Morphological and Cytochemical Studies of Peripheral Blood Cells of Schizothorax prenanti

The morphological and cytochemical studies of peripheral blood cells of Schizothorax prenanti were studied by light and electron microscopy. Erythrocytes, thrombocytes and three types of leucocytes, lymphocytes, neutrophils and monocytes, were distinguished and characterized. In addition to mature erythrocytes, immature and dividing erythrocytes were observed. A few organelles such as mitochondria were distributed in the cytoplasm of erythrocytes. Lymphocytes with heavily clumped heterochromatic nucleus and minimal cytoplasm were classified into small and large lymphocytes. Three different populations of granules, with distinctive ultrastructural aspect, were observed in neutrophils. Monocytes were the fewest leucocytes possessing rich organelles, phagocytized materials and vacuoles. Thrombocytes with various types were the most abundant blood cells among leucocytes and contained a prominent nucleus with dense bands of heterochromatin and many cytoplasmic vacuoles. Periodic acid‐Schiff staining was positive in neutrophils, monocytes, lymphocytes and thrombocytes, but not in erythrocytes. Peroxidase‐positive staining was observed in neutrophils and monocytes, but not in erythrocytes, lymphocytes and thrombocytes. Only neutrophils were positive for oil red O. Except for erythrocytes, the other blood cells stained positively for acid phosphatase. Only neutrophils and monocytes were positive for α‐naphthyl acetate esterase. None of the cells studied were positive for alkaline phosphatase. The morphologic and cytochemical features of blood cells of S. prenanti are similar to those of other fish. This investigation may be helpful as a tool to monitor the health status of cultured S. prenanti and will grant early detection of clinical pathology.     

Macrophages of the bovine heifer mammary gland: morphological features during initiation and resolution of the inflammatory response

This work characterizes macrophage morphological features during initiation and resolution of an inflammatory response by the bovine mammary gland. The study has been carried out in 20 mammary glands of five virgin heifers by using light microscopy of natural and stained cells and by using transmission electron microscopy (TEM). The inflammatory reaction was induced by an intramammary administration of phosphate-buffered saline (PBS). It has been found that both the initial as well as the resolution phases of the inflammatory reaction are characteristic of the presence of various morphologically different macrophage forms. During the initial phase of the inflammatory response, the major proportion of the macrophage population consisted of monocyte-like macrophages, which represented newly migrated cells. These macrophages were 12-15 mum in size, with spherical or ovoidal shapes, and contained homogenous, fine-granular cytoplasm rich in Golgi complexes, numerous mitochondria, and no lysosomes. The nuclei of the macrophages were kidney-shaped, and surrounded by dark chromatin along the peripheries. Macrophages with phagocytosed apoptotic neutrophils in the cytoplasm were detected already during the initial phase. These macrophages reached the highest proportion 48-72 h after the influx induction and participated in the resolution of the inflammatory reaction. Other cells, also detected during the resolution of the inflammatory reaction, were vacuolized macrophages that formed the largest cells in the lavages of the mammary glands and that were structurally characteristic for the presence of vacuoles in the cytoplasm. In TEM the macrophage vacuoles formed both phagolysosomes with residues of pre-digested material of phagocytosed apoptotic neutrophils and vacuoles that were less electon-dense. Morphologically different forms of macrophages reflected their real-time functions in the inflammation process.     

