll-Hypaque discontinuous gradients and ammonium chloride lysis of contaminating erythrocytes. Heterophils can be isolated from multiple blood samples within 3 to 4 hours. Using this technique, 66.4 +/- 18.4% (mean +/- standard deviation) of blood heterophils were harvested. Final cell isolates averaged 96.0 +/- 2.9% heterophils with few contaminating eosinophils (2.5 +/- 2.3%) or basophils (1.6 +/- 1.8%). Cell viability, as determined by trypan blue dye exclusion, was 98.0 +/- 1.4%.     

Separation of avian heterophils from blood using Ficoll-Hypaque discontinuous gradients

Rapid separation of avian heterophils from anticoagulated whole blood was achieved using Ficoll-Hypaque discontinuous gradients. An average of 14.4% of blood heterophils was harvested with a mean purity exceeding 99%. Heterophil viability, as determined by trypan blue dye exclusion, averaged 99.8%. The integrity of isolated heterophils was evaluated by cytochemical staining and ultrastructural examination. Cytochemical staining reactions of heterophils in whole blood and of isolated cell suspensions were similar. No ultrastructural abnormalities were observed. Using this procedure, viable intact heterophils were rapidly isolated from blood with an acceptable cell yield and purity for cell function studies.     

Differential leukocyte counts determined in chicken blood using the Cell-Dyn 3500

Background: Automated hematology instruments commonly are used for mammalian blood analysis, but there is a lack of accurate automated methods available for avian leukocyte analysis. Objective: The aim of this study was to validate differential leukocyte counts in blood from chickens using the Cell-Dyn 3500 hematology system and avian-specific software.
Methods: Blood samples were collected in lithium-heparin tubes from 2 groups (n = 84 and n = 139) of laying hens. Manual 200-cell differential counts were done on routinely-stained blood smears, and manual total granulocyte counts (heterophils and eosinophils) were done using an eosinophil stain in a counting chamber. Automated differential counts were done using VET 2.3, a research and development version of avian-specific software for the Cell-Dyn 3500. Results were analyzed using Pearson's correlation and difference plots.
Results: Automated granulocyte counts from the Cell-Dyn were in good agreement with manual granulocyte counts ( r = 0.93 and 0.80 for the 2 study groups). No correlation was found between automated and manual lymphocyte counts. Correlation coefficients for monocyte counts were 0.70 and 0.43. Conclusion: Automated leukocyte results from the Cell-Dyn using VET 2.3 software were not fully accurate. Total granulocyte counts may be of clinical usefulness, but results obtained for other parameters were unreliable.     

南极企鹅外周血细胞初步研究

2012年8—10月,在天津海昌极地海洋世界采用Wright—Giemsa’s染色和Natt—Herrick’s染色方法研究了南极企鹅（Pygoscelisantarcticus）外周血细胞。研究表明,外周血液分类为：红细胞、白细胞和血栓细胞。其中自细胞包括：异嗜性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、淋巴细胞和单核细胞。测得红细胞平均大小（长径短径）为15．32μm×7．83μm,异嗜性粒细胞直径为（11．98±0．97）μLm,嗜酸性粒细胞直径为（11．61±0．78）μm,嗜碱性粒细胞直径为（11．87±0．69）μm,淋巴细胞直径为（9．93±1．29）Ixm,单核细胞直径为（15．04±0．93）μm,血栓细胞直径为（6．96±0．79）μm。统计红细胞总数为（1．85±0．17）×1012／L,白细胞总数为（6．37±1．68）×109／L。     

Sub-cellular localisation of alkaline phosphatase activity in the cytoplasm of tammar wallaby (Macropus eugenii) neutrophils and eosinophils

Alkaline phosphatase (ALP) has been used in studies of neutrophil morphology and function as a marker for identifying different granule populations. In human neutrophils, ALP is found within secretory vesicles, a rapidly mobilisable vesicle population important for upregulating membrane receptors during early activation. Intra-cellular ALP activity in the heterophils of rabbits and guinea pigs, in contrast, is found only in secondary granules. The neutrophils and eosinophils of tammar wallabies (Macropus eugenii) have previously been reported to contain large amounts of ALP activity when stained using routine cytochemical techniques. To define the subcellular location of ALP in this species, cell suspensions were examined using cerium chloride cytochemistry and transmission electron microscopy (TEM). ALP was found in 2 distinct cytoplasmic compartments. One compartment displayed morphology consistent with a subpopulation of secondary granules while a second tubulo-vesicular population appeared similar to the secretory vesicles of human neutrophils. Thin tubular vesicles containing ALP were also identified within the cytoplasm of tammar wallaby eosinophils. Large numbers of ALP-containing vesicles have not been recognised previously in eosinophils and this may represent a novel cytoplasmic compartment. In both cell types, ALP-containing structures showed alteration in morphology following stimulation with N-formyl-Met-Leu-Phe (fMLP) and PMA.     

