IMMUNOGLOBULIN CHANGES IN BOARS EXPOSED TO ADMINISTRATION OF LEVAMISOLE AND EXOGENOUS ADRENOCORTICOTROPIC HORMONE

The objective of this paper is to determine the effect of levamisole (LEV) immunoglobulin concentration in the serum of boars exposed to three-day stress induced by the adrenocorticotropic hormone (ACTH). Boars were assigned to four groups (n=7). The first group received LEV for three days (2.5 mg/kg BW), the second group received ACTH (10 g/kg BW) for three days and the third group received LEV for three consecutive days and ACTH on the three following days (2.5 mg/kg BW ; ACTH 10 g/kg BW). The control group received saline solution (1 mL) during the experiment (six days). Concentrations of total protein, globulin, albumin and immunoglobulin concentration (IgG, IgA and IgM) were determined during levamisole, ACTH and levamisole + ACTH combined treatment administration, and during the 16-day post- administration period. Total protein levels were significantly increased during the stress period and over the next sixteen days (p0.01 to p0.0001, respectively) in the ACTH group of boars. However, total protein levels were significantly elevated in the LEV+ACTH group on day 1 and 2 of ACTH injection (p0.05 and p0.001) and on day 5 and the last day of measurement (p0.001 and p0.01). However, the three-day administration of levamisole (LEV) stimulated a statistically significant increase of protein concentrations compared to control values after levamisole treatment, on days 2 and on days 11, 15 and 24 (p0.01, all). Levels of serum albumin were not affected with levamisole or ACTH treatment. According to determined total protein concentrations, globulin concentrations were significantly increased throughout and after the ACTH treatment in the second group (p0.01 to p0.0001, respectively). Also, globulin concentrations were significantly elevated in the LEV+ACTH group throughout the three-day ACTH treatment and in the 16-day post-administration period (p0.05 to p0.01, respectively). The three-day levamisole administration resulted in an increase of globuline concentrations (LEV). However, there were no statistically significant differences in comparison to control pigs. ACTH treatment significantly elevated serum IgG concentration during the stress period (p0.001 to p0.0001, respectively) and over the next 16 days (p0.01 to p0.0001, respectively). However, in the LEV+ACTH group of boars, IgG levels were significantly elevated on days 1 and 3 of ACTH injection (p0.05 and p0.01) and days 1 and 5 in the post-treatment period (p0.01 and p0.05). Administration of levamisole (LEV) had no significant impact on IgG levels compared to the control boar group. However, a statistically significant increase in IgA concentrations in boars treated with levamisole (LEV) was determined on day 2 (p0.02) and day 11 (p0.05) after administration of levamisole compared to animals of the LEV+ACTH group. Furthermore, boars receiving levamisole before stress induction (LEV+ACTH) on day 5 after stress induction had IgM concentrations statistically significantly higher (p0.05) than animals treated only with ACTH. These results show that levamisole may stimulate the immune system in swine and immunoglobulin in the serum of boars exposed to stress.


