Relationship between mitogen receptors in peripheral blood lymphocytes and blastogenic responses to mitogen

The relationship between the optimum concentration of mitogen which induces lymphocyte blastogenic response and the receptor occupancy by mitogen was investigated. The receptor occupancies which induced maximal blastogenic activity in equine, bovine and canine peripheral blood lymphocytes (PBL) were 31.1 per cent, 26.5 per cent and 38.4 per cent with phytohaemagglutinin-P, and 48.2 per cent, 17.9 per cent and 24.5 per cent with concanavalin A, respectively. The data clearly show that each animal species had its own optimum concentration of mitogen for stimulation of PBL. Optimum concentration for blastogenesis and number of binding sites of each mitogen had a good correlation with each other for all three species.     

In vitro blastogenic responses of peripheral lymphocytes from marmosets to phytohemagglutinin and to autologous lymphocytes transformed by Epstein-Barr virus

White-lipped marmosets were evaluated for their cell mediated immune (CMI) response to EBV to determine the feasibility of CMI studies in marmoset models for EBV oncogenesis. The mitogen, cell concentrations, the length of incubation period and serum requirements were defined for in vitro lymphocyte stimulation tests. The level of response of each animal was dependent on the concentration of phytohemagglutinin-P (PHA-P) and was independent of cell densities employed. The rate of tritiated-thymidine incorporation by mononuclear cells due to PHA-P increased exponentially between 2–4 days. This test was reproducible for a given batch of PHA-P when the cells were cultured in the presence of 10% heat inactivated fetal bovine serum. The five white-lipped marmosets were seronegative for EBV antigens and did not show lymphocyte stimulation with EBV particles and EBV soluble antigen, but two of these animals exhibited significant stimulation with autologous lymphocytes transformed in vitro by B95-8 virus. Despite the limited amount of blood (3–4 ml) that could be obtained from each animal in a single bleeding, it was possible to perform multiple lymphocyte stimulation assays with the protocol used.     

Suppressed lymphocyte blastogenic responses and enhanced in vitro growth of infectious bovine rhinotracheitis virus in stressed feeder calves

Hereford steers were stressed on a large-animal treadmill operating at speeds of 1.8 to 2.2 m/s. Blood samples were collected from indwelling jugular catheters before, during, and after exercise. Peripheral blood lymphocytes from stressed calves at 5 and 30 minutes after exercise had less (P less than 0.01) mitogen-induced blastogenic responses when compared to pre- or 60-minute postexercise values. Serum from stressed calves incorporated into lymphocyte cultures from nonstressed steers resulted in less (P less than 0.01) lymphocyte blastogenic responses. Infectious bovine rhinotracheitis viral growth in bovine kidney cell cultures was enhanced 4-fold when cultured with serum from stressed calves. These data indicate that acute physical exertion may cause physiologic alterations in calves that modulate cellular immunity and viral replication.     

Changes in blastogenic responses of lymphocytes and delayed type hypersensitivity responses after vaccination in dogs.

To clarify the immunologic effects of vaccination in dogs, we monitored total leukocyte and lymphocyte counts, humoral antibody responses, blastogenic responses of lymphocyte, and delayed type hypersensitivity (DTH) responses after vaccination. Mixed vaccines were administered on day 0 except for canine parvovirus (CPV) vaccine which was readministered on day 21. The puppy and adult dogs had a significant decrease in leukocyte and lymphocyte counts on day 7. The puppies showed a significant increase in the blastogenesis of lymphocytes after each vaccination, whereas the adult dogs had no significant changes. However, the adult dogs were divided into two groups, high responders and low responders in blastogenesis of lymphocytes. The dogs with higher or lower response in SI values on day 0 tended to show decrease or increase after the first vaccination, respectively. Since almost all dogs developed high titers of humoral antibody, it is considered that vaccination acts in an immunomodulative fashion. DTH responses to phytohemagglutinin (PHA) and CPV vaccine monitored at 0, 3, and 8 weeks after the first vaccination produced strong reactions, in particular those to CPV vaccine rose significantly after vaccination and maintained the higher responses for at least 2 months. These results suggest that DTH responses to PHA and CPV vaccine are helpful to monitoring non-specific and specific immune functions in vivo, therefore, DTH could be used as simple and rapid immunologic tests in canine practice.     

Analysis of pokeweed mitogen-induced in vitro proliferative and antibody responses of bovine lymphocytes.