Fine structural and cytochemical studies of eosinophils from fowls and ducks with eosinophilia

The ultrastructure and cytochemistry of eosinophils from adult fowl and ducks with either spontaneous or experimentally induced eosinophilia were examined. The results showed that a high proportion of the eosinophils in the peripheral blood of eosinophilic birds had ultrastructural features different from those of normal eosinophils. In both species, there was a reduction in cell size. Fowl eosinophil granules showed similar morphological changes to those seen in the quail with many crescentic and vacuolated forms being present. In eosinophilic ducks, the crystalline interna of the specific granules were often fragmented or were either partially or completely lysed. Cytochemically, peroxidase activity in both species was generally unaltered in abnormal eosinophils compared with those from normal birds. This correlates with the findings in man. However, amounts of acid phosphatase and trimetaphosphatase were reduced in many cells, with a large proportion of granules being non-reactive. The latter observation corresponds with that in quail but differs from man, in which stimulated eosinophils have increased enzymatic activity.     

Plasma histamine concentration and histamine detection in peripheral blood eosinophils in cats

Plasma histamine levels were measured in 11 clinically healthy cats and 15 cats with allergic dermatitis. Histamine levels were markedly elevated in 5/15 allergic cats. A calcium ionophore, A23187, stimulates histamine release from feline peripheral blood cells. Immunostaining of blood smears from clinically healthy cats revealed that approximately 10% of eosinophils possessed histamine-containing granules. These results indicate that some peripheral eosinophils in cats contain histamine and can release histamine by appropriate stimulation.     

Hematology of the West Indian Manatee (Trichechus manatus)

Hemograms on blood obtained from 10 clinically normal West Indian Manatees (Trichechus manatus) were studied. The red cells were large and in lower number than in most terrestrial species. The manatee does not have a neutrophil as is present in most species, but it has a heterophil whose granules stain pink with the Wright-Giemsa stain. The eosinophil has uniform red-staining granules that make its differentiation from heterophils difficult. Both large and small lymphocytes, monocytes and basophils are present. The total number of white blood cells was comparable to that of common domestic species as were the platelet numbers. No reticulocytes were found.     

Morphologic, cytochemical staining, and ultrastructural characteristics of blood cells from eastern diamondback rattlesnakes (Crotalus adamanteus)

OBJECTIVE: To evaluate light microscopic, cytochemical, and ultrastructural characteristics of blood cells from eastern diamondback rattlesnakes. ANIMALS: 10 healthy snakes. PROCEDURE: Various stains, including Wright-Giemsa, benzidine peroxidase, Sudan black B, chloroacetate esterase, alpha-naphthyl butyrate esterase, acid phosphatase, leukocyte alkaline phosphatase, periodic acid-Schiff with diastase, and toluidine blue, were used to stain leukocytes differentially on multiple blood smears. Electron microscopy also was performed. RESULTS: Lymphocytes were the most commonly observed leukocyte and could be distinguished from thrombocytes, using periodic acid-Schiff stain with diastase. Azurophils also were commonly observed; their granules stained with peroxidase. Eosinophils were not identified; however, 2 morphologic variations of heterophils were seen in the blood of all snakes and were considered the same cell type at different stages of cytoplasmic granule development. Heterophil granules were better preserved, using a one-step Wright-Giemsa method that did not require alcohol fixation prior to staining. Degranulated heterophils were observed in all preparations. CONCLUSIONS: Most leukocytes of eastern diamondback rattlesnakes can be identified easily on Wright-Giemsa-stained preparations. However, hematologic stains that do not require alcohol fixing prior to staining may be preferred for leukocyte evaluation in certain reptiles. A limited degree of heterophil maturation may continue in the blood of healthy snakes. This, along with degranulation of heterophils, may result in a variable staining pattern in this cell type, regardless of the stain used. CLINICAL RELEVANCE: Results provide baseline data for use in hematologic testing in diagnosis of disease and monitoring of treatment of sick or injured snakes.     

The ultrastructural morphology of the camel eosinophil

The ultrastructural morphology of the eosinophil was studied in specimens of peripheral blood from normal adult camels and those with eosinophilia. Specific granules were extremely polymorphic. The specific granules exhibited the basic structure of an electron-dense crystalloid core surrounded by a lighter, homogeneous matrix. The crystalloid cores were extremely variable in size and shape, often were segmented and demonstrated a variety of lamellated patterns that were transverse, longitudinal or concentric to the long axis of the core. It was not uncommon to observe multiple crystalloid cores in a single granule. In addition to large specific granules, microgranules and specific microgranules were observed. The extreme polymorphism of the specific granules and variety of lamellated patterns differentiate camel eosinophils from those of other species.     