Animals and Treatments
Swedish Landrace boars aged 6 months (n=28) and weighing between 110 and 145 kg were included in the study.Boars were housed in individual pens on farms.Stable temperatures varied between 9 o C and 15 o C during the experiment.The animals were given 3 kg of standard corn-soy ration per day.Water was available ad libitum.
Boars were randomly assigned to four experimental groups.During the 6 experimental days, LEV, LEV and ACTH, ACTH and saline were administered at 10:00 a.m.All animals were handled using restraint with a snare during the procedures of LEV, ACTH and saline administration, and blood collection.The control group (CONTROL, n=7) received 1 mL of placebo (sterile 0.9% saline) intramuscularly during the 6 experimental days.The first experimental group (LEV, n=7) intramuscularly received LEV (Nilverm, Veterina d.o.o., Zagreb, Croatia; preparation contains 75 mg LEV hydrochloride/mL) at the dose of 2.5 mg/kg BW on 3 consecutive days and then received 1 mL of placebo intramuscularly on days 4, 5 and 6.The second group (ACTH, n=7) was injected intramuscularly with 1 mL of placebo on days 1, 2 and 3, and then with ACTH (ACTH, from porcine pituitary, 80 IU/mg, Sigma-Aldrich Co., St. Louis) in a dose of 10 mg/kg BW on days 4, 5 and 6 into the ear vein.The third experimental group of animals (LEV+ACTH, n=7) was given LEV (preparation of 75 mg LEV hydrocloride/mL; Nilverm ® , Veterina d.o.o., Zagreb, Croatia) intramuscularly on days 1, 2 and 3 (2.5 mg/kg BW), and animals were then injected with ACTH (ACTH, from porcine pituitary, 80 IU/mg, Sigma-Aldrich Co., St. Louis) in a dose of 10 mg/kg BW on days 4, 5 and 6 into the ear vein.

Blood sampling
All boars were frequently handled and used to the blood collection procedure to be performed via the jugular vein 90 min after administration of LEV, ACTH or saline on treatment days 1, 3, 4, 5 and 6 and throughout the 16 days post-treatment (day 7, 11, 14, 18 and 22).Blood samples were collected using a sterile syringe (Becton Dickinson SA, Fraga, Spain) and immediately transferred to ice-cold glass tubes for serum separation (SST, DB Vacutainer ® , Preanalytical Solutions Belliver Industrial Estate, Plymouth, UK).Subsequently, blood samples were centrifuged for 10 min at 750 x g, the serum was separated and stored at -20 o C until analysis.

Cortisol analysis
Serum cortisol concentration was determined by radioimmunoassay using commercially available RIA Coat-A-Count Kit (Diagnostics Products Corp., Los Angeles, USA) according to the manufacturer's instructions.Samples were quantified in two assays, with average intra-and interassay coefficients of variation of 7.5% and 12.0%, respectively.The assay sensitivity was 0.1 nmol/L.

Serum proteins analysis
Technicon RA-1000 System Spectrophotometer (Technicon ® Instruments Corporation, Tarrytown, New York) was used for total protein and albumin analyses.
Total protein concentration was determined by the Biuret method using commercially available kits (Randox Laboratories Ltd., Crumlin, Co. Antrim, United Kingdom).Albumin content was determined by bromocresol green method using Randox Laboratories Ltd. kits.Intra-and inter-assay variation coefficients were 5.5% and 7.6%, respectively, for total protein and 4.3% and 5.1%, respectively, for albumin.
Globulin levels in serum samples were calculated as the difference between total protein and albumin concentrations.

Immunoglobulins analysis
Radial immunodiffusion (RID) was used for the determination of pig immunoglobulins (Mancini et al., 1965).Test plates for the determination of IgG were prepared by diluting 2 g of agarose in 100 mL of barbiturate buffered saline (0.1 M) with the addition of anti-pig IgG antiserum (1:10) and 0.1% sodium azide.Five microlitres of reference standard solutions of IgG and diluted serum samples (1:20) were pipetted to a separately identified well of test plates.The plate was securely covered and incubated for 48 to 72 hours at room temperature.Following incubation, the plates were removed and placed over a source of illumination to clearly see the precipitation rings.The external diameters of the rings were measured to the nearest 0.1 mm by using an ocular scale.A reference curve was plotted using the diameters measured from standard solutions.From the reference curve, the IgG concentration of each diluted test sample was calculated by multiplying the concentration read from the curve by the dilution factor to obtain the actual concentration.Intra-and inter-assay variation coefficients were 3.5% and 5.4%, respectively.
The concentrations of immunoglobulins A and M were determined using commercially available Pig IgA and IgM VET-RID kits (Bethyl Laboratories, Inc., Montgomery, Texas).Intra-and inter-assay coefficients of variation were 1.3% and 2.5%, respectively, for IgA and 0.5% and 1.2%, respectively, for IgM.

