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1.
A rapid method is described for parallel isolation of white blood cells, granulocytes and purified neutrophils from peripheral blood of normal cattle. The mean recovery (+/- S.D.) of white blood cells, granulocytes and purified neutrophils isolated from peripheral blood of 13 cows was 66.4 +/- 12.6%, 68.7 +/- 20.0% and 38.0 +/- 20.9%, respectively. The mean purity of the isolated granulocyte and neutrophil suspensions was 94.0 +/- 3.8% and 95.0 +/- 6.0%, respectively. Viability of isolated cells was more than 97%.  相似文献   

2.
Morphologic features of heterophils, eosinophils, and basophils of wild adult Pygoscelid penguins (Pygoscelis adeliae; 1 male, 4 females) of Antarctica were studied. Granulocytes were described on the basis of analysis of stained blood films and of 0.5-micron sections of centrifuged resin-embedded blood. Morphometric analysis of granulocytes included average cell diameter, cell shape, and nuclear configuration as well as the number, size, and shape of intracytoplasmic granules. Large round heterophils were the most prevalent granulocyte and possessed numerous rod- and spindle-shaped granules and a polymorphous nucleus. The second most prevalent granulocyte was the eosinophil. These eosinophils contained a bilobed nucleus and an average of 29.52 homogeneous granules/cell. The smallest and least encountered granulocyte was the basophil. Basophils possessed a single eccentric nucleus surrounded by round metachromatic granules (12.20 granules/cell) of varying size.  相似文献   

3.
As a basis for other experiments using flow cytometry of porcine peripheral blood leukocytes, cell fractions were isolated by various methods and analyzed by forward angle light scatter and 90 degree light scatter. Cytospin smears of cell samples were also studied by leukocyte differential counts and nonspecific esterase staining. Three main populations of peripheral blood leukocytes [lymphocytes, monocytes, and granulocytes (primarily neutrophils)], were defined in the log 90 degree light scatter by forward angle light scatter histogram. Partial overlap was observed between lymphocyte and monocyte, and between monocyte and granulocyte domains. Correlation between leukocyte differential counts and flow cytometric quantification based on bitmap statistics of appropriate domains was between r = 0.872-0.892 for lymphocyte and granulocyte. Percoll density gradients were used for subfractionation of leukocyte populations, especially for the enrichment of granulocytes. The specific densities were calculated for lymphocytes (1.0585-1.0819 g/cc), monocytes (1.0585-1.0702 g/cc), granulocyte (1.0819-1.0936 g/cc), and erythrocytes (greater than 1.0952 g/cc). We suggest that light scatter characterization is a basis for future studies of porcine blood by flow cytometry.  相似文献   

4.
The target cells of classical swine fever (CSF) virus in the peripheral blood of pigs infected with recent field isolates from Germany were studied. Eight weaned pigs were inoculated oronasally with the CSF virus field isolate Visbek/Han 95 and three weaners were inoculated with the isolate Losten/Freese 98. All pigs showed severe clinical signs typical of CSF and died or had to be euthanized between 9 and 24 days post‐infection (dpi). The first cells in the peripheral blood which became infected with CSF virus were mixed granulocytes (a combination of low‐ and high‐density granulocytes). These cells yielded the highest infectivity for PK 15 cell cultures. On day 7 post‐infection, the peripheral blood mononuclear cell (PBMC) fraction was virus positive, while the peripheral blood leucocyte (PBL), peripheral blood T lymphocyte (PBT) and high‐density granulocyte fractions were either negative or their infectivity was lower than the infectivity of the PBMC fraction. These results indicate that PBMC contain more virus‐positive cells than other fractions of leucocytes. These findings may also have diagnostic implications for the detection of CSF virus in blood samples. Because PBMC showed the highest infectivity in the early stages of CSF, it should be the sample of choice for CSF virus isolation.  相似文献   

5.
A new method for the isolation of bovine neutrophilic granulocytes from peripheral blood based on centrifugation in a discontinuous metrizamide gradient has been developed. The procedure is rapid, taking only about 2 h, and gives highly purified (greater than 90%) neutrophils in a high yield (approximately 85%). The function of the cells, as measured by chemiluminescence and migration assays, is not significantly influenced by the isolation procedure. Eosinophils can also be isolated by a slight variation of the method. Initial applications of the separation procedure indicate its usefulness in clinical studies of bovine neutrophil function. A variation between individuals in the function of the cells was thus demonstrated. Moreover, the chemiluminescence of neutrophils from infected animals was found to be greatly increased, and neutrophil migration was shown to be stimulated by in vivo ACTH treatment.  相似文献   