Ultrastructure of phagocytes from mammary glands of non-pregnant heifers

Results of ultramicroscopic investigations of phagocytes isolated from non-secreting and aberrantly secreting juvenile mammary glands of non-pregnant heifers are presented. The two types of phagocytes observed in cell suspensions obtained by lavage of mammary gland cavities were polymorphonuclear leukocytes and macrophages. Polymorphonuclear leukocytes were spherical or irregular in shape and contained segmented nuclei. Azurophilic and specific electron-dense granules, mitochondria, glycogen particles, phagosomes and phagolysosomes in cytoplasma and characteristic pseudopodia on the cell surface were observed. In addition to these normal polymorphonuclear leukocytes, degenerating cells, characterized by spherical nuclei, total absence of pseudopodia, merged nuclear segments and altered granules, other cellular organelles and plasmalemma were present. Two types of macrophages, i.e. vacuolized and non-vacuolized, could be distinguished. Typical of the non-vacuolized type was a kidney-shaped nucleus, a rich Golgi complex and a large amount of lysosomes in the cytoplasm. The vacuolized macrophages contained a large amount of electron-dense vacuoles in the cytoplasm. Unlike non-secreting glands, the cell suspensions collected from aberrantly secreting juvenile mammary glands contained only vacuolized macrophages. The vacuolization results from phagocytosis of corpuscular particles of aberrant milk plasma.     

Ultrastructure and stereology of leukocytes and platelets of normal foxes and a fox with a Chediak-Higashi-like syndrome

Peripheral blood leukocytes and platelets from five normal foxes (Vulpes vulpes) and a fox with phenotypical characteristics of Chediak-Higashi syndrome (CHS) were examined by electron microscopy. Lymphocytes, monocytes, neutrophils, eosinophils, and platelets from the affected fox contained giant membrane-bound granules that resembled lysosomes. In eosinophils and neutrophils from the affected fox and a normal fox, relative cell volume occupied by granules and number of granules per unit area were calculated. Relative cell volume occupied by granules was the same in both foxes, but there were significantly fewer granules per unit area in the affected fox. This result is consistent with the idea that the giant granules arose from fusion of pre-existing, normal-sized granules, as occurs in CHS. In platelets from the affected fox, no osmiophilic granules were seen. Our findings agree with those from studies of CHS-affected blood cells in other species.     

缺锌对雏鸭免疫功能影响的研究

1日龄天府肉鸭 10 0只分为 2组 ,喂以缺锌 (Zn 2 2 9mg)和对照 (Zn 10 0mg)日粮 7周 ,观察缺锌对免疫功能的影响。缺锌组免疫器官的绝对重量、生长指数和淋巴细胞生长周期以及外周血T淋巴细胞ANAE+阳性率、血清免疫球蛋白含量和红细胞C3b受体花环、免疫复合物花环率显著降低 (P <0 0 1或P <0 0 5 ) ;病理组织学观察 ,免疫器官淋巴细胞显著减少并呈退行性变 ,网状细胞变性坏死 ,电镜下淋巴细胞胞核固缩或溶解 ,细胞器消失 ,网状细胞粗面内质网扩张 ,线粒体肿胀 ,出现大小不等的溶酶体、吞噬体和自噬体。结果表明 ,缺锌严重抑制免疫器官生长发育和外周血T淋巴细胞活化 ,显著降低血清免疫球蛋白含量和红细胞免疫功能 ,并对免疫器官造成明显的病理损伤 ,导致免疫功能下降。     

The image of exocytosis during neutrophils and macrophages phagocytic activities in inflammation of mammary gland triggered by experimental Staphylococcus aureus infection

The experiments were carried out in five clinically normal virgin heifers. Before the experimental infection and at 24, 48, 72 and 168 h after the infection, respective mammary glands were rinsed with phosphate buffered saline. Neutrophils as well as macrophages underwent a classic exocytosis accompanied by translocation of lysosomal granules. The granules filled the protuberances of the plasmalemma and after the protuberances separated from the cell, they entered the extracellular space in the shape of round bodies of different sizes. After exocytosis, neutrophils displayed a smaller nucleus/cytoplasm ratio, a greater chromatin density of the nucleus, and an overall smaller size. Macrophages phagocytosed bacteria and/or neutrophils with and without signs of apoptosis (early and late apoptotic respectively) and neutrophils after exocytosis. Macrophages underwent cytolysis that was accompanied by extrusion of granules, phagosomes and phagolysosomes containing phagocytosed bacteria or neutrophils. Confluences were formed in which the process of digestion continued. Apoptosis of neutrophils gradually appeared and intensified in resolution of inflammation. The macrophages contributed to the inactivation of bacterial noxa as well as of histotoxic contents of neutrophils. Nevertheless, macrophages often underwent cytolysis at the site of inflammation.     

