Cell titration assay for measuring blastogenesis of bovine lymphocytes

The blastogenic response of bovine peripheral blood lymphocytes to phytohemagglutinin (PHA) and to microbial antigens was measured using a lymphocyte titration assay. Culture conditions, including lymphocyte concentrations, incubation periods and medium formulation, were established which produced linear or nearly linear responses over a range of cell concentrations. These conditions were established by testing lymphocytes from unimmunized cattle and from heifers infected with Brucella abortus with PHA and a B. abortus extract. Four cell concentrations in 2-fold increments were selected for measuring responses to PHA (3.125 X 10(3) to 2.5 X 10(4) cells/well) and to antigens (5.0 X 10(4) to 4.0 X 10(5) cells/well). The strength of response varied among animals and also over time for individual animals, but the titration assay allowed exponential proliferation to be distinguished from decline, which may have been due to overcrowding of microtiter wells, exhaustion of nutrients or induction of regulatory events. This assay provided a more reliable and discriminating method of evaluating lymphocyte proliferation responses than that achieved by single point assays. The displacement of the titration curves could be used to estimate the relative frequency of lymphocytes responding to antigens or mitogens.     

Human recombinant interleukin-2 augments in vitro blastogenesis of bovine and porcine lymphocytes

The recent cloning of the human gene encoding interleukin 2 (IL-2) has provided the means for economical production of large quantities of the pure lymphokine for clinical studies. Human recombinant interleukin 2 (HrIL-2) has been reported to have in vitro and in vivo immunomodulating effects in the murine system, suggesting the cloned gene product has cross-species activity. Bovine and porcine peripheral blood lymphocytes were tested for responsiveness to HrIL-2 in a lymphocyte blastogenesis assay. Not only was the HrIL-2 highly stimulatory but it also reconstituted lymphocyte responsiveness to maximal values following incubation with suboptimal concentrations of mitogen plus exogenous lymphokine. These studies suggest that HrIL-2 has the potential of serving as an in vivo modulator of immunoresponsiveness in domestic species. The contribution to food animal medicine will be considerable if administration of the lymphokine results in augmentation of antigen-specific immune responses when applied as an adjuvant, non-specific booster of pre-existing immunity, or for therapy of immunosuppression.     

Induction of blastogenesis in bovine peripheral blood lymphocytes by oxidation with sodium periodate

Suitable treatment and culture conditions are defined for the induction of blast transformation in bovine peripheral blood lymphocytes by oxidation with sodium metaperiodate (NaIO4). Stimulation with NaIO4 required slight modification of techniques used routinely for activation of lymphocytes in vitro with lectins and antigens. Gradient-separated mononuclear leukocytes responded with maximal [3H]TdR incorporation after oxidation with 0.50 to 1.0 mM NaIO4 for 30 minutes at 25 C. Oxidized cells cultured at 1 to 2 X 10(6)/ml responded better than cells cultured at any other concentration, when compared with untreated cells. Blastogenesis in response to oxidation reached its maximum rate within 48 hours of treatment, after which it declined rapidly. Partial removal of glass wool-adherent cells reduced periodate-triggered blastogenesis by 95%, but did not significantly affect activation with phytohemagglutinin, concanavalin A, pokeweed mitogen, or purified protein derivative. Reintroduction of macrophages restored responses to their precolumn level. Oxidation with NaIO4 provided a simple, rapid means of inducing blastogenesis in bovine lymphocytes. Manipulation of the well-defined triggering conditions may help to explain the mechanisms involved in lymphocyte activation.     

Effects of steroids on bovine T-lymphocyte blastogenesis in vitro

Day 13 to 24 bovine conceptuses convert C-19 and C-21 neutral steroids to 5 beta-reduced steroids with great efficiency. Pregnancy steroids have been reported to be immunomodulatory in several species. This study examined the possibility that 5 beta-reduced products of bovine conceptus steroid metabolism, precursors, related steroids, progesterone or cortisol might affect bovine T-cell blastogenesis. The steroids tested were 5 alpha-androstan-17 beta-ol-3-one, androstene-3, 17-dione, 5 beta-pregnane-3,20-dione and 5 beta-pregnane-3 alpha,20 alpha-diol. The ability of each steroid, over a dose range of 10(-2) to 10(4) nM, to affect phytohemagglutinin (PHA)-induced or concanavalin A (Con A)-induced bovine T cell blastogenesis, or the mixed lymphocyte reaction (MLR) was evaluated. Five lactating Holstein cows served as lymphocyte donors for mitogen studies and two, that exhibited strong MLR, were donors for the MLR evaluation. Of the seven steroids tested none affected blastogenesis in a dose-related manner except for cortisol, which suppressed Con A-induced lymphocyte transformation as well as the MLR. Cortisol did not affect PHA-induced blastogenesis. Thus, of the pregnancy steroids tested at the concentrations noted, none had significant immunomodulatory effects.     

