Effect of ammonia on viability and blastogenesis of bovine lymphocytes

The effect of addition of ammonia into the tissue culture on viability and functions of bovine lymphocytes was studied. The concentrations of ammonia in the tissue cultures represented toxic, subtoxic, and normal concentrations of ammonia in the bovine blood during clinical and subclinical urea toxicosis. Lymphocytes separated from peripheral bovine blood were incubated in control medium and test medium with various concentrations of ammonia and/or PHA or Con A. Viability of the lymphocytes was measured by trypan blue exclusion test and their mitogenic reactivity by incorporation of ³H thymidine into DNA of lymphocytes. Approximately 30% bovine lymphocytes were killed by ammonia in medium during 72 hours of incubation. Ammonia also affected the response of lymphocytes to stimulation with PHA or Con A as well as mixed lymphocyte culture reaction. The mitogenic response of lymphocytes was also reduced when lymphocytes were preincubated with ammonia for even 1 hour. The mitogenic response was not restored when the number of lymphocytes preincubated with ammonia was reconstituted to the initial concentration to compensate for the killed lymphocytes before stimulation with PHA. Therefore, addition of ammonia to the culture either killed lymphocytes or permanently impaired their functions.     

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.     

A rapid, quantitative assay for titration of bovine virus diarrhoea-mucosal disease virus

An end point dilution microtitration assay is described that can be used for the titration of both cytopathic and non-cytopathic isolates of bovine virus diarrhoea-mucosal disease virus. Indirect immunofluorescence is used to detect infected MDBK cells in the wells of Terasaki plates. The virus titre is derived from the number of uninfected wells, using the Poisson distribution. The assay is simple, fast and economical. Titres of cytopathic virus determined by the microtitration assay and standard plaque assay are equivalent.     

In vitro stimulation of bovine milk lymphocytes standardization of the assay for bovine brucellosis

Lymphocytes of bovine milk origin were investigated by immunostimulation in vitro to standardize the assay for measuring the immune responses of the cells which might be useful in further understanding the immunopathology and diagnosis of bovine brucellosis. The lymphocytes were separated from whole freshly collected milk by centrifugation. The pellet of lymphocytes was washed in RPMI-1640 medium, cultured at different concentrations for different days and with Brucella abortus soluble antigen strain 1119-3 and Concanavalin A. Each culture was labelled with 1.0 μCi of methyl-[³H]thymidine 16–18 hours prior to termination of incubation at 37 C. Termination was done by cooling to 4 C. The cells were harvested for liquid scintillation counting spectrometry. In the groups of calfhood vaccinated cows and nonexposed milkers, a milk lymphocyte concentration of 2.0 × 10⁶/ml of medium yielded a statistically significant blastogenesis. The Brucella abortus soluble antigen concentration of 4.4 μg of protein/well was found optimal to induce significant immunostimulation. A period of 4 days of incubation of the milk lymphocyte in the test was found optimal in inducing statistically significant blastogenesis in this system.     

Rapid assay for repair of damage induced by ultraviolet light in bovine lymphocytes

Bovine peripheral blood lymphocytes exhibit a peak of deoxyribonucleic acid (DNA) repair, measured after exposure of cells to ultraviolet light, three to four days after phytohaemagglutinin addition to diluted blood cultures. This peak of DNA repair is induced coincidentally with that of DNA replication. DNA repair is determined in the presence of hydroxyurea which inhibits DNA replication. This assay provides a rapid screening method for deficits in DNA repair synthesis in cattle.     

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.     

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 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.     

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.     

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.     

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.     

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.     

Enzyme-linked immunosorbent assay for bovine apolipoprotein A-IV

The present report describes an enzyme-linked immunosorbent assay for bovine apolipoprotein (apo) A-IV. This assay was applied to the determination of its concentration and distribution in sera from cattle. The distribution of apoA-IV in lipoprotein fractions separated by ultracentrifugation was mostly recovered in the non-lipoprotein fractions (d>1.21 g/ml, 90%), but, in the case of gel filtration chromatography, apoA-IV was mainly eluted in HDL and non-lipoprotein fractions. The apoA-IV concentrations during early, mid- and late lactating stages in cows were significantly higher than during the nonlactating stage (p<0.05). From early to late lactating stages, the concentration of apoA-IV was unaltered. After 4 days of fasting, the concentration of plasma apoA-IV had decreased significantly (p<0.05) at days 3 and 4, and was returned to the basal level by 3 days of refeeding. These results suggested that the concentration of apoA-IV is modified by nutritional conditions.     

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.