The functional role of subpopulations of bovine cells in the regulation of pokeweed mitogen (PWM)-induced proliferative and antibody responses of peripheral blood mononuclear cells (PBM) was analysed. Subpopulations of bovine PBM identified by specific monoclonal antibodies (mAbs) were isolated by sorting them through the fluorescence activated cells sorter (FACS). The depletion from PBM of T cells bearing the CD4 marker, recognised by mAb IL-A12, resulted in a reduction of PWM-induced responses, which could be partly reversed by the addition of CD4 positive T cells. The depletion of cells belonging to the macrophage/monocyte lineage also resulted in a reduction of PWM-induced proliferative responses. PBM depleted of CD8 positive T cells, recognised by mAb IL-A51, showed increased PWM-induced responses, which in turn were reduced by the addition of mAb IL-A51 positive cells. Proliferative and antibody responses were also obtained by PWM stimulation of FACS-purified B cells, in the presence of bovine T cell growth factor.     

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 corticosteroids on responses of bovine peripheral blood lymphocytes cultured with phytohemagglutinin.

Corticosteroids given in vivo altered the response of lymphocytes in the peripheral blood of calves. Lymphocytes were cultured and stimulated in vitro with phytohemagglutinin (PHA). After an initial suppression of lymphocyte responses to PHA, there was a rapid return to normal. It is concluded that, in calves, short-term, high-dose immunosuppressive therapy with corticosteroids produces a population of lymphocytes resistant to corticosteroids, possibly by destruction of corticosteroid-sensitive lymphocytes.     

In vitro stimulation of bovine peripheral blood lymphocytes: suppression of phytomitogen and specific antigen lymphocyte responses by aflatoxin.

The effects of aflatoxin B1 on responses of the peripheral blood lymphocytes (PBL) of 2 normal animals to phytohemagglutinin, concanavalin-A, and pokeweed mitogen and of the PBL of 2 Mycobacterium bovis-infected animals to phytohemagglutinin and purified protein derivative of M bovis (PPD) were studied. Aflatoxin concentrations of greater than or equal to 10 microgram/ml significantly suppressed the lymphocyte response of normal animals to the phytomitogens. Lymphocyte response of M bovis-infected animals to specific antigen PPD was significantly suppressed at aflatoxin concentration of 0.5 microgram/ml. Fifty- to 100-fold higher concentrations of aflatoxin were required to produce 50% suppression of lymphocyte response to phytomitogens, as compared with that produced to PPD.     

Specificity of the anti-inflammatory effect of a staphylococcal cell wall extract in the bovine udder

The cell wall of Staphylococcus aureus (strains 21 and Glaxo) was treated with deoxycholate and the insoluble residue was solubilised with lysozyme. The effect of the extract in modulating the inflammatory response due to infection of the lactating bovine udder was evaluated. Cows were infected with S. aureus strain 21 or Streptococcus agalactiae, with or without the cell wall extracts. The clinical response to infection was assessed, and milk samples collected up to 30 h were assayed for antitrypsin and NAGase levels, somatic cell count, and for the ability of whey to support bacterial growth. The extracts markedly reduced the inflammatory response elicited by both S. aureus and S. agalactiae, indicating the effect was non-specific. The extract from strain 21 was generally more effective than that from strain Glaxo.     

Effect of prostagladin F2 alpha on the bovine fetal ruminal wall in vitro

Strips of rumen wall from bovine fetuses were incubated in an organ bath with prostaglandin F₂ alpha (0.13 to 33.76 g/ml). The highest reactivity with a submaximal dose (17.03 g/ml) was observed in the period between 3.0 and 7.9 months of fetal age. A smaller response, but higher than in 1.0 to 2.9 months old fetuses, was observed in the 8.0 to 8.9 months fetuses. The period of the highest reactivity to prostaglandin F₂ alpha coincides with the age of onset of papillary morphogenesis and the period of highest reactivity to autonomic and putative transmitter drugs.     

