Bull. Vet. Inst. Pulawy, 287-292, 2005
INFLUENCE OF SUBCHRONIC EXPOSURE TO FUNGICIDE TOLYLFLUANID ON HUMORAL AND CELL-MEDIATED IMMUNE PARAMETERS IN SHEEP IMMUNIZED WITH COXIELLA VACCINE NATÁLIA KOVALKOVIČOVÁ, JURAJ PISTL, DANIEL ELIAŠ1, ANNA JACKOVÁ, JAROSLAV NOVOTNÝ, SABINA HLINČÍKOVÁ, JAROSLAV LEGÁTH, VIERA RÉVAJOVÁ AND GABRIEL KOVÁČ University of Veterinary Medicine, 041 81 Košice, Slovakia 1 Mevak a.s., 949 91 Nitra, Slovakia e-mail:
[email protected] Received for publication July 07, 2005.
Abstract The effect of subchronic exposure to fungicide tolylfluanid on humoral and cell-mediated immune parameters in Merino sheep was studied. Fungicide was administered by rumen tube at a daily dose of 1/20 LD50 for 90 days. Both exposed and control animals were immunized with vaccine Bodibion inj. a.u.v., containing inactivated bacteria Coxiella burnetti on day 45 and 60 of tolylfluanid exposure. The cell-mediated immune response was studied by assays evaluating the functional activity of phagocytes and lymphocytes. A significant decrease in metabolic activity of blood phagocytes was recorded on day 30 (P < 0.01) and day 60 (P < 0.05) of the exposure. Proliferation of lymphocytes activated by phytohaemagglutinin (PHA) was significantly decreased on day 30 of the exposure (P 1000 guinea pigs, rabbits 250 − 500 cats > 500 sheep 937.5 ± 312.5 (purity 99.2%)
289 Lympho-proliferative test (LPT). A colorimetric immunoassay was used to quantify lymphocyte proliferation, based on the measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA synthesis (Cell Proliferation ELISA Kit, BrdUcolorimetric, Roche, Germany). Suspensions of sheep lymphocytes (100 µl of 106 cells/well in RPMI 1640 with 10% FCS) were cultured for 72 h with 10 µg/ml of mitogen (phytohaemagglutinin, PHA, Sigma, Germany), and without mitogen in a 96-well microtitre test system at 37oC in a humid atmosphere at 5% CO2 for 4 d. Each culture was tested in triplicate. Eighteen hours before the end of cultivation, BrdU in concentration of 100 µmol was added and the cells were reincubated. After removing the medium, denaturation of DNA and fixation of the cells on the bottom of wells, 100 µl of anti-BrdU-peroxidase labelled conjugate was added and allowed to react for 90 min at room temperature. The immune complexes were detected by the subsequent substrate reaction (100 µl of substrate solution) for 30 min at room temperature. The reaction was stopped by 25 µl of 1 mol H2SO4 and the absorbance was measured in an ELISA reader at 450 nm. The cell activation rate was calculated as a stimulation index (SI) of lymphocytes according to a formula: SI = OD450 (PHA stimulated cells) / OD450 (non stimulated cells). Leukocyte migration-inhibition assay (LMIA). The LMIA, characterizing the reactive capacity of lymphocytes to mitogenic activation, was carried out according to the method described previously (13). Leukocyte suspensions (2.108/ml) were prepared in a culture medium RPMI 1640 with 25 mmol HEPES, 0.3 mg/ml of L-glutamine (Gibco, Germany), supplemented with 100 µg/ml of streptomycin, 100 IU/ml of penicillin (Gibco, Germany), and 10% FCS (Gibco, Germany). Cell suspensions (50 µl) were placed to glass microcapillaries (Sklárny Kavalier, Czech Republic). One end of the capillaries was carefully closed by heat. After centrifugation for 2 min at 400 g, the capillaries were cut at the interface of sediment and supernatant was placed into the wells of plastic plates (24-well tissue culture plate, Sarstedt, USA) and incubated at 37oC for 18 h in 5% CO2 atmosphere with 1 ml of RPMI 1640 medium with or without 10 µg/ml of PHA. All the tests were made in triplicate for each animal. PHA-activated T-lymphocytes produce higher levels of lymphokines including the migration inhibition factor (MIF). MIF causes an inhibition of leukocyte migration (monocytes, polymorphonuclears). The intensity of this inhibition depends on the quantity of MIF produced by the activated lymphocytes. The results were expressed as a migration index (MI), measured as the ratio of mean leukocyte migration areas with PHA and areas of leukocyte migration without PHA. In the case of routine interpretation of LMIA, MI