Grey eosinophils in a Miniature Schnauzer with a poorly differentiated mast cell tumor

A 10‐year‐old female spayed Miniature Schnauzer was presented for investigation of an intra‐nasal mass. The mass was diagnosed by histopathologic examination as an undifferentiated round cell neoplasm with an infiltrate of segmented leukocytes, interpreted as neutrophilic inflammation. The mass was treated with palliative radiation and systemic chemotherapy due to the presence of regional lymph node metastasis. During subsequent monitoring over several months, the peripheral leukocyte concentration was repeatedly within reference intervals to slightly increased with low numbers of toxic neutrophils. Four months after the initial diagnosis, there was a significant leukocytosis of 66 100 cells/μL, and 39 700 cells/μL of the leukocytes had variably mature, lobulated, and hypolobulated nuclei, and grey cytoplasm with clear vacuoles, resembling grey eosinophils. To further characterize these cells, peripheral blood smears from the patient and a canine control with eosinophilia were stained for alkaline phosphatase (AP), peroxidase, and esterase activities, and with Luxol fast blue (LFB). Histopathologic sections of the nasal mass were stained with LFB and immunohistochemically for tryptase. On blood smears, the cytoplasm of the suspected grey eosinophils stained for AP and granules stained with LFB confirmed that there was an eosinophilic lineage. Peroxidase staining was weak, and esterase staining was absent. On histopathologic sections from the nasal mass, the segmented leukocytes contained LFB‐staining granules, indicating an eosinophilic infiltrate was present. Neoplastic cells expressed tryptase, which confirms a mast cell lineage. Our findings suggest that grey eosinophils might be under‐recognized and interpreted incorrectly as toxic neutrophils. This report expands the canine breeds in which these eosinophils have been identified.     

Light microscopic and ultrastructural characterization of cells recovered by respiratory-tract lavage of 2- and 6-week-old chickens

This study characterized the cell population recovered by respiratory-tract lavage of 57 two-week-old and 59 six-week-old specific-pathogen-free chickens as a prerequisite to study the response of the avian respiratory tract to infectious agents. The respiratory tract of each bird was lavaged through the trachea with a series of three lavages of 10 ml of room-temperature, neutral phosphate-buffered saline per lavage. The three lavages per bird were pooled for analysis. Total recovery volumes were measured, lavage fluid cellularity was determined, and a 200-cell differential count of non-erythrocyte cells was performed. Lavage fluid recovery was greater from 2-week-old birds (91.3 percent) than from 6-week-old birds (86.3 percent). Total cells recovered were greater for 6-week-old chickens (6.79 x 10(5)) than for 2-week-old chickens (5.03 x 10(5)). Cells of epithelial origin included squamous cells, goblet cells, and both ciliated and non-ciliated columnar epithelial cells. Cells of non-epithelial origin consisted of heterophils, lymphocytes, macrophages, eosinophils, basophils, and erythrocytes. Cells of epithelial origin were the predominant cell type recovered from the 2-week-old chickens, followed by heterophils. In 6-week-old chickens, heterophils were the predominant cell type recovered, followed by cells of epithelial origin. In descending order of prevalence, the remainder of cell types recovered from chickens of both ages were lymphocytes, macrophages, eosinophils, and basophils.     

Morphological Observations on the Erythrocytes, Leukocytes and Thrombocytes of Blue Tongue Lizards (Lacertilia: Scincidae, Tiliqua)

Blood cell appearances and sizes varied little between T. scincoides and T. rugosa. Both mature and immature erythrocytes were present in circulating blood. They were oval and, ultrastructurally, had prominent marginal microtubules. Thrombocytes were oval and often showed vacuolation. On TEM examination they had parallel arrays of microtubules and scattered dense granules. Lymphocytes were small and sometimes difficult to distinguish from thrombocytes. Ultrastructurally, they had few organelles in their cytoplasm. Monocytes were commonly packed with fine azurophilic granules which, ultrastructurally, corresponded to variably-shaped dense granules. Type I granulocytes had eccentric, irregularly shaped nuclei, and cytoplasm which contained many orange-brown oval to round granules. Ultrastructurally, the granules were membrane bound, dense, and round to elongate oval. Type II granulocytes had eccentric, oval to round nuclei and numerous purple red, round to irregularly oval granules. On TEM examination the granules were commonly large, light grey and reticulate, and often contained a variably sized fibrillar rod. Type III granulocytes had a centrally located nucleus and numerous round, purple blue granules. The granules were round and homogeneously dense at the ultrastructural level.