Ultrastructural and Cytochemical Properties of Peripheral Blood Cells of Piebald Naked Carp (Gymnocypris eckloni)

The ultrastructural and cytochemical properties of peripheral blood cells of Gymnocypris eckloni were investigated by transmission electron microscopy and a range of cytochemical techniques to provide clear insight into the structure and function of blood cells from this fish. Ultrastructurally, erythrocytes, leucocytes (neutrophils, eosinophils, lymphocytes, monocytes), thrombocytes and plasma cells were identified in the peripheral blood of G. eckloni. The most special ultrastructural characteristics of blood cells in this fish were that neutrophils exhibited only one type of cytoplasmic granules containing an eccentric, spherical or oval electron‐dense core, and eosinophils presented two types of granules with non‐uniform electronic density and without crystalloids in their cytoplasm. Neutrophils, eosinophils, lymphocytes, monocytes and thrombocytes were positive for periodic acid–Schiff and α‐naphthyl acetate esterase staining. Intense peroxidase positive staining was observed in neutrophils and monocytes, but not in eosinophils, lymphocytes and thrombocytes. Neutrophils, eosinophils and monocytes were stained positively for acid phosphatase, whereas lymphocytes and thrombocytes did not stain. Leucocytes and thrombocytes were negative for alkaline phosphatase and Sudan black B staining. Erythrocytes were negative for all cytochemical staining. The cytochemical and ultrastructural features of peripheral blood cells of G. eckloni were similar to those of other fish species. However, some important differences were identified in G. eckloni.     

Activation-induced mobilization of secretory vesicles in bovine neutrophils.

OBJECTIVE: To characterize mobilization of secretory granules in bovine neutrophils. SAMPLE POPULATION: Neutrophils obtained from four 6- to 18-month-old Holstein cattle. PROCEDURE: Mobilization of secretory granules in bovine neutrophils was determined by measuring changes in cell-surface alkaline phosphatase activity on cells treated with various inflammatory mediators. Subcellular distribution of the alkaline phosphatase activity was determined by analysis of bovine neutrophil homogenates fractionated on density gradients. RESULTS: Alkaline phosphatase-containing secretory granules of bovine neutrophils were readily mobilized by a number of inflammatory agents, including platelet-activating factor, interleukin-8, tumor necrosis factor-alpha, lipopolysaccharide, leukotriene B4, and zymosan-activated plasma. In contrast, N-formyl-methionyl-leucyl-phenylalanine did not have a significant effect. Phorbol myristate acetate induced a biphasic response with up-regulation of cell-surface alkaline phosphatase at low doses and a return to baseline or even a reduction in cell-surface alkaline phosphatase at higher doses (> or = 10 ng/ml). Subcellular fractionation of bovine neutrophil homogenates revealed that alkaline phosphatase activity resided in light-density membrane vesicles (ie, location of secretory granules), which were distinct from specific, azurophil, and large granules. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine neutrophils respond to various inflammatory mediators by mobilizing alkaline phosphatase-containing secretory granules. This suggests that the process is an important early step in the host-defense response of bovine neutrophils.     

Effect of EDTA on morphology of neutrophils of healthy dogs and dogs with inflammation

Increased basophilia and foamy vacuolation in the cytoplasm of neutrophils in smears made within 1 hour of blood collection suggest inflammatory disease. Due to the mild increase in foamy vacuolation which occurred with time in EDTA, the significance of mildly vacuolated neutrophils in smears prepared a few hours after blood collection is questionable. However, moderate or severe foamy vacuolation should be considered clinically significant. In vitro morphologic changes in neutrophils from healthy dogs in EDTA include clear, discrete vacuoles in the cytoplasm, uneven distribution of cytoplasmic granules, irregular cell membrane and pyknosis. These neutrophils generally lack basophilia, have minimal foamy vacuolation of the cytoplasm and are morphologically different from neutrophils associated with severe inflammation.     

