Immune Response of Sheep at Subchronic Intoxication by Pyrethroid Insecticide Supercypermethrine

Mikula I., J. PistI, P. Kacmar: Immune Response of Sheep at Subchronic Into­ xication by Pyrethroid Insecticide Supercypermethrine. Acta vet. Brno, 61, 1992: 57-60. Testing of the influence of subchronic (6 weeks) load of sheep organism caused by the pyrethroid insecticide supercypermethrine on functionality of cells of the immune system was carried out. Insecticide was administered daily in molasses feed in the doses 50,200 and 300 mg . kg-1 of live weight. During the last two weeks (5th to 6th weeks) of the experiment we recorded a decrease of the phagocytic index dependent on the supercypermethrine dose. Percentage of E rosette forming cells and the migra­ tion index were either unchanged or displayed insignificant dose-dependent values. Pyrethroid insecticide, supercypermethrine, sheep, phagocytic acti7Jity, E rosette test, leucocyte migration index Outer environment represents the source of a great number of different chemical factors influencing the immune response of humans and animals (Moore and Faith 1976). Many chemical preparations have been developed and applied in free nature during the recent decades. This refers most often to herbicides and insecticides. Their wide-area application brings along the toxicological hazards as well. Characteristics of pesticides from the viewpoint of their influence on animal health represents a highly topical aspect of the present. Besides the classical


Experimental animals
Sheep of Slovak merino breed of both sexes, aged 7 months, 21-28 kg of live weight, were used as model animals in the study of subchronic (6 weeks) intoxication.They were divided into three groups, five animals in each.Insecticide substance supercypermethrine added to the molasses feed was administered to sheep of the first experimental group in the daily dose 50 mg .kg-1 of live weight.Sheep of the second experimental group were offered the tested insecticide in the daily dose 200 mg .kg-1 of live weight, which was increased to 300 mg .kg-1 of live weight after four • weeks of experiment.The third group of sheep served as a control.

Blood sampling
Blood samples were obtained in one-week intervals during 7 weeks, in morning hours.
3. Immunological testing a) Determination of phagocytic activity (PA) Phagocytic activity and the phagocytic index of peripheral blood polymorphonuclears were determined using microspherical hydrophilic particles (MSHP kit, Institute for Research, Production and Utilization of Radioisotopes, Prague, according to V C!tvicka et al. 1982).
b) Phagocytic index (PI) Mean number of engulfed particles per one potentially phagocytic cell was determined as follows: PI = number of engulfed particles/100 potential phagocytes c) Determination of E rosettte forming lymphocytes (E rosette test, ER).
• Lymphocytes were isolated by means of Lymphoprep (Nyegaard, Oslo) using Harris and Ukaejiofo method (1969).E rosette test was carried out according to Binns (1978) method.Percentage of rosette forming lymphocytes was calculated as follows: % of rosettes = number of rosettes x 100/200 lymphocytes A lymphocyte with 3 or more attached lymphocytes was considered as rosette.• d) determination of inhibition of leucocyte migration (capillary migration-inhibition test MIT) (Bendixen et al. 1976).
Leucocytes were isolated from calf blood using the method of osmotic lysis of erythrocytes in deionized water (Karlson and Kaneko 1973).Each leucocyte sample was tested 3-times with mitogene (Phytohemagglutinin, HA-15, Wellcome, England) and without mitogene.Test was evaluated after 24-hrs incubation at 37 °e.Migration index (MI) was calculated on the basis of ratios of areas of leucocyte migration with mitogene (Mx) and areas without mitogene (Mo): MI = = Mx/Mo.Evaluation of MIT was carried out according to Wilson et aI. (1982).

Evaluation of tests
Mean values (x ± sd) of immunological tests in animal groups were evaluated by the Student's • t-test to determine the significance at 95% level.

Results
Immunological determinations were conducid in weekly intervals and the summary of results is presented in Table 1.Phagocytic activity did not display more pronounced differences of values in any group of sheep.In the 5th week of the experiment sheep of the second group~ which received the highest dose of preparation~ showed a marked decrease of the phagocytic index (5.2± 0.5) compared to the first (8.3 ± 0.9) and the control groups (8.2 ± 0.5).Statistically significant difference of PI values was recorded in the second group during the 5th and 6th week of the experiment (p < 0.05).
In the 6th week~ we observed a marked decrease of number of E rosette forming lymphocytes (23.6 ± 1.6 %), in the second group of sheep compared to the control group (32.2 ± 3.1 %)~ however~ this difference was not statistically significant.Mean values of the leucocyte migration index obtained in sheep of the second experimental group were on the positivity margin expressed by capability of lymphocytes to react to the mitogenic impulse of phytohemagglutinine.Mentioned differences of migration index values were not statistically significant.

Discussion
Mechanism of action of photostabile synthetic pyrethroids differs from that of organophosphates mainly by the fact that they do not integrate with the acetylcholine-esterase system (Elliot et al. 1978).Their oral~ dermal and inhalation toxicity is low since the metabolizing enzymes prevent their penetration into the nervous system (Elliot et al. 1978).Desi et al. (1980~ 1984Desi et al. (1980~ , 1985Desi et al. (1980~ , 1986) ) studied the effect of pesticide cypermethrine on the immune system of rats and rabbits.They discovered a dose-dependent significant (p > 0.05, p < 0.01) decrease of specific antibody titre (anti-ovoalbumine blood serum titre) at subacute intoxications of rats (1/40, 1,/20, 1/10 LD50)' This decrease was accompanied by decreased hypersensitivity reaction of the delayed type and peripheral blood lymphocyte decline (tested by the E rosette test) after 6 to 12 weeks of cypermethrine application.
We recorded in sheep a supercypermethrine dose-dependent statistically insignificant decrease of PI during the last two weeks (5th and 6th weeks) of our experiment.Other parameters (ER~ MI) remained either unchanged or showed dose-depen-dent~ although statistically insignificant differences.These results make it possible to state~ that longer lasting intake of supercypermethrine in relatively high doses can results in the damage of the immune state of experimental sheep.