6.
按标准方法提取制备了猪的转移因子（transfer factor, TF）,用吞噬杀伤试验MTT法检测了供试杂种牧羊犬肌肉注射猪TF后外周血中性粒细胞吞噬杀伤活性的变化。试验摸索出MTT法测定犬外周血中性粒细胞吞噬杀伤大肠杆菌的最佳条件为：中性粒细胞浓度1.3×106个/ml、大肠杆菌浓度6×105个/ml 时,大肠杆菌和中性粒细胞混合培养2 h,加入MTT后继续培养4 h。体外试验结果表明,猪TF浓度在0.052～1.56 mg/ml范围内,能够明显促进中性粒细胞吞噬杀菌作用,当猪TF浓度为1.56 mg/ml时,对中性粒细胞杀菌活性的影响最大。体内试验结果表明,注射猪TF后第2 d,外周血中性粒细胞数量最高,中性粒细胞吞噬杀菌能力最强。  相似文献   

7.
A flow cytometric method was developed to perform differential leukocyte counts on bovine blood. Blood specimens from 50 healthy Holstein cows were analyzed by use of a flow cytometer. The method entailed diluting blood with phosphate-buffered, hypotonic saline solution containing acridine orange, and performing a step-wise, 3-parameter analysis on the bases of cell size, cellular granularity, and granulocyte fluorescence. Initially, proportions of monocytes, granulocytes, and lymphocytes were determined by creating appropriate windows on dot plots of cell size (determined by forward light scatter) vs cellular granularity (determined by the logarithm of side light scatter). Eosinophils were resolved by analysis of granulocytes as dot plots of logarithms of green vs red fluorescence ascribed to acridine orange. Proportions of eosinophils and neutrophils were computed from data so generated. Microclumps of platelets spuriously affected counts of some granulocytes, particularly eosinophils. Differential leukocyte counts determined by flow cytometry generally compared favorably with those obtained by use of the conventional microscopic method, using Wright-stained blood films. Mean neutrophil and eosinophil counts determined by the 2 methods did not differ significantly, but lymphocyte counts determined by flow cytometry were significantly higher than those determined by microscopy (P less than 0.01). Correlation coefficients for counts of neutrophils, eosinophils, and lymphocytes determined by the 2 methods ranged from 0.519 to 0.833. Correlation between monocyte counts was low (r = 0.147), although mean monocyte counts determined by the 2 methods did not differ significantly.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

8.
A method for the separation of pure and viable lymphocytes and granulocytes from the same blood sample in horses was reported. By centrifuging equine heparinized blood at 100 xg for 10 min at room temperature (r.t.), the resulting supernatant plasma was an almost pure (97.71 +/- 0.30%; n = 15) suspension of highly viable (98.72 +/- 0.28%) lymphocytes. When sodium citrate was used as an anticoagulant, lymphocyte suspensions collected in the same manner showed lower purity (87.89 +/- 1.59%; n = 9) and higher yields (56.56 +/- 3.89%, n = 9 versus 36.11 +/- 2.23%, n = 15). Where needed, a further centrifugation at 250 xg for 3 min (r.t.) of heparinized lymphocyte preparations removed an average of 87.39% (n = 15) contaminating platelets. A suspension of 85.96 +/- 2.20% pure granulocytes (93.23 +/- 1.74% neutrophils; n = 14) with minimal contamination by erythrocytes and high viability (93.11 +/- 1.26%) was obtained by performing a flash red blood cell lysis on the white-greyish layer resulting from the centrifugation of the heparinized blood samples. Among the several methods available, the procedure described herein is easy, rapid, cheap and reproducible.  相似文献   

9.
Tritiated thymidine uptake in response to Concanavalin A was recorded as stimulated counts per minute (SCPM) in equine whole blood cultured with an optimum concentration of 3 X 10(4) mononuclear cells/culture. A significant negative correlation was found between log10 SCPM and granulocyte level in culture, in a group of 27 adult horses (r = -0.745, P less than 0.001). Addition of isolated autologous granulocytes to such cultures resulted in a reduction of log10 SCPM of a magnitude similar to that predicted by the gradient of the log10 SCPM/granulocyte level regression line in the group of adult horses. It is concluded that the presence of granulocytes in culture directly depresses lymphocyte reactivity, and that a large part of the variability of SCPM between horses is due to variation of granulocyte levels.  相似文献   

