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.     

Influence of conditioned media from bovine cotyledon tissue cultures on function of bovine neutrophils.

Bovine fetal placental (cotyledon) tissue obtained from pregnant cows on days 255, 265, and 275 of gestation, as well as immediately after parturition (n = 5) was incubated in media for 48 hours, and the incubation media were collected. Neutrophils from 4 ovariectomized nonpregnant cows were incubated for 2 hours with conditioned media from placental tissue cultures or medium (control). Immediately after incubation, the neutrophils were subjected to the following leukocyte function assays: chemotaxis against zymosan-activated serum, chemotaxis against undiluted conditioned media (only neutrophils that were incubated in medium only), random migration, ingestion of 125I-iododeoxyuridine Staphylococcus aureus (125I-IdUR-S aureus), iodination of proteins, cytochrome C reduction, and antibody-independent and -dependent cell-mediated cytotoxicity. Conditioned media from cultured cotyledon tissue was chemoattractant for bovine neutrophils, and increased chemotactic response of neutrophils against zymosan-activated serum by 13%. The following neutrophil functions were decreased: random migration by 25%, iodination of proteins by 44%, cytochrome C reduction by 13%, and antibody-dependent cell-mediated cytotoxicity by 5%. Ingestion of 125I-IdUR-S aureus and antibody-independent cell-mediated cytotoxicity were not influenced by coincubation of neutrophils and conditioned media. Time of gestation did not alter the effects of conditioned media on neutrophil function. It was concluded that chemotactic properties of cotyledon tissue extracts, as has been reported earlier, may be attributable to substances released by fetal placental tissue. Those substances might also locally or systemically influence the oxygen-dependent antimicrobial system of neutrophils, thereby causing an increased susceptibility to bacterial infections in the peripartum period.     

Chemotactic properties and absence of the formyl peptide receptor in ferret (Mustela putorius furo) neutrophils

This study describes a chemotaxis assay of ferret polymorphonuclear cells (PMNs). The optimal conditions for this chemotaxis assay were investigated for three chemoattractants: zymosan activated serum (ZAS), recombinant human interleukin-8 (rhIL-8) and N-formyl-Met-Leu- Phe (fMLF). In this study, ferret polymorphonuclear cells (PMNs) reacted to ZAS and rhIL-8, but not fMLF. The optimal concentration of ZAS and rhIL-8 were 5% and 100 ng/ml, respectively. The optimal incubation time of each reagent was 60 min. Due to the lack of response shown from fMLF, the existence of formyl peptide receptors (FPR) on ferret PMNs was investigated by evaluating FPR binding using flow cytometry. The receptor was not detected, implying that ferret neutrophils may lack FPR. This study confirms the fundamental experimental conditions for ferret PMNs chemotaxis and elucidates new findings concerning FPR in ferret neutrophils.     

Defective in vitro motility of polymorphonuclear leukocytes of homozygote and heterozygote Chediak-Higashi cats.

The in vitro migratory responses of neutrophils of homozygote and heterozygote Chediak-Higashi cats were defective in an under-agarose assay when compared to the behavior of phagocytes of control cats. The linear distances traversed by the leading front of migrating Chediak-Higashi neutrophils toward streptococcal culture supernatant, zymosan-activated serum or buffer were reduced and smaller numbers of Chediak-Higashi phagocytes populated the resulting migration areas than did cells of control animals. The relative migration parameters of the Chediak-Higashi phagocytes, however, did not differ from the corresponding parameters of control neutrophils in the presence of streptococcal culture supernatant. Therefore, phagocytes of homozygote and heterozygote Chediak-Higashi cats recognized and responded equally well to the bacterial stimuli as did cells of control animals but traveled shorter distances primarily because of a reduced inherent motility. Similar results were also obtained when the feline phagocytes were attracted by zymosan-activated serum. In addition the relative migration parameters of the neutrophils of homozygote Chediak-Higashi cats were reduced and the normalized spatial distributions of their migrating cells were significantly different in the presence of 100% and 20% zymosan-activated serum when compared to the corresponding migration parameters of carrier and control animals. Defective recognition or responses to the higher concentrations of these host-derived attractants complicated, therefore, the already reduced inherent motility of the phagocytes of homozygote Chediak-Higashi cats.     