Functional and ultrastructural evaluation of neutrophils from foals and lactating and nonlactating mares

Neutrophils from 4 pony foals, 3 lactating pony mares, and 3 nonlactating mares were evaluated ultrastructurally and by in vitro function tests. Neutrophils from foals had significantly (P = 0.05) less random migration than neutrophils from mares; values in tests for iodination and Staphylococcus aureus ingestion were also lower with foal neutrophils. Neutrophils from lactating mares had lower responses to iodination, antibody-dependent cell-mediated cytotoxicity, and random migration tests than did neutrophils from nonlactating mares. Ultrastructurally, granule concentration did not differ significantly among groups. A slight decrease in primary granules and a corresponding increase in granules with a flocculent matrix indicates partial spontaneous neutrophil degranulation in foals and lactating mares.     

Lesions in Brangus cattle with Chediak-Higashi syndrome

Hair, peripheral blood leukocytes, and other tissues from two related Brangus calves with phenotypic characteristics of Chediak-Higashi syndrome were examined by light and electron microscopy. Enlarged, pleomorphic, cytoplasmic granules, morphologically compatible with lysosomes, were seen in several neutrophils, many eosinophils, renal tubular epithelial cells, and Kupffer cells. Hair shafts of the calves showed irregular distribution and clumping of melanin granules. Severe infection and a possible hemorrhagic tendency were recognized. These Brangus calves represent the third breed of cattle affected with this genetic disease.     

Morphology,cytochemical staining and ultrastructural characteristics of reindeer (Rangifer tarandus) leukocytes

Peripheral blood smears from four adult reindeer (Rangifer tarandus) were examined after staining with Romanowsky's stain and cytochemical stains, including alpha-napthyl butyrate esterase (alpha-NBE), Sudan black B (SBB), chloroacetate esterase (CAE) and alkaline phosphatase (ALP). Romanowsky-stained eosinophils, neutrophils, lymphocytes and monocytes resembled those of cattle, sheep and goats. Basophils had two different staining patterns with Romanowsky's stain. Basophils that we termed "grey basophils" were similar in appearance to grey eosinophils in Greyhound dogs, with medium blue-grey to lavender-grey cytoplasm containing varying numbers of clear vacuoles or granules and variable numbers of small, intensely basophilic, perinuclear granules. The second basophil staining pattern was more typical of ruminant basophils, with uniform, pale to dark basophilic cytoplasmic granules. Basophils stained positive for alpha-NBE, SBB, CAE, and ALP. Eosinophils stained positive for SBB, and were negative for alpha-NBE, CAE, and ALP. Neutrophils were negative for SBB, CAE, and ALP. Monocytes stained positive for alpha-NBE, were rarely positive for CAE and SBB, and were negative for ALP. Transmission electron microscopy revealed matrix within all granulocytes granules, including those of basophils.     

Tamoxifen induces apoptotic neutrophil efferocytosis in horses

Macrophages and neutrophils are important cellular components in the process of acute inflammation and its subsequent resolution, and evidence increasingly suggests that they play important functions during the resolution of chronic, adaptive inflammatory processes. Exacerbated neutrophil activity can be harmful to surrounding tissues; this is important in a range of diseases, including allergic asthma and chronic obstructive pulmonary disease in humans, and equine asthma (also known as recurrent airway obstruction (RAO). Tamoxifen (TX) is a non-steroidal estrogen receptor modulator with effects on cell growth and survival. Previous studies showed that TX treatment in horses with induced acute pulmonary inflammation promoted early apoptosis of blood and BALF neutrophils, reduction of BALF neutrophils, and improvement in animals’ clinical status. The aim of this study was to describe if TX induces in vitro efferocytosis of neutrophils by alveolar macrophages. Efferocytosis assay, myeloperoxidase (MPO) detection and translocation phosphatidylserine (PS) were performed on neutrophils isolated from peripheral blood samples from five healthy horses. In in vitro samples from heathy horses, TX treatment increases the phenomenon of efferocytosis of peripheral neutrophils by alveolar macrophages. Similar increases in supernatant MPO concentration and PS translocation were observed in TX-treated neutrophils, compared to control cells. In conclusion, these results confirm that tamoxifen has a direct effect on equine peripheral blood neutrophils, through stimulation of the engulfment of apoptotic neutrophils by alveolar macrophages.     