Regulation of phytohemagglutinin-induced bovine lymphocyte blastogenesis by leukotrienes

Leukotriene (LT) B4, a 5-lipoxygenase metabolite of arachidonic acid, is a potent inducer of suppressor cells in phytohemagglutinin-stimulated cultures of bovine peripheral blood mononuclear cells. In contrast, LTC4 and LTD4 have little activity. Incubation of T lymphocytes with LTB4 at concentrations as low as 1 X 10(-12)M rendered these lymphocytes suppressive of [3H]thymidine incorporation in subsequent phytohemagglutinin-stimulated cultures of fresh autologous lymphocytes. This LTB4-induced cell was radiosensitive to irradiation at 2,000 rads. Leukotriene B4 may have an important part in immunoregulation during hypersensitivity reactions.     

Effects of ACTH administration on bovine polymorphonuclear leukocyte function and lymphocyte blastogenesis

Yearling steers were treated with ACTH to determine the effect of increased plasma cortisol concentration on bovine lymphocyte and polymorphonuclear leukocyte (PMN) function. The administration of ACTH caused a significant (P less than 0.01) increase in serum cortisol concentration and depression of lymphocyte blastogenesis in response to phytohemagglutinin and concanavalin A. The response to pokeweed mitogen was also depressed, but not significantly. Random migration by PMN was significantly enhanced by ACTH treatment, but there was no effect on ingestion of Staphylococcus aureus, nitroblue tetrazolium reduction, or antibody-dependent cell-mediated cytotoxicity by PMN. The iodination reaction, which evaluates the activity of the myeloperoxidase-hydrogen peroxide-halide antibacterial system of the PMN, was significantly impaired after ACTH treatment. These data indicate that specific parameters of lymphocyte and neutrophil function were impaired directly or indirectly by elevated in vivo concentrations of plasma cortisol.     

不同浓度防冻剂和投液氮温度对牛体外受精胚胎冷冻效果的影响

比较了3种不同浓度的甘油（1.4,1.0,0.7mol/L）和投液氮温度（-25℃、-30℃和-35℃）对牛体外受精胚胎冷冻效果的影响。结果发现,最终投液氮温度为-25℃时,胚胎冻后存活率以1.4mol/L甘油组为最高,同1.0mol/L甘油组相比,差异显著（78.5%vs 64.5%,P〈0.05）;与0.7mol/L甘油组相比,差异极显著（78.5%vs53.5%,P〈0.01）。以1.0mol/L的甘油冷冻,-30℃投液氮的效果优于-25℃的,它们之间冻后胚胎的孵化率差异显著（77.1%vs 64.5%,P〈0.05）。用0.7mol/L甘油冷冻,投液氮温度以-30℃的为最好,冻后胚胎的孵化率（73.3%）极显著（P〈0.01）高于-25℃的（53.5%）。用1.4mol/L甘油冷冻,以-25℃投液氮为最好,冻后胚胎的孵化率（78.5%）显著（P〈0.05）高于-35℃的（65.7%）。结果表明,不同的甘油浓度和投液氮温度对牛体外受精胚胎的冷冻效果有显著的影响。     

Optimal conditions for in vitro blastogenesis of feline peripheral blood lymphocytes

A microculture technique was developed for the in vitro blastogenesis of feline lymphocytes. Blastogenesis of ficoll-diatriazoate gradient separated mononuclear cell, washed blood and whole blood were compared. In general the whole blood cultures yielded higher stimulation indices (SI) than the washed blood or separated mononuclear cell cultures.The effect of several variables on the stimulation of lymphocyte cultures was examined. A cell concentration of 3 × 10⁵ cells/well and a 1:20 dilution of washed and unwashed whole blood gave optimal stimulation with concanavalin A (Con A). Phytohaemagglutinin-P (PHA-P) did not give significant levels of stimulation. Inactivated fetal calf serum (FCS) at levels of 2.5% (for washed blood) and 5% (for separated mononuclear cell and whole blood) gave highest SI. Supplementation with FCS was preferable to autologous, homologous or horse sera for all cultures. Optimal SI was obtained in all cultures incubated for 3 days and labelled with 1 μCi tritiated thymidine (³H-TdR) for the last 16 hours. The highest SI were in the range of 70 to 105 (18,764 to 42,681 counts per minute (CPM) for separated mononuclear cell culture, 100 to 165 (28,403 to 45,334 CPM) for washed blood culture and 105 to 186 (41,076 to 69,999 CPM) for whole blood culture.     

