Effect of levamisole administration on immunogenic and protective capacity of Brucella abortus [RB.sub.51].
Brucellosis affects many animals' species as well as human in most countries. Brucella abortus is the most important cause of bovine brucellosis, while both Brucella melitensis and Brucella ovis cause brucellosis in sheep. 
Vaccination represents an essential element in the control of bovine and ovine brucellosis. Live attenuated B. abortus strain 19 and B. melitensis Rev 1 have served as efficacious vaccine strain for cattle and sheep, respectively. [2,3] However, both vaccines have the disadvantages of inducing O-polysaccharide (OPS)-specific antibody responses that interfere with the serological diagnosis of disease.  They may cause abortion in vaccinated animals under some circumstances  and of being pathogenic for humans. 
In recent years, Schurig and his coworkers produced a stable rough variant of virulent B. abortus 2308 that was designated [RB.sub.51].  Strain [RB.sub.51] diminished virulence in comparison with strains 2308 and  and did not induce the formation of OPS-specific antibodies. Recent experiments have indicated that strain [RB.sub.51] may serve as an alternative vaccine for cattle;  however, there is interest in attempting to enhance its efficacy using immune-stimulating agents.
Levamisole is an immunopotenciator drug which is used as an anthelmintic drug as well as very effective remedy on cellular immunity compared with humoral immunity.  Levamisole, a potent anthelmintic that is widely used in veterinary medicine, is also known as an immune stimulant. Because levamisole act at the cellular level, cells of immune-regulatory mechanisms are affected.  Under selective condition, levamisole can enhance immune responses to viral antigens.  Levamisole is thought to have its greatest immunostimulating effect in immunosuppressed animals and reduced the frequency of some diseases. 
The aim ofthis study is to determine the capability of levamisole to enhance the humoral antibody response and cell-mediated immunity as well as protective capacity of [RB.sub.51] vaccine in mice against challenge with virulent B. melitensis strain.
MATERIALS AND METHODS
B. abortus [RB.sub.51] vaccine (Professional Biological Company, Denver, Co, USA).
Vaccination, Treatment, and Challenge Infection of Experimental Animal
Six groups of 7-8-week-old female Bulb/C mice obtained from VACSERA (Helwan) weighing 21 [+ or -] 5 g were used.
1. Group I was vaccinated I/P with 5 X [10.sup.8] colony-forming units (CFU) of [RB.sub.51] strain (vaccinated control group).
2. Group II was vaccinated I/P with 5 X [10.sup.8] CFU of [RB.sub.51] strain simultaneously injected (at 0 day) s/cu with 12.5 mg/Kg levamisole (levamisole hydrochloride ICI Company, England). 
3. Group III was vaccinated I/P with 5 X [10.sup.8] CFU of [RB.sub.51] strain and injected at 7-day postvaccination with 12.5 mg/Kg levamisole.
4. Group IV was vaccinated I/P with 5 X [10.sup.8] CFU of [RB.sub.51] strain and injected at the day of vaccination (0 day) and 7-day postvaccination with 12.5 mg/Kg levamisole.
5. Group V was injected with levamisole alone.
6. Group VI was kept as control group (negative control). 8-week postvaccination; all vaccinated as well as control animals groups received I/P challenge of 2 ml dose of 2 X [10.sup.4] CFU/ml of virulent strain of B. melitensis biovar 3.
Collection of Blood and Tissue Samples
1. Blood was collected for the separation of sera from mice in all groups. The first samples were taken prevaccination, then, at intervals of 2, 4, 6, and 8 weeks postvaccination for serological examination.
2. Spleens, livers, kidneys, and lungs were collected by sacrificed 10 animals 2 weeks after challenge from each group and exposed to bacteriological examination.
Evaluation of Humoral Immune Response using ELISA Test
Brucella-specific antibodies were detected by the use of whole cell [RB.sup.51] ELISA. Microtitration plates for the whole cell [RB.sup.51] ELISA were coated with killed B. abortus [RB.sup.51] organisms which were prepared from plate (Trypticase soy agar) grown cells that were killed with methanol,  and mice sera were serially diluted in phosphate-buffered saline solution (PBS) - 0.05% tween 80 were tested using goat anti-mouse IgG labeled with horseradish peroxidase for reactivity against rough antigens. A test result was considered positive if the sample gave a reading equal twice that obtained of known negative control serum samples.
Evaluation of Cell-mediated Immunity by Delayed Type Hypersensitivity (DTH) Test
DTH reaction was determined by the method described by Arya et al.,  using B. abortus soluble antigen (BASA) which prepared from live cells of strain [RB.sub.51] that were suspended in PBS and autoclaved to extract BASA.  14-day postvaccination, 10 mice from each group were injected with BASA (20 mg in 20 of PBS) in the right footpad. Thicknesses were measured 24, 48, and 72 h later with Hauptner dial caliper. A difference in footpad thickness of [greater than or equal to]2 U (Iu = 0.1 mm) was regarded as positive reaction.
Bacteriological Examination for Isolation of B. melitensis Biovar 3 from Mice
The isolation of B. melitensis biovar 3 from spleen, liver, kidney, and lung was carried out according to the method explained by. 
For evaluation of the humoral immune response in mice groups vaccinated with [RB.sub.51] vaccine and groups vaccinated with [RB.sub.51] vaccine and treated with levamisole as well as control group, the ELISA test was performed.
Mice vaccinated I/P with [RB.sub.51] strain (5 X [10.sup.8c] CFU) showed high serum antibody titers to the surface antigen of intact [RB.sub.51] bacteria from week 1 to week 8 postvaccination [Table 1].
It could be seen also from the data presented in Table 1 that levamisole treatment in addition to vaccination with [RB.sub.51] vaccine either 7-day postvaccination or 0 and 7 postvaccination (Groups III and IV) had an immune-potentiating effect where the mean antibody levels as measured by ELISA all over the experimental period were higher than those in the levamisole-untreated vaccinated mice (Group I). Mice treated with levamisole simultaneously with vaccination (Group II) showed mild elevation in antibody titer compared with vaccinated (Group I). Results of cellmediated immunity by DTH elicited in mice vaccinated with [RB.sub.51] strain are illustrated in Table 2. The use of BSA as an eliciting antigen resulted in developing of DTH 14-day postvaccination (Group I). The highest reaction was observed 48 h postelection.
Group IV treated with levamisole 0 and 7 days postvaccination revealed the highest level of DTH followed by mice group treated with levamisole 7-day postvaccination (Group III), and then, mice in Group II treated with levamisole simultaneously with vaccination. The isolation percentage of B. melitensis was recovered from 100% of non-vaccinated challenged mice (Groups V and VI); the highest isolation rate was recovered from spleen. Meanwhile, the vaccinated groups revealed various percentage of B. melitensis biovar 3 isolation, Group II simultaneously treated with levamisole at 0 day, Group III treated with levamisole 7-day postvaccination, and Group IV treated with levamisole 0 and 7 days postvaccination resulted in 30%, 10%, and 10%; isolation rate, respectively.