Association among filamentous actin content, CD11b expression, and membrane deformability in stimulated and unstimulated bovine neutrophils

OBJECTIVE: To investigate rheologic properties of bovine neutrophils that may result in adhesion molecule-independent sequestration of neutrophils in inflamed lungs of cattle. ANIMALS: Healthy 2- to 4-week-old male Holstein calves. PROCEDURES: Neutrophil deformability, filamentous actin (F-actin) content, and CD11b expression was determined for unstimulated bovine neutrophils and bovine neutrophils incubated with the inflammatory mediators tumor necrosis factor-alpha (TNF), platelet-activating factor (PAF), interleukin-8 (IL-8), zymosan-activated plasma (ZAP), Pasteurella haemolytica-derived lipopolysaccharide (LPS), and P haemolytica leukotoxin. Neutrophils were separated into 3 subpopulations on the basis of size. The Factin content and CD11 b expression were evaluated by use of flow cytometry. Leukocyte deformability was evaluated by filtration of dilute whole blood. RESULTS: The subpopulation of the smallest-sized neutrophils (>90% of neutrophils) contained little F-actin. A subpopulation of slightly larger neutrophils had a profound increase in F-actin content and CD11 b expression. The subpopulation of the largest neutrophils had increased F-actin content and CD11b expression, compared with those for both subpopulations of smaller neutrophils. Incubation of neutrophils with PAF and ZAP but not TNF, IL-8, LPS, or leukotoxin, resulted in decreased neutrophil deformability and increased F-actin content. Incubation with PAF and TNF induced an increase in size of neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: Size can be used to identify subpopulations of large and rigid neutrophils in blood samples from healthy calves. Platelet-activating factor and activated complement fragments are potent inducers of F-actin formation and neutrophil rigidity. Physical changes in neutrophils may impede their transit through lung microvasculature and result in leukocyte trapping independent of adhesion molecule interactions with endothelial cells.     

Bovine neutrophils treated with chemotactic agents: morphologic changes

Neutrophils were isolated from the peripheral blood of cattle. After the neutrophils were incubated with zymosan-activated serum, the neutrophils changed from spherical to a bipolar shape. Ninety percent of the neutrophils became bipolar in 5 to 10 minutes. Bacterial cell filtrate and casein also induced bipolar shape changes in neutrophils, but N-formyl-L-methionyl-L-leucinyl-L-phenylalanine did not. The neutrophil-shape-change response was a rapid in vitro assay to evaluate early chemotactic events.     

Neutrophils contact to plasma membrane of keratinocytes including desmosomal structures in canine pemphigus foliaceus

Pemphigus foliaceus (PF) is an autoimmune blistering skin disease that affects certain mammals including dogs. In canine PF, neutrophils are infiltrated intensely into pustular lesions including acantholytic cells, although neutrophilic infiltration is not characterized in human PF. The roles of the neutrophils in the cutaneous lesions of canine PF have not yet been understood. The purpose of this study was to characterize the ultrastructural features underlying the acantholysis with pustule formation in canine PF. Four dogs diagnosed as PF on the basis of clinical signs, histopathological findings, and direct and indirect immunofluorescence examinations were performed. Electron microscopy revealed that the acantholytic cells were adjacent to multiple neutrophils in the pustules. At the contact points between neutrophils and acantholytic keratinocytes, half-desmosomes of acantholytic keratinocytes with intact attachment plaques were observed within invaginations of neutrophils. Furthermore, on the surface of acantholytic cells in the pustules, neutrophil granules seemed to be secreted to the surface of acantholytic cells and to degenerate the half-desmosome structures. Neutrophils were also observed within the epidermis adjacent to the pustule. At the intercellular gap between two dissociated keratinocytes, neutrophils inserted its pseudopodia into the gap between the two half-desmosomes of keratinocytes. These findings taken together suggested that, at least in the areas where we analyzed ultrastructurally, neutrophils contact desmosomal structures and seem to play some parts in separation of keratinocytes and degeneration of split-desmosomes in pustules of dogs with PF.     