Lymphocyte blastogenesis in bluetongue virus or Mycobacterium bovis-inoculated bovine fetuses

Bovine fetuses were inoculated with Mycobacterium bovis, bluetongue virus or placebo at approximately 125 days of gestation, and blastogenic responses of peripheral blood lymphocytes and lymph node cells were determined at various time intervals after inoculation. Lymphocytes from all fetuses were stimulated by phytohemagglutinin, concanavalin A, and pokeweed mitogen, and peripheral blood lymphocytes gave consistently greater stimulation indices than did prescapular lymph node cells. Bluetongue virus infection did not consistently suppress mitogen induced lymphocyte blastogenesis. Lymphocytes taken from fetuses at 20 or 50 days after Mycobacterium bovis inoculation were not stimulated by purified protein derivative (PPD), whereas lymphocytes taken from adult cattle at similar intervals after Mycobacterium bovis inoculation were stimulated by PPD. Although lymphocytes from bovine fetuses may be stimulated by mitogens, antigen specific blastogenesis to a known inducer of cellular immunity was not detected by 175 days of gestation.     

Lymphocyte blastogenesis and cellular cytotoxicity in a congenital infection of bovine fetuses related to epizootic bovine abortion

The disease referred to as epizootic bovine abortion (EBA) was experimentally induced in bovine fetuses. Dark-field microscopy was used to detect congenital infection with an unclassified spirochaete-like organism. Some of the fetuses collected at abattoirs were also found to be naturally infected with a morphologically similar microorganism. Blood counts and organ weights were correlated with the presence of the microorganism. Lymphocyte blastogenesis increased, the result of in vivo stimulation among the infected fetuses. Phytomitogens (phytohaemagglutinin, concanavalin A and lipopolysaccharide) also stimulated greater responses in infected fetuses when compared to results in normal fetuses. Cellular cytotoxicity was examined by the single cell assay and results indicated that there were fewer cytotoxic lymphocytes among the diseased fetuses. The infected abattoir-collected specimens were obtained from clinically normal adult cattle, and the immunological changes in these fetuses were closely characterised with those of the EBA diseased fetuses. These naturally infected fetuses showed signs of a mild infectious disease.     

Ochratoxin A as a suppressor of mitogen-induced blastogenesis of porcine blood lymphocytes

The in vitro effect of ochratoxin A on pig lymphocytes stimulated by the mitogen Concanavalin A was studied by measuring the rates of ³H-thymidine incorporation in DNA.Ochratoxin A inhibited the mitogenic response to Concanavalin A in a dose dependent way. An almost total inhibition was obtained with ≥ 1 mg ochratoxin A/1, approximately 60 % inhibition was produced by 0.5 mg ochratoxin A/1 and approximately 10 % inhibition by 0.06 mg ochratoxin A/1.The immunosuppressive effect of ochratoxin A was not altered much by different contents of bovine serum albumin, 0.1 or 0.3 %, in the cell culture medium.     

Membrane IgM of bovine lymphocytes

The membrane immunoglobulins of peripheral blood lymphocytes (PBL) obtained from four bovine-leukemia-virus infected calves and from a normal cow were isolated and characterized. They were found to consist of an IgM exhibiting a μ-chain of an apparent molecular weight (AMW) of 95,000 daltons, which is 10,000 daltons more than found for the μ-chain of serum IgM. It thus seems that this is a property of membrane-bound IgM of bovine origin.     

Effect of levamisole on lymphocyte blastogenesis and neutrophil function in dexamethasone-treated cattle

Levamisole was evaluated at 6 dose levels for its ability to prevent the dexamethasone-induced suppression of in vitro lymphocyte blastogenesis or neutrophil function in cattle. Dexamethasone (0.4 mg/kg of body weight, IM) and levamisole hydrochloride (0.5, 1.0, 2.0, 4.0, or 8.0 mg/kg orally) were administered to groups of 4 cattle daily for 3 days. Another group of 4 cattle were given the 3-day dexamethasone treatment and 6.0 mg/kg of levamisole (the recommended anthelmintic dose) was given only once on the 1st day that dexamethasone was given. Results obtained from the dexamethasone-levamisole-treated cattle were compared with results obtained from cattle that were given only dexamethasone. Levamisole had no apparent consistent ability to enhance lymphocyte blastogenic responsiveness (to the mitogens phytohemagglutinin, concanavalin A, or pokeweed mitogen or in a 1-way mixed lymphocyte reaction) or to enhance neutrophil function (random migration, nitroblue tetrazolium reduction, iodination, or antibody-dependent cell-mediated cytotoxicity) in dexamethasone-treated cattle.     