Replication of bovine respiratory syncytial virus in bovine and ovine peripheral blood lymphocytes and monocytes and monocytic cell lines

The present study compared the replication of bovine respiratory syncytial virus (BRSV) in bovine and ovine peripheral blood mononuclear cells, ovine and bovine monocytic cell lines and ovine alveolar macrophages. Low titres of virus were detected in ovine and bovine lymphocytes and monocytes 24–96 h post-exposure to the virus but there was no apparent replication of the virus in ovine alveolar macrophages during the culture period. The virus replicated to higher but statistically insignificant titres in ovine and bovine peripheral blood monocytes than in lymphocytes, with lymphocytes yielding peak titres significantly earlier. The secondary cell lines obtained from ovine liver and bone marrow also supported the replication of BRSV to high titres. The titres of BRSV in ovine and bovine lymphocytes and monocytes were significantly lower than in secondary cell lines. The addition of human recombinant tumour necrosis factor alpha after exposure to the virus or pre-incubation of ovine or bovine monocytic cells with either human recombinant interleukin 2 or phorbol myristate acetate before exposure to BRSV, did not significantly affect virus titre. Pre-incubation of cells with indomethacin or actinomycin significantly lowered virus titre (p<0.05).     

In vitro activation of bovine macrophage and peripheral blood mononuclear cells by Nocardia rubra cell wall skeleton (N-CWS)

Nocardia rubra cell wall skeleton (N-CWS) was used for immunotherapy to bovine leukemia virus (BLV)-positive cattle with enlarged subcutaneous lymphatic nodules. Electron microscopical observations showed the infiltration of macrophage and T cells around N-CWS treated lesions. Antitumor effect induced by N-CWS was examined in vitro. The maximum cytolytic activity of macrophage was observed, when the cells were incubated with 10 micrograms/ml of N-CWS. Chemiluminescence response of peripheral blood mononuclear cells (PBMC) using N-CWS as stimulant was observed with a high level of activity for a long period, 5 hr. Mitogenic effect of N-CWS to PBMC was observed in the presence of macrophages but not without macrophages. Furthermore, interleukin 2 activity was recognized in supernatant of PBMC cultured with N-CWS. Maximum cytotoxic T lymphocyte response was induced when PBMC were cultured with 0.1 micrograms/ml of N-CWS.     

Effect of peripartal administration of mycobacterium cell wall fraction on health and fertility of Holstein cows under organic-certified management

The main objective of this study was to evaluate the effect of peripartal administration of a commercially available nonspecific immune stimulant (mycobacterium cell wall fraction; MCWF [Amplimune, NovaVive Inc., Napanee, ON, Canada]) on the incidence of disease during early lactation and subsequent fertility of dairy cows. A second objective was to characterize the dynamics of circulating white blood cells (WBC) and metabolic markers following treatment administration. Cows in an United States Department of Agriculture (USDA) organic-certified dairy herd were blocked by parity and, based on sequential calving dates, randomly assigned to receive two injections (5 mL s.c.) of either a placebo (saline solution) as a control (CON; n = 71) or MCWF (n = 65) at enrollment (7 d before expected calving) and within 24 h after calving. Blood samples were collected from a subsample of the study population (MCWF = 16; CON = 18) for WBC count at enrollment, at day 2 post enrollment, and at days 1, 3, 7, and 14 after calving. Serum fatty acids, beta-hydroxybutyrate, and Ca concentrations were determined at days 1 and 7 postpartum (MCWF = 21; CON = 21). Main outcome variables included incidence risk of peripartal and early lactation health disorders and pregnancy at first artificial insemination (AI), at 100, and at 150 days in milk (DIM). In addition, the average daily milk yield up to 90 DIM and death and live culling before 305 DIM were compared. Treatment effects were assessed using multivariable logistic regression, time-to-event analyses, and repeated measures analysis of variance (ANOVA). A treatment effect on the incidence risk of some of the health disorders in the study was established. Incidence risk of metritis and clinical mastitis <28 DIM was smaller in MCWF than in CON cows (36.9% vs. 50.7% and 6.3% vs. 19.7%, respectively). On the contrary, the incidence risk of respiratory disease <28 DIM was smaller in CON (0%) than in MCWF (7.7%). Reproductive performance of multiparous cows was affected by MCWF administration: pregnancy at first AI and pregnancy at 100 and 150 DIM were greater in MCWF than in CON (35.6% vs. 19.2%; 51.1% vs. 25.0%; and 64.4% vs. 40.4%, respectively). Overall, median intervals from calving to pregnancy were 90 vs. 121 d in MCWF and CON cows, respectively. No treatment effects on the dynamics of circulating WBC or in postpartum metabolic status were established. No differences for milk yield or for the proportion of cows that survived up to 305 DIM were determined, although cows in MCWF left the herd earlier than cows in CON. In conclusion, incidence risks of metritis and mastitis in early lactation were smaller in cows receiving MCWF, whereas the incidence risk of respiratory disease was smaller in CON. Fertility significantly improved in MCWF compared with CON cows. As this study was performed in an organic-certified dairy, specific health and reproductive management practices may affect the external validity of the current findings.     