Statistical analysis
Data were analyzed using the Statistica software package 6.0 (StatSoft ® Inc., Tulsa, USA).Results were expressed as mean ± SE.Differences in studied cortisol levels and immunoglobulin parameters in the control and treated groups of boars with LEV or ACTH and the combined treatment by LEV and ACTH were examined using analysis of variance (ANOVA).To evaluate the differences in means between the control and treated groups of boars on specific days of measurement, the t-test for independent samples was used.Probability values of 0.05 or less were considered to be statistically significant.

Cortisol levels
The mean serum cortisol response in experimental groups of boars is shown in Table 1.The administration of 10 mg/kg body weight of ACTH in the experimental groups (ACTH and LEV+ACTH) induced an increase in serum cortisol concentration after 90 minutes on all three days of ACTH administration.The administration of saline and LEV had no effect on cortisol concentrations.
In both ACTH treated groups of boars the increase in cortisol concentrations was highly significant as compared with cortisol concentration after saline infusion on each treatment day (p<0.05).Also, the concentrations of cortisol were significantly higher in both ACTH treated groups 24 hours after the last ACTH dosage in comparison with control and levamisole treated boars.Thereafter, the concentration of cortisol returned to normal levels on days 11, 14, 18 and 22 after the last drug dosage.There was no significant difference in cortisol concentration between the two groups treated with ACTH.

Serum proteins
The effects of LEV, ACTH and the combined treatment LEV and repeated ACTH administration in the experimental group of boars (ACTH and ACTH+LEV) on serum concentrations of total protein are shown in Table 2. Total protein levels were elevated during the stress period (p<0.05) and over the next 16 days (p<0.05) in the ACTH group of boars compared to control animals.However, in comparison to the control group total protein levels were elevated in LEV+ACTH group on day 1 and 2 of ACTH injection (p<0.05) and day 5 (day 11) and on the last day of follow-up (day 22) (p<0.05).There were no differences in total protein levels between LEV+ACTH and LEV group of boars.However, administration of LEV during 3 days induced an increase in protein concentration compared to control values on day 2 (day 5) and on days 11, 15 and 24 (day 14, 18 and 22) after LEV treatment (p<0.05).
According to the determined total protein concentrations, globulin concentrations (Table 3) were increased throughout and after the ACTH treatment (ACTH) in relation to the control group of boars (p<0.05).Globulin concentrations were elevated in the LEV+ACTH group in comparison to the control group during all 3 days of ACTH treatment and in the next 16 days after the cessation of treatment (p<0.05).Higher globulin levels were measured in the ACTH group than in LEV treated animals on day 1 and 2 during treatment (day 4 and 5) and after treatment on day 7 (p<0.05).There were no differences in globulin levels between LEV+ACTH and LEV group of boars and also between LEV and control groups of boars (p>0.09).
There were no differences in serum levels of albumin (Table 4) among all four groups of boars throughout the study period (p>0.07).concentrations were elevated in the ACTH group during the stress period (p<0.05) and over the next 16 days (p<0.05).However, IgG levels were not changed in the ACTH group compared to the LEV group of boars (p>0.09).In the LEV+ACTH group, IgG levels were elevated on day 1 and 3 of ACTH injection (p<0.05) and day 1 (day 7) and day 5 (day 11) after treatment (p<0.05).There were no differences in IgG levels between LEV+ACTH and LEV group (p>0.08).

Immunoglobulins
LEV had no impact on IgG levels compared to the control group (p>0.08).There were no differences in serum IgA and IgM concentrations between treated groups and the control group throughout the study period (p>0.1).Also, there were no differences in IgA and IgM levels between ACTH and LEV group of animals (p>0.09).Higher IgA concentrations were determined in boars treated with LEV on day 2 (p<0.05) and day 14 (p<0.05)after administration of LEV compared to animals of LEV+ACTH group.