In vitro migration responses of neutrophils from cows and calves

The directional (chemotactic) and random migration activities of neutrophils from cows and newborn and 2-week-old calves were determined by use of the chemotaxis-under-agarose assay. Blood samples were stored for 2, 24, or 48 hours and at 4 or 25 C before testing. During the assay, cells were incubated at 17, 27, or 37 C. The assay was found suitable for testing the directional and random migration activities of neutrophils from cattle. Directional migration of neutrophils was diminished (P less than or equal to 0.05) when cells were incubated at 17 or 27 C, compared with data from incubation at 37 C. Random migration of neutrophils was unaffected by test incubation temperature. Significant (P less than or equal to 0.05) differences were found between cows and calves regarding the percentage number and viability and the directional and random migration activities of neutrophils. Neutrophils from cows were adversely affected to a greater extent by prolonged sample storage times or low storage temperature than were neutrophils from calves. Results indicate that a sample storage time of up to 24 hours, a sample storage temperature of 25 C, and a test incubation temperature of 37 C provided optimal conditions for testing the migratory activities of neutrophils from cattle.     

Neutrophil chemotaxis in rhesus macaques (Macaca mulatta)

Chemotactic responses of isolated peripheral blood neutrophils from rhesus macaques (Macaca mulatta) were studied, using a micropore filter method. Cell migration toward zymosan-activated serum was similar to that of human cells, whereas the response to N-formyl methionyl leucyl phenylalanine (FMLP) was weaker than was that in human cells, requiring higher concentrations of FMLP for maximal migration. Optimal FMLP concentrations for attraction of rhesus neutrophils and human neutrophils were 5.0 X 10(-7)M and 1.0 X 10(-8)M, respectively. The chemotactic responses of the 2 neutrophils to complement (zymosan-activated serum) were similar. However, rhesus neutrophils required a higher concentration of the formyl peptide, FMLP, for maximal migratory response.     

Effects of human IL-8 isoforms on bovine neutrophil function in vitro

Interleukin 8 (IL-8) is a potent chemotactic and activating agent for human neutrophils and bovine IL-8 is chemotactic for bovine neutrophils; however, it is unclear whether IL-8 activates bovine neutrophils. Two isoforms of human recombinant (hr) IL-8 protein (77 and 72 amino acid) were used to stimulate bovine neutrophils in vitro. Bovine neutrophils exhibited significant migration in the presence of 0.1, 0.5, 1.0 and 5.0ngml(-1) hr IL-8 when incubated for 30min at 37 degrees C in a modified Boyden chamber assay. Both the 77 and 72 aa forms were equally effective in inducing migration in this assay. At the highest doses of IL-8 examined (1 and 5ngml(-1)), migration was similar to migration in the presence of 20% zymosan-activated serum (ZAS) or 12h lipopolysaccharide (LPS)-stimulated blood monocyte supernatants (CM). Significant (p<0. 05) release of alkaline phosphatase (ALK-P) (from specific granules) occurred but myeloperoxidase (MPO) release and superoxide anion production were not enhanced in bovine neutrophils by either form of hrIL-8 at any of the doses tested. Significant (p<0.05) alkaline phosphatase release was observed in the presence of 10 and 100ngml(-1) for the 72 aa form of IL-8 and only at the higher dose for the 77 aa form of IL-8. The ZAS and CM significantly enhanced neutrophil degranulation of ALK-P and MPO as well as inducing superoxide anion production. These results suggest that IL-8 may play a role in both neutrophil recruitment and activation during bovine inflammatory processes.     