10.
The influence of free radicals generated by polymorphonuclear leukocytes from bronchopneumonic calves on lipid peroxidation in red blood cells was studied. Incubation of granulocytes with red blood cells causes a rise in malondialdehyde production in these cells in linear dependence on the granulocyte number. Incubation of red blood cells with malondialdehyde causes a generation of adduct moiety of this compound with phosphatidylserine and phosphatidylethanolamine. This adduct is also present in erythrocytes which have been incubated with neutrophils of bronchopneumonic calves. Cytochrome c loaded erythrocyte ghosts showed a high reduction rate of this compound during incubation of ghosts with neutrophils of diseased calves. It is suggested that neutrophils of bronchopneumonic calves are in an activated state in the blood-stream, and free radicals generated by these cells are capable of peroxidizing the red blood cell membrane lipids and causing cross-links between phospholipid moieties in erythrocyte membranes.  相似文献   

11.
Since the teleost pronephros is an important source of diverse immunocytes, suspensions of pronephric cells from young adult carp have been characterized. In freshly prepared suspensions, adherent, spreading cells (macrophages?) constituted less than 3% of the total population. Granulocytes and lymphocytes were co-dominant (less than 80%) leucocyte types. Continuous Percoll density gradient centrifugation yielded discrete subpopulations with these rho values and cytological characteristics: Fraction I & II rho = 1.055-1.070 thrombocytes, monocytes, macrophages, and lymphocytes. Fraction III rho = 1.080-1.090 granulocytes, type 1. Fraction IV rho = 1.105-1.110 erythrocytes and granulocytes, type 2. Fraction V rho = 1.118-1.125 granulocytes, type 3. Fraction VI rho = 1.140-1.150 granulocytes, type 4. Granulocyte motility increased markedly over the first 24 hr in vitro, and was enhanced by components washed from intact yeast. The subtypes of granulocytes were distinguishable by not only the rho values, but also on the basis of cell size, ultra-structure of the granules, and their histochemical and phagocytic characteristics. After simultaneous in vivo injection of Bacillus megaterium (Gram + ve), Aeromonas hydrophila (Gram - ve) and Saccharomyces cerevisiae (yeast), individual pronephric leucocytes were found capable of phagocytosing all three types of particle. Granulocytes which had phagocytosed B. megaterium were slower than macrophages in their ability to kill the bacteria. Encounter with B. megaterium or S. cerevisiae in vitro elicited a clumping reaction which involved mostly the larger leucocytes [granulocytes]. Both adherent cells and non-adherent cells were phagocytic in vitro.  相似文献   

12.
The objective of this study was to investigate if occurrence of clinical disease was related to granulocyte traits in sows. Functional capacity of granulocytes and plasma steroid hormone concentrations were assessed before inoculation with Escherichia coli in the mammary glands in sows at parturition. Blood samples were taken for 3 days approximately 1 week before parturition, and granulocyte migration, phagocytic capacity and expression of CD 18 adhesion molecules were determined. Inoculation was done within 36 h before partus. Thereafter, daily thorough clinical examinations were performed including udder health, habitus, appetite and rectal temperature, to assess the severity of disease. Based on the clinical findings four sows were classified as affected and eight as non-affected by clinical mastitis within 48 h after parturition.No difference (p>0.10) in pre-inoculation chemotaxis, phagocytosis or CD 18 expression was found between granulocytes from the sows resisting and developing clinical mastitis, respectively. However, there was an effect by the individual sow (p=0.001) on the numbers of granulocytes and white blood cells, and on plasma concentrations of estradiol-17beta and progesterone. In conclusion, these data does not suggest that impaired chemotaxis or phagocytosis by blood granulocytes contribute to the development of clinical coliform mastitis in the periparturient sow.  相似文献   

13.
A quantitative buffy coat (QBC) analysis was evaluated for 175 canine, 125 feline, and 125 equine blood samples. The method used centrifuged whole blood and yielded rapid results expressed as respective band lengths for RBC, granulocytes, nongranulocytes, and platelets. Simple regression analysis of band lengths and reference laboratory methods yielded correlation coefficients (r) ranging from 0.72 to 0.99. The PCV, granulocyte count, and total WBC count, as determined by the 2 methods, correlated well (r greater than or equal to 0.93 in all cases). Platelet and nongranulocyte counts were less well correlated. The QBC system provided a means of performing rapid hematologic screening. The principal problem encountered was poor separation of the RBC-granulocyte interface in 17% of canine samples, which interfered with measurement of band lengths. Evaluation of the QBC tube for detection of Dirofilaria immitis microfilaremia revealed 100% sensitivity to counts as low as 160 microfilariae/ml of whole blood.  相似文献   