DISCUSSION
Different stress situations in animals can suppress the non-specific defence mechanism and cause increased susceptibility to disease and infection (Blecha, 1988;Griffin, 1989).In an intensive husbandry system, stressful events for pigs such as weaning, bacterial infections or a new social environment can change the behaviour or physiology, and also decrease the cellular and humoral immune response (Morrow-Tesch et al., 1994; Nagy and Fekete, 1999).Also, prenatal stress affects humoral and cellular immune responses of neonates and is able to alter the postnatal response of the immune system to stress (Tuchscherer et al., 1998).Protection from stress, or at least reduction of its effects, is significant for specific stages of breeding and reproduction of pigs.LEV has been shown to increase serum antibody titers after immunization, the number of leucocytes, phagocyte activity, expression of cytokines by monocytes/macrophages, lymphocyte proliferation and antitumor responses (Kimball et al., 1992;Szeto et al., 2000;Holcombe et al., 2001;Janjatovi} et al., 2008).
In this study the measured baseline cortisol levels were comparable to previously obtained concentrations in pigs (Becker et al., 1985;Hessing et al., 1994;Wallgren et al., 1994).Adrenocortical response to ACTH administration in this study induced a significant increase in serum cortisol concentration 90 min after application on all 3 days of ACTH treatment as shown previously (Biland`i} et al., 2006).Used dose of ACTH has been reported to induce a high cortisolresponse in treated animals (Janssens et al., 1994;Haussmann et al., 2000;Mwanza et al., 2000).In the present study, the significant increase in cortisol concentration persisted for 24 hours after the last ACTH dose as compared with the control group and levamisole treated animals.
Results obtained in this study have shown increased protein and globulin concentrations in both treated groups of boars during ACTH administration and during the two week period following completion of treatments.Previous studies had shown that the biosynthesis of immunoglobulins in lymphocytes depends on the duration of the stress situation and on the individual perception of stress in pigs (Hessing et al., 1995;Tuchscherer et al., 1998).Acute stress does not cause changes in the concentrations of total protein, globulin, albumin and IgG in boars (Hicks et al., 1998).However, in chronic stress situations, increased total protein concentrations were determined in sheep (Antunovi} et al., 2002) and pigs (Tuchscherer et al., 1998;De Groot et al., 2001).Chronic lice irritation in pigs was followed by an increase in total protein serum, globulin and albumin (Davis and Williams, 1986).Stress caused by transfer stimulates a lower proliferation of lymphocytes and synthesis of IgM, cytokine IL-10 and interferon g in boars than in sows (De Groot et al., 2001).Also, mixing of animals from various litters caused an increase in total protein and IgG concentrations in the dominant pigs in the group (Tuchscherer et al., 1998;De Groot et al., 2001).In pigs exposed to prenatal stress, immunoglobulin concentrations were reduced in the postnatal period (Otten et al., 2000).It was shown that increased cortisol excretion causes changes in the effects of macrophages, neutrophils, lymphocytes, and other immune system components.Changes in the lymphocyte subpopulations were observed, including changes in the biosynthesis of cytokines of helper T-cells (a type of Tlymphocyte) T h 1 or T h 2 (Yang and Glaser, 2002).Increases in the number of T h 2 cells were observed.These induce the activity of B-lymphocytes and production of immunoglobulin through biosynthesis of cytokines (IL-4, IL-5, IL-6) (DeKruyff et al., 1998; Yang and Glaser, 2002).