Evaluation of Cell Culture Methods for Detection of Largemouth Bass Virus

Abstract

Largemouth bass virus (LMBV), a recently discovered iridovirus found in the eastern United States, is usually detected by isolation in cell culture. Although LMBV will replicate in several cell lines, optimal cell culture methods for the detection of this virus have not been determined. We tested inoculation method, adsorption time, incubation temperature, and various cell lines to determine the conditions that would provide the most sensitive cell culture assay for LMBV. The optimal inoculation procedure tested was to remove the culture medium from the culture well before the addition of the inoculum, and the optimal adsorption procedure tested was to allow the virus to adsorb for 40 min while the plates were on an orbital shaker. Following inoculation, incubation at 30°C resulted in a higher number of viral plaques than incubation at 25°C or 32°C. Four cell lines (bluegill fry (BF-2), fathead minnow (FHM), epithelioma papulosum cyprini, and channel catfish ovary cells) inoculated with LMBV had similar susceptibility to infection. Similar percentages of LMBV-positive samples were detected in BF-2 and FHM cell cultures inoculated with homogenized organ samples from largemouth bass Micropterus salmoides; however, the use of two cell lines increased the number of infected samples discovered. A blind passage also increased the number of positive samples detected in cell culture. Subcultivation to confirm virus-positive samples was useful for reducing false-positive results.     

The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) colorimetric assay for the quantitation of Actinobacillus pleuropneumoniae cytotoxin.

Using swine neutrophils as target cells, two MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) colorimetric assay systems, one with and one without phorbol 12-myristate 13-acetate (PMA) stimulation were established for the quantitation of Actinobacillus pleuropneumoniae cytotoxin. The MTT assays were optimized for the number of neutrophils, incubation time, and PMA concentration by a series of experiments. The optimal conditions were 25 x 10(4) cells/well incubated for four hours for the assay system without PMA stimulation, and 12.5 x 10(4) cells/well incubated for two hours for the assay system with PMA stimulation. One culture supernatant of a toxigenic Pasteurella multocida strain and five A. pleuropneumoniae cytotoxin preparations produced from three A. pleuropneumoniae strains were used to test assay reproducibility. Results showed both assays were reproducible with a coefficient of variation ranging from 7.8 to 18% for the assay system without PMA stimulation and from 10.7 to 18.2% for the assay system with PMA stimulation. The PMA-stimulated assay had 40 to 60-fold higher sensitivity than the nonstimulated MTT assay. The MTT assay also was applied to the measurement of neutralizing antibody titers against A. pleuropneumoniae cytotoxin.     

Optimization of culture media of pathogenic Mycoplasma hyopneumoniae by a response surface methodology

Composition of culture medium for mass production of Mycoplasma hyopneumoniae was optimized using a response surface methodology (RSM). Initially, the influence of glucose, thallium acetate, fresh yeast extract, horse serum, and porcine serum on the production of mycoplasmal protein was assessed using a ''one factor at a time'' technique. Next, factors with a significant effect, including fresh yeast extract, and horse and porcine sera, were selected for further optimization using a central composite design (CCD) of RSM. The experimental results were fitted into a second order polynomial model equation. Estimated optimal condition of the factors for maximum production of mycoplasmal protein (i.e., triple-fold increase from 0.8 mg/L produced by basal mycoplasma media to 2.5 mg/L) was 10.9% fresh yeast extract, 15% horse serum, and 31.5% porcine serum (v/v). For the optimized conditions, a 2.96 mg/L experimental result was observed, similar to the estimated optimal conditions result of the CCD.     