14.
The specificity of guinea pig erythrocyte (GPE) rosettes for feline peripheral blood lymphocytes was studied. Of the GPE rosette-positive cells from peripheral blood, 54% were monocytes, 29% were granulocytes, and only 17% were lymphocytes. Results were similar for rosettes incubated at 4 C and those incubated at 37 C. Mononuclear cells separated with polyvinylpyrrolidone-coated silica formed fewer monocyte rosettes (49%) and more granulocyte rosettes (34%) than did cells separated with sodium diatrizoate-Ficoll (60% monocyte rosettes and 18% granulocyte rosettes), whereas the percentage of lymphocyte rosettes was similar for both media. Mononuclear cells suspended in Eagle's minimum essential medium had a higher percentage of monocyte rosettes (75%) and a lower percentage of granulocyte rosettes (12%) than did cells suspended in RPMI 1640 medium (59% monocyte rosettes and 27% granulocyte rosettes). The percentage of lymphocyte rosettes was similar in the 2 media. Two sequential 45-minute plastic adherent cell depletions decreased monocyte rosettes to 51% and increased lymphocyte rosettes to 23% compared with 63% monocyte rosettes and 12% lymphocyte rosettes before adherent cell depletion. The granulocyte rosettes were unchanged by plastic adherent cell depletion. The percentage of rosette-positive cells (9%) was not significantly affected by incubation at 4 C or 37 C, cell separation with polyvinylpyrrolidone-coated silica or lymphocyte separation medium, or suspension in Eagle's minimum essential medium or RPMI 1640 medium. Plastic adherent cell depletion decreased the percentage of rosette-positive cells. Feline thymocytes were 38% to 80% GPE rosette-positive and a feline leukemia virus-infected lymphoblastic cell line (F422) was 88% GPE rosette-positive.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

15.
Turkey rotaviruses from the intestinal contents of poults were isolated and serially propagated in MA104 cell monolayers by a simple procedure. The initial virus isolation was done by low-speed centrifugation of the inoculum onto the monolayers, and subsequent passages were accomplished in roller-tube monolayers using trypsin-treated virus suspensions. Each of the turkey rotavirus isolates possessed the morphologic, antigenic, and genomic attributes characteristic of turkey group A rotaviruses. Attempts to isolate and serially propagate turkey rotavirus-like viruses in MA104 cell monolayers by this procedure were unsuccessful. Turkey reoviruses also did not serially propagate in MA104 cell monolayers by this procedure.  相似文献   

16.
Hyposegmentation of granulocyte nuclei was diagnosed in an adult dog after unsuccessful attempt at finding an occult chronic infection. Intermittent signs of prostatitis were temporarily observed, but the immature nature of circulating granulocytes persisted nine months. Electron microscopic examination and neutrophil function studies revealed no abnormalities. Since previous blood smear evaluation had been normal, it is proposed that the granulocyte anomaly was acquired and may have resulted from an idiosyncratic reaction to one of several chemotherapeutic agents administered.  相似文献   

17.
The procedure used for the cultural isolation of morbillivirus from seals as well as some growth characteristics of isolates is described briefly. Emphasis was laid upon cytopathic changes typical for morbillivirus and observed in seal kidney cell culture subsequent to inoculation of organ tissue suspensions or buffy coat leucocytes. The modalities which resulted in the identification of 21 morbillivirus isolates from 16 seals by using direct fluorescent antibody (FA) or peroxidase linked antibody (PLA) techniques are outlined and illustrated.  相似文献   

18.
A simple discontinuous Percoll density-gradient technique was adapted for isolation of granulocytes and mononuclear cells from cats, dogs, horses and cattle. Separation was accomplished at low speeds using a standard tabletop centrifuge. Cell purity was 100% for both granulocytes and mononuclear cells and cell viability exceeded 95%. Percent recovery of leukocytes ranged from 69 to 83%.  相似文献   

19.
Arachidonic acid-induced granulocyte activation was studied in calves. Both in healthy and in bronchopneumonic animals, neutrophil stimulation with 67 microM arachidonic acid caused a rise in granulocyte exocytosis. This event, however, was much more intense in granulocytes from diseased calves. Fatty acids were found to stimulate granule exocytosis from granulocytes. Oleic acid possessed the weakest while dihomo-gamma-linoleic acid showed the strongest activity of this type. It is suggested that arachidonic acid may be an important mediator of calf bronchopneumonia.  相似文献   

20.
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.  相似文献   

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