In this study, LEV was applied to investigate the effects of immunomodulating substances on the immune system and physiological processes in pigs exposed to 3 days of stress.Previous studies have shown varying effects of LEV on immune cells (Symoens and Rosenthal, 1977;Van Wauwe and Janssen, 1991).When applied in doses for prevention and treatment of parasites in pigs, it has an immunosuppressive effect by reducing cell immune response, i.e. migration of leukocytes (Symoens and Rosenthal, 1977;Blecha, 1988;Jenkins and Hurdle, 1989).However, a stimulation of the immune response, increase of leukocyte maturation and lymphocyte differentiation is achieved with a dose of 2 to 3 mg/kg BW (Renoux and Renoux, 1974).LEV affects the immune response more efficiently when applied at regular intervals, and its administration is recommended during 3 consecutive days or as one weekly dose (Brunner and Muscoplat, 1980).Administration of LEV prior to stress induction in the previous study resulted in a balanced ratio of neutrophyls and lymphocytes in the blood of boars (Biland`i} et al., 2005).
In the present study, administration of LEV in a dose of 2.5 mg/kg BW in boars stimulated an increase in total protein concentrations in the serum of boars by 9.6% and IgG concentrations by 13.8 to 24.6% compared to control animals, while IgA and IgM concentrations were unchanged.Administration of 6 mg/kg BW LEV reduced the level of antibodies in herpes virus infection in cows (Blecha, 1988).However, where cows were exposed to stress during transport, LEV increased the concentration of antibodies.At a dose of 2.5 mg/kg BW, LEV increased IgM concentrations in rats aged up to 10 days (Hunter et al., 1981).Administration of the same dose in mares in the final stage of gravidity increased the concentrations of total protein, IgG and IgM in the colostrum (Flesh et al., 1982;Karakowski et al., 1999).In the serum of foals of treated females, a significant IgG concentration increase was observed compared to foals of mothers that had not received LEV (Karakowski et al., 1999).
In this study, administration of LEV in boars for 3 days prior to ACTH induction reduced total protein levels and values were similar to those in boars receiving only LEV.The applied treatment with LEV and ACTH had no impact on albumin concentrations and was in compliance with control concentrations.However, globulin concentrations were increased, which may be explained by increased globulin concentrations caused by ACTH effects.Accordingly, IgG concentrations were significantly increased during and two weeks after the treatment with ACTH in both groups of boars.LEV used before ACTH administration stimulated the reduction in IgG concentrations particularly in the week after stress, which confirmed its impact on balancing the stress-induced immune response.During stress, LEV had no impact on albumin, IgA and IgM concentrations.
LEV acts primarily on T-cells (Symoens and Rosenthal, 1977).It may act directly or indirectly on B-cells by affecting T-lymphocytes and macrophages, which in turn activate B-cells and immunoglobulin synthesis through specific cytokine synthesis (Kühlmann-Rabens et al., 1987;Jenkins and Hurdle, 1989).One of the possible mechanisms of the impact of LEV on the immune system is an increase in the synthesis of cytokine IL-1 in macrophages as determined in in vitro research on mice macrophages (Kimball et al., 1991;Szeto et al., 2000).IL-1 is the main agent in T-lymphocyte activities and in the synthesis of cytokine IL-2 and IL-6 which stimulate the activity of B-cells.LEV reduces gene expression for cytokine IL-4 (T h 2 cells) in rats and induces the activity of T h 1 cells (Szeto et al., 2000).It acts selectively on the increase of synthesis of cytokine IL-18 in macrophages by inducing biosynthesis of INF-g in T h 1 cells, which then stimulate the activity of Blymphocytes.Recent investigations have suggested that LEV may act as an adjuvant for preventive vaccines and stimulate both humoural and cell-mediated immune responses (Bo`i} et al., 2002;Jin et al., 2004;Bo`i} et al., 2006).
In conclusion, the presented results indicate that administration of LEV may have a practical application regarding its impact on immune response.LEV stimulates the production of immunoglobulins in boars exposed to stress.Application of LEV in investigated concentrations may protect boars from the negative influence of stress and provoke improved non-specific immunity.Protection of stress could also reduce economical losses in pig production.

Table 2
cLEV and ACTH