Ingestion and killing of Pasteurella haemolytica A1 by bovine neutrophils in vitro

In this study, various parameters affecting the ability of bovine neutrophils to ingest and kill a virulent strain of Pasteurella haemolytica A1 in vitro were examined. Ingestion of P. haemolytica was serum dependent (optimal serum concentration 10%) and was mediated principally by heat-stable opsonins, presumably antibodies, that could be removed by absorption with formalin-killed P. haemolytica. Ingested P. haemolytica were killed by neutrophils within 1-4 h incubation; the magnitude of killing being directly dependent on the number of neutrophils present. The number of viable P. haemolytica was reduced by approximately 1.5 log at bacterial concentrations of 0.01-100 P. haemolytica per neutrophil; a concomitant reduction in neutrophil viability was observed at the highest bacterial concentration (100:1). Bovine neutrophils underwent a vigorous luminol-enhanced chemiluminescence response after ingesting opsonized P. haemolytica, thus indicating that reactive oxygen intermediates were being formed that could have contributed to the intracellular killing of P. haemolytica.     

Phagocytosis induced by yeast cells in canine granulocytes: a methodological study

A phagocytic function assay of canine granulocytes was established. This method allows the proportion of active granulocytes to be estimated as well as the number of adhered and ingested yeast cells. The influence of different factors on phagocytosis was studied. Temperature variation within the interval 36-41 degrees C did not affect phagocytosis. The incubation time for optimal phagocytosis of yeast cells was 35 min. The opsonization procedure giving the optimal phagocytosis was purified IgG and serum together.     

检测猪伪狂犬病病毒gE抗体红细胞凝集试验的建立及应用

以纯化的抗人红细胞单链抗体(ScFv)—猪伪狂犬病病毒(PRV)gE蛋白双功能融合蛋白为抗原,建立了检测猪伪狂犬病毒gE抗体的红细胞凝集试验。利用方阵滴定试验筛选出最佳抗原工作浓度为55μg/mL,血清最佳稀释度为1∶20,作用时间15 min,与猪瘟(CSF)、猪细小病毒(PPV)、猪繁殖与呼吸综合征(PRRS)、猪乙型脑炎(JE)、猪布氏杆菌病(Brucellosis)阳性血清和PRV gE缺失疫苗接种的猪免疫血清均不出现红细胞凝集现象,与PRV标准阳性血清反应出现肉眼可见的凝集圈。与美国进口的PRV抗体检测gE-ELISA诊断试剂盒检测结果比较,50份猪血清的阴、阳性检出符合率均为100%。红细胞凝集试验检测方法具有操作简便、敏感性和特异性较高的特点,可用于PRV野毒感染的快速筛查。     

Influence of arachidonic acid metabolites and steroids on function of bovine polymorphonuclear neutrophils.

Polymorphonuclear neutrophils (PMN) from 4 ovariectomized healthy cows were incubated with 0 (control), 10(-8), 10(-7), and 10(-6) M arachidonic acid metabolites of the cyclo- and lipoxygenase pathways for 30 minutes, and with steroids for 2 hours. Immediately after incubation, PMN were subjected to the following function assays: chemotaxis against zymosan-activated serum, chemotaxis against arachidonic acid metabolite or steroid at the doses given (only control PMN were tested), random migration, ingestion of 125I-iododeoxyuridine-labeled Staphylococcus aureus (125I-IdUR-S aureus), iodination of proteins, cytochrome C reduction, antibody-independent and -dependent cell-mediated cytotoxicity (AICC and ADCC). Prostaglandin F2 alpha was chemoattractant and stimulated ingestion of 125I-IdUR-S aureus. Prostaglandin E2 stimulated cytochrome C reduction, whereas prostacyclin inhibited iodination of proteins. Thromboxane B2 stimulated ADCC. Leukotriene B4 was chemoattractant for bovine PMN and stimulated random migration and AICC. 5-Hydroxyeicosatetraenoic acid was also chemoattractant, but inhibited ingestion of 125I-IdUR-S aureus. 15-Hydroxyeicosatetraenoic acid was chemoattractant and decreased ADCC. Lipoxin A4 stimulated random migration, whereas lipoxin B4 inhibited chemotaxis against zymosan-activated serum, but was chemoattractant and stimulated cytochrome C reduction. 12-Hydroxyhepadecatrienoic acid and 12-hydroxyeicosatetraenoic acid did not influence any of the PMN functions tested. Of the steroids tested, cortisol increased ADCC, and progesterone stimulated cytochrome C reduction, but decreased ADCC. 17 beta-Estradiol and estrone were chemoattractant and stimulated cytochrome C reduction. In addition, estrone also stimulated random migration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pathogenesis of porcine Actinobacillus pleuropneumonia: Part I. Effects of surface components of Actinobacillus pleuropneumoniae in vitro and in vivo.

To understand the role of non-secreted components of Actinobacillus pleuropneumoniae in virulence, we investigated in vitro cytotoxicity and in vivo pulmonary changes in pigs due to various A. pleuropneumoniae (serotype 1) fractions. Following 1.5 h incubation, lipopolysaccharide (LPS), 2 crude extracts and bacterial culture supernatant (BCS) at high concentrations were cytotoxic to porcine pulmonary alveolar macrophages (PAM), peripheral blood mononuclear leucocytes, neutrophils and a cultured porcine bone marrow cell line. Heat-killed bacteria were cytotoxic to PAM after 24 h incubation. The 2 crude extracts were prepared by shaking either intact bacteria after removing culture supernatants (crude surface extract, CSE), or whole bacterial culture (crude surface plus culture supernatant extract, CSSE) with glass beads in saline at 60 degrees C. Further experiments showed that proteins from the bacterial membrane were partially involved in cytotoxicities of these 2 extracts. Both BCS and CSSE caused multivocal hemorrhage and neutrophil infiltration when inoculated into porcine lungs, but CSE did not. The lung:whole body weight ratios of the pigs treated with CSSE were significantly higher (P < 0.05) than those of pigs treated with BCS, CSE, or control solution. It is concluded that beside the secreted proteins, bacterial surface components including LPS and non-secreted proteins were cytotoxic in vitro; and secreted and non-secreted components act synergistically to cause lung lesions.     

Phagocytic function of equine neutrophils exposed to Mycoplasma felis in vitro and in vivo

Neutrophils were isolated from the peripheral blood of adult equids (group 1) and were purified on a density gradient of polyvinylpyrrolidone-coated silica gel. A bactericidal assay was developed, using an equine skin isolate of Staphylococcus epidermidis as target bacterium in medium containing pooled fresh equine serum for opsonization. Significant (P less than 0.05) killing was observed after 60 or 120 minutes' incubation. Reduction in bactericidal function of blood neutrophils was not found after incubation with a virulent strain of Mycoplasma felis for 30 or 60 minutes. Similarly, the function of blood and pleural neutrophils of ponies with M felis-induced pleuritis (group 2) was not different from the function of similar neutrophils from sham-inoculated control ponies (group 3). The number of viable M felis organisms was markedly reduced in the presence of fresh equine serum and equine-specific antibodies, but not when the serum was heat inactivated. Equine neutrophils did not phagocytize M felis. Seemingly, M felis did not impair bactericidal activity of equine neutrophils. Therefore, this mechanism does not predispose equids to bacterial pleuritis.     

Development and Application of a PEDV Nsp7-based Indirect ELISA for Antibody Detection

This study was aimed to establish an indirect ELISA to detect antibodies against different strains of porcine epidemic diarrhea virus (PEDV). Puried nonstructural protein 7(Nsp7) was used as coating antigen, and the indirect ELISA was established by optimizing the ELISA reaction conditions. The results showed that the optimal reaction conditions were as follow:The amount of coating antigen was 0.20 μg/well, and the coating condition was at 37℃ incubation for 1 h then 4℃ overnight; The working dilution of serum samples and HRP-labelled secondary antibody were 1:300 and 1:10 000, and the incubation time were 2 and 1.5 h, respectively; TMB substrate incubation time was 15 min. Serum sample was determined as positive when its S/P>0.1694 and negative when its S/P<0.1398. The ELISA was specific, reproducible and sensitive. Forty samples of suspected PEDV serum samples were tested by the established ELISA, and the coincidence rate between the ELISA and the commercial kit was 95%. The ELISA established in this study could be used clinically to detect the antibody level of different strains of PEDV, and it also had the potential for early diagnosis of PEDV, providing a basis for the development of effective measures to control PEDV.     