Ultrastructure of Developing and Mature Caprine Leukocytes

Transmission electron microscopy demonstrated that, based on the structural uniqueness of the granules, caprine granulocytes are easily distinguishable from each other from the promyelocyte stage onwards. The neutrophils had the smallest granules which varied in size and were, in mature cells, either spherical to dumb-bell in shape; in mature cells the granule contents were compact and finely granular. The primary granules were smaller than the secondary granules. The eosinophil granules were large and typically had internal crystalloid structures; a second group of spherical granules with moderately coarse non-crystalloid sub-structure was present in smaller numbers in promyelocytes and myelocytes only. The basophil granules were also large, lackes crystalloids but showed variation in coarseness of granule substance, ranging from finely granular to markedly coarse. Mature granulocytes lackes Golgi apparatus and rough endoplasmic reticulum and ribosomes which were present in promyelocytes, myelocytes, metamyelocytes and bands. The monocytes had moderate numbers of spherical granules, rough endoplasmic reticulum, mitochondria and ribosomes, as well as prominent Golgi apparatus, and the cytoplasm had many small vacuoles.     

Morphology of leukocytes from cats affected with alpha-mannosidosis and mucopolysaccharidosis VI (MPS VI)

The morphology and ultrastructure of circulating white blood cells from six Persian and from five Russian Blue/Siamese cats deficient in lysosomal activity of alpha-mannosidase and arylsulfatase B, respectively, were studied and compared to cells from corresponding normal and carrier cats. In cats with mannosidosis, light microscopic examination revealed vacuoles in lymphocytes and monocytes, whereas electron microscopic studies demonstrated additional vacuoles in neutrophils, eosinophils, and basophils. In cats with mucopolysaccharidosis VI (MPS VI), vacuoles containing metachromatic granules were observed in lymphocytes, neutrophils, eosinophils, and monocytes. Ultrastructural studies of these cells identified the accumulation of fibrillar material, which often was associated with lamellated membrane structures.     

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.     

The Thymus of the Guinea Fowl from the Eighteenth Day of Incubation until Maturity

The thymus gland of the guinea fowl appeared as a series of pink, irregular-shaped lobes along the jugular veins. These lobes were 13 in number with seven on the right and six on the left side. The third and sixth lobes on the right side were the largest. The largest lobe measured about 20.0 × 5.0 mm while the smallest measured about 5.0 × 4,0 mm. Histologically, it was encircled by a thin layer of poorly stained connective tissue from which strands penetrated the thymic tissue giving incomplete lobules peripherally as early as day 18 of incubation. Lymphocytes and reticular epithelial cells were the two types of cells occurring at this time. Reticular epithelial cells were observed to be vacuolated especially towards the centre of the gland. Macrophages were observed in the thymus on day 20 of incubation for the first time. The blood vessels had accompanied the interlobular septa into the thymus. By day 21 of incubation, blood vessels had actually penetrated the thymic tissue but most of these vessels were of the capillary size. The cortex also became quite distinct from the medulla. At day 22, vacuolation ceased to appear in the cells at the cortical zone. Macrophages were still less than 10 in the slide. At day 23, most blood vessels were within the medulla and they displayed compressed lumi-na. Mitotic figures became a common site amongst the large lymphocytes and reticular epithelial cells. By day one post-hatch, lobulation of the thymus was still poor and macrophages became difficult to identify. The small and medium lymphocytes had outnumbered the large lymphocytes throughout the thymic tissue except at the most peripheral zone of the cortex. Fine vacuoles had reappeared within the cytoplasm of the reticular epithelial cells in the cortex. By day five, the distribution of blood vessels was almost uniform between the cortex and medulla. At three weeks post-hatch, plasma cells and red blood cells were seen in the stroma of the gland for the first time and most of the red blood cells occurred in the medulla.