ASSOCIATION BETWEEN HANDLING STRESS IN THE CORRAL AND RABIES ANTIBODY TITERS IN SELENIUM-SUPPLEMENTED CATTLE

This study determined the correlation between serum cortisol levels and rabies antibody titers in cattle primo-vaccinated against rabies and supplemented with dietary selenium (Se). Sixty Nelore male calves (10 to 12 months old) received daily and individual dietary supplementation with 0, 3.6, 5.4 and 6.4 mg Se (groups Gc, G3.6, G5.4 and G6.4, respectively). The animals were vaccinated against rabies (day 0) and subjected to handling stress in the corral for 120 days. Blood sampling procedures were performed on days 0, 15, 30, 60, 90 and 120. Cortisol levels increased until day 90, but had dropped significantly by day 120 (P < 0.01). Rabies antibody titers on days 30 and 90 were similar among Sesupplemented groups; in the control group, rabies antibodies decreased significantly from day 30 to 60, and 90 to 120. Serum cortisol levels and antibody titers were not correlated in most of the groups or blood sampling days. A positive correlation among these variables was found only in G6.4 on days 60 (R = 0.513; P = 0.05) and 120 (R = 0.644; P = 0.009). In conclusion, repeated handling in the corral stresses cattle, but without compromising rabies humoral immune response.