Partial characterization of phagocytic activity in neutrophils of the nine-banded armadillo Dasypus novemcinctus

Though the armadillo is important as a research model in leprosy studies, the activity of armadillo's neutrophils is an aspect of little research. The aim of this study was carried out to partially characterize the chemotaxis, endocytosis and bacteriocidal ability of the neutrophils found in the nine-banded armadillo (Dasypus novemcinctus). Results showed that the chemotactic activity of the neutrophils, evaluated by the movement of the neutrophils through a nitrocellulose membrane (5 microm) in response to a chemo-attractive substance, was greater towards the armadillo serum (5.16+/-1.35 migration index, p<0.05) than towards the formil methionyl leucil phenylalanine (fMLP, 1.43+/-0.18 migration index) or human serum (0.56+/-0.18 migration index). Regarding endocytic capacity of the neutrophils and the monocytes against Escherichia coli was evaluated by a flow cytometry and using opsonized and non-opsonized E. coli-FITC at the following incubation times: 5, 10, 20, 30 and 60 min. The largest percentage of endocytosis by the neutrophils was 92.32+/-0.12% with opsonized bacteria and 77.73+/-14.33% with non-opsonized bacteria at 10 min incubation time, while the largest percentage of endocytosis by monocytes was 89.94+/-1.40% with opsonized bacteria and 73.07+/-15.6% with non-opsonized bacteria at 20 min incubation time. Evaluation of the bacteriocidal capacity of neutrophils using the methyl-thiazol-tetrazolium salts (MTT) reduction color-measurement assay showed an 89.0+/-10% mortality rate of non-opsonized E. coli and 89.0+10% of opsonized E. coli. In conclusion, the armadillo neutrophils show a good phagocytosis and bacteriocidal activity; however, a deficiency in the migration towards the fMLP was observed. This deficiency could be a cause so that the armadillo neutrophils do not respond quickly to invading microorganism.     

Ultrastructure of equine endothelial cells exposed to endotoxin and flunixin meglumine and equine neutrophils

An in vitro system of cultured equine endothelial cells was evaluated as a model for endotoxin (ET) exposure in the horse. Primary cell lines from pulmonary vessels and aortas were cultured from tissues of 6 horses. Effects of ET alone with and without serum and in combination with the cyclo-oxygenase inhibitor flunixin meglumine and isolated equine neutrophils were evaluated by transmission electron microscopy. Cells plus serum were incubated with 10, 25, 50, or 100 micrograms of ET/ml of incubation medium for 1, 3, 8, or 24 hours. Cells without serum were cultured for 1 and 3 hours. Flunixin meglumine was used at a concentration of 20 micrograms/ml. Cells also were incubated in the presence of 1,000, 5,000, or 20,000 neutrophils/ml plus ET and in the presence of a combination of ET and flunixin meglumine for 1 or 3 hours. Endotoxin alone did not cause cell damage, and the only evidence of an effect was an increased number of secondary lysosomes at incubation hour 8. At incubation hour 24, cells appeared normal. Endotoxin plus neutrophils caused cells to become round and detach from the growth substrate. Cell pathologic changes included swollen and distorted mitochondria and cytoplasmic vacuolization. Response to the ET plus neutrophil combination was variable and ranged from 5% to 50% of the cells being affected. The variability appeared to have some correlation with cell age, as well as individual preparation of neutrophils.