Effect of phenylephrine, ergonovine, oxytocin and norepinephrine as an extender ingredient on viability of bovine spermatozoa

We evaluated effects of three concentrations of phenylephrine, ergonovine, oxytocin and norepinephrine (myometrial stimulants) on viability of spermatozoa when they were included in a seminal extender. Using a split ejaculate technique, ejaculates from each of 10 bulls were extended in egg-yolk citrate to a final concentration of 35 x 10(6) sperm/ml, including 20 mg/ml, 2 mg/ml and .2 mg/ml, of phenylephrine or ergonovine, 20 IU/ml, 2 IU/ml and .2 IU/ml oxytocin or 200 micrograms/ml, 20 micrograms/ml and 2 micrograms/ml norepinephrine prior to freezing. Extended semen without a myometrial stimulant served as the control. Percentage of intact acrosomes was determined prior to freezing for all treatments. Motility and percentage of intact acrosomes were determined immediately after thawing (0 h) and again after 4 h incubation at 37 degrees C. Percentage of intact acrosomes was reduced (P less than .05) prior to freezing by phenylephrine (20 mg/ml) and ergonovine (20 mg/ml) (phenylephrine = 56%; ergonovine = 63%; control = 74%). The same doses of phenylephrine and ergonovine reduced (P less than .05) post-thaw motility and percentage of intact acrosomes at both 0 and 4 h compared with controls. Sperm exposed even to the intermediate concentration (2 mg/ml) of ergonovine had lower (P less than .05) motility 4 h post-thaw. No other compound or concentration of compound or concentration of compound affected percentage of intact acrosome or motility. There were no two or three-way bull x compound and concentration interactions (P greater than .2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Lymphocyte blastogenesis and neutrophil function in cattle persistently infected with bovine viral diarrhea virus

Neutrophil function and mononuclear cell proliferative responses to mitogens were determined in healthy cattle and in cattle persistently infected with bovine viral diarrhea (BVD) virus. Uptake of [3H]thymidine by resting and mitogen-stimulated peripheral blood mononuclear cells was significantly lower in cattle persistently infected with BVD virus than in healthy cattle. Neutrophils from cattle persistently infected with BVD virus had significantly impaired capability to ingest Staphylococcus aureus, but were normal in respect to random migration under agarose, cytochrome C reduction, iodination, and antibody-dependent cell-mediated cytotoxicity. Impairment of neutrophil function in cattle persistently infected with BVD virus differs from impairment of neutrophil function reported in healthy cattle mounting an immune response to recent BVD virus infection.     

Effect of group thawing on post-thaw viability of bovine spermatozoa packaged in .5-milliliter French straws

The objective of this study was to determine the effects of thawing groups of 2, 5, 10, 15, or 20 .5-ml French straws on post-thaw spermatozoal viability. Thermostatically controlled and nonthermostatically controlled thawing baths were compared. Using a split-plot design, semen from 10 bulls was extended in egg yolk citrate, frozen, and then thawed (in the respective groups) at 36 degrees C in two types of thawing baths. Motility and percentage of intact acrosomes were determined immediately after thawing (0 h) and again after 4 h of incubation at the respective temperature of each thawing bath. Neither percentage of intact acrosomes nor motility was influenced by the number of straws thawed at 0 h (P greater than .05). Thawing bath had no effect (P greater than .05) on motility or percentage of intact acrosomes at 0 h. Bull variation was significant in both the 0- and 4-h evaluations. After 4 h of incubation, there was a significant (P less than .05) straw number x thawing bath interaction. When 15 or 20 straws were thawed in the thermostatically controlled bath there was a reduction (P less than .05) in motility and percentage of intact acrosomes. However, in the nonthermostatically controlled bath there was no reduction in motility and percentage of intact acrosomes as the size of straw group increased. Our results indicate that, when using a nonthermostatically controlled thawing bath, semen packaged in .5-ml straws can be thawed in groups of 20 without an effect on post-thaw sperm viability.