The large-scale and regular vaccination of cattle is an efficient low-cost method that prevents and controls rabies infection, minimizing economic losses (1,4,5).
However, inappropriate stimulation is employed during cattle vaccination, such as driving animals from pasture to corral, physical exercises forced during handling in the corral, exposure of animals to the corral environment, the presence and shouts of the stockman as well as pain from vaccine application (6)(7)(8)(9)(10)(11).
Stressors activate the hypothalamus-pituitary-adrenal (HPA) axis that stimulates the adrenal glands to release hormones including cortisol, epinephrine and noradrenalin (12,13).High levels of blood cortisol provoke immunosuppression, rendering the cattle more susceptible to infectious diseases (12,14).Moreover, vaccinated animals may be vulnerable to infectious diseases if minimum antibody protection levels are not reached.
In addition, the lack of nutrients, such as selenium (Se), account for cattle susceptibility to these infections.Selenium deficiency is associated with low resistance, i.e., decreased antibody production and lymphocyte proliferation (15,16).
Selenium is scarce globally, although it is essential to many organic functions, based on its antioxidant properties and its incorporation in selenoproteins such as glutathione peroxidases (GSH-Px) that protect cell membranes against free radical damage and eliminate peroxides from blood and extracellular space (15,16).The present study investigated the effect of Se-supplemented diets on anti-rabies humoral response of Nelore (Zebu) cattle under handling stress.This was achieved by correlating serum cortisol levels and rabies antibody titers.
The experiment was carried out from February to June 2007, in Lutécia, São Paulo state, Brazil.Sixty Nelore cattle (Bos taurus indicus), aged between 10 and 12 months, were maintained on an extensive pasture system in which they grazed on Brachiaria decumbens forage.The cattle were randomly divided into four groups (15 animals each).All groups were fed ad libtum Top Line Recria® protein mineral mixture (Matsuda Sementes e Nutrição Animal Ltda., Brazil), approved by the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA).The control group (Gc) was fed exclusively this diet while the other groups received this mixture Reis LSLS et al.Association between handling stress in the corral and rabies antibody titers in seleniumsupplemented cattle.J Venom Anim Toxins incl Trop Dis.2009;15(4):780 supplemented with daily concentrations of 3.6, 5.4 or 6.4 mg of Se (groups G 3.6 , G 5.4 and G 6.4 , respectively).Given that each animal consumes about 200 g of mineral mixture per day (as measured during the experiment), diets for groups G 3.6 , G 5.4 and G 6.4 included, respectively, 18, 27 and 32 mg of selenium per kilogram.
Each experimental group was kept in a paddock (100 kg live weight/ha) in a continuous grazing system with groups rotated among the four paddocks every 30 days.The fields had similar topography and were covered with Brachiaria decumbens.The mineral mixture was offered in covered wooden feeders (13 cm length, available for each animal), while water was placed 50 m from the feeders.
After 30 days of cattle adjustment to management conditions and to experimental diets, animals were primo-vaccinated against rabies on day 0.Over the next 120 days, the cattle underwent five handling sessions for blood collection (on days 15, 30, 60, 90 and 120).Blood samples were employed to determine serum cortisol and rabies antibody titers.
Handling stress was imposed on cattle in the morning, while animals were being prepared for vaccination and blood sampling.The stressors consisted of the following stimuli commonly used in livestock practices: driving from pasture to the corral, time spent in the corral, the presence of a shouting stockman, rabies vaccination or blood sampling, forced movement in the corral, restraint of animals in a stun box for five minutes.
The rabies vaccine was subcutaneously injected in individual 2-mL doses.This inactivated commercial vaccine (Alurabiffa®, Merial, Brazil), approved by MAPA, is indicated for cattle and consists of a Pasteur-fixed rabies virus (PV) suspension, replicated in BHK 21 clone-13 cells.
Blood was collected by puncture from the jugular vein into 10-mL vacuum tubes, without anticoagulant.Blood samples were centrifuged at 2,500 rpm for 10 minutes; subsequently, serum samples were stored in 1.5-mL tubes at -20°C.Commercial kits  On day 0, forage samples from all paddocks were collected and stored at -5°C.
Selenium concentration was determined from these samples through graphite furnace atomic absorption spectrophotometry.
Serum cortisol concentrations were compared among groups (Gc, G 3.6 , G 5.4 and G 6.4 ) and days of observation (T 0 , T 15 , T 30 , T 60 , T 90 and T 120 ) by repeated measure analysis of variance followed by the least significant difference (LSD) test to compare significant differences (19).An alpha error of 0.05 was set for all tests.
Data on rabies antibody titers contradicted normal predictions (Kolmogorov-Smirnov test) and were, therefore, analyzed using Friedman non-parametric test that examined these titers temporally within each group (19).An alpha error of 0.05 was established for the tests.
Serum cortisol levels and rabies antibody titers were correlated using Spearman's non-parametric correlation (R) (19).Sixteen correlations were tested for the different groups and blood sampling days.An alpha error of 0.05 was set for all the tests.Serum cortisol concentrations were not affected by selenium supplementation (F (3,65) = 0.35; P = 0.79; data pooled for each day of observation) nor by any interactions between selenium concentration and time (F (15,280) = 1.41;P = 0.14).Concerning the overall temporal effect, serum cortisol levels increased gradually in all groups throughout the study (ANOVA; F (5,280) = 32.65;P = 0.01).Cortisol levels had peaked by day 90 and had decreased by day 120 (Figure 1).Rabies antibody titers were constant from day 30 to 90 in groups G 3.6 , G 5.4 and G 6.4 , but decreased in Gc (P < 0.05) from day 30 to day 60 onward (Figure 2).Only two positive correlations between serum cortisol levels and rabies antibody titers were detected, on days 60 and 120, in group G 6.4 .No other correlation was found among the groups or on blood sampling days (Table 1).The selenium concentration in paddock forage was 0.04 mg Se/kg.
for solid phase radioimmunoassay (Coat-A-Count®, Diagnstic Products Corporation, USA) and Cobra II Gamma Counter® (Packard Bio Sciences, USA) were utilized to determine serum cortisol levels.Serum neutralization of BHK 21 clone-13 cells was used to determine rabies neutralizing antibody titers, according to the rapid fluorescent focus inhibition test (RFFIT) and the fluorescent inhibition microtest (FIMT)(17,18).

Table 1 .
Reis LSLS et al.Association between handling stress in the corral and rabies antibody titers in seleniumsupplemented cattle.J Venom Anim Toxins incl Trop Dis.2009;15(4):784 Spearman's correlation coefficient (R) for serum cortisol levels and rabies antibody titers