Involvement of calcium in mitogenic and immunoglobulin responses in bovine lymphocytes

Removal of calcium from cultures of bovine splenic lymphoid cells with ethyleneglycol-bis-(2-amino ethylether)-N,N'-tetraacetic acid (EGTA) caused significant suppression of pokeweed mitogen-induced immunoglobulin (Ig) biosynthesis. It was found that removal of calcium for 48 hours of a 5-day culture significantly altered Ig biosynthesis. Re-addition of calcium after 48 hours did not restore Ig synthesis. The EGTA caused significant (P < 0.05) suppression of DNA synthesis when calcium was removed from concanavalin A- and purified protein derivative-stimulated peripheral blood lymphocyte cultures. Further, removal of calcium for 48 hours of 4-day cultures significantly altered the DNA synthesis in mitogen- and antigen-stimulated cultures.     

Influence of sarcoptic mange and cold and ambient temperature on blastogenic responses of lymphocytes and serum cortisol concentrations of pigs

Blood samples from sarcoptic mite-infested pigs were evaluated for effects of mite infestation and cold and ambient temperatures on lymphocyte blastogenic responses and for effects of mite infestation on serum cortisol concentrations. In experiment 1, sarcoptic mite-infested and noninfested pigs were housed in cold (5 to 15 C fluctuating) and thermoneural (25 C) environmental chambers for 5 weeks. Differences were not observed (P greater than 0.10) in blastogenic responses to phytohemagglutin or pokeweed mitogen between lymphocytes from infested and noninfested pigs on postinfestation days (PID) 7, 21, 28, and 35 in either environmental chamber. When lymphocytes from noninfested pigs were cultured with sera from infested pigs, alterations of blastogenic responses were not detected. Cortisol values were higher (P less than 0.05) in sera from sarcoptic mite-infested pigs, compared with those from noninfested pigs, at 4 PM on PID 14 and 4 AM and 10 AM on PID 15. Cortisol values were higher (P less than 0.05) in sera obtained at 10 AM on PID 14 and at 10 AM on PID 15 from pigs housed in cold chambers, compared with those from pigs housed in thermoneutral chambers. Interactive effects between sarcoptic mite infestation and cold ambient temperatures were not observed. At 4 AM on PID 15 (experiment 2), cortisol values were higher (P less than 0.05) in sera of infested pigs, compared with those in noninfested pigs. Seemingly, sarcoptic mange in pigs did not alter mitogen-induced lymphocyte blastogenic responses, but did increase serum cortisol concentrations, indicating that sarcoptic mange may be a stressor in pigs.     

The inhibitory effects of colchicine, vinblastine and cytochalasin D on lymphocyte blastogenic responses

The inhibitory effects of colchicine, vinblastine and cytochalasin D on blastogenic responses of equine, bovine and canine peripheral blood lymphocytes (PBL) were investigated. These drugs inhibited blastogenic responses of each PBL. The concentrations for 50% recovery in PBL blastogenic response of colchicine and vinblastine were lower in equine and canine PBL than in bovine PBL. This suggested that microtubules may be more concerned in blastogenic response of equine and canine PBL than of bovine PBL. On the other hand, the concentration for 50% recovery in PBL blastogenic response of cytochalasin D was almost the same in the PBL of each animal. This meant that microfilaments made only a small contribution to the lymphocyte blastogenic response.     

Comparison of protection induced in lambs by Corynebacterium pseudotuberculosis whole cell and cell wall vaccines

Colostrum-deprived lambs were vaccinated IM with 10 mg (dry weight) of Corynebacterium pseudotuberculosis whole cells (WC) or cell walls (CW) and their immunity was challenged by IV injection of 3.1 X 10(4) colony-forming units of C pseudotuberculosis. Before challenge exposure, the logarithmic mean antibody titers were 2.0837 in lambs that were vaccinated with WC, 2.6858 in lambs that were vaccinated with CW, and 1.4214 in control lambs. Significant protection was demonstrated by fewer abscesses and organisms in the lungs of lambs vaccinated with WC or CW (P less than 0.05) than in control lambs. By the same criteria, more protection was provided to lambs vaccinated with CW than to lambs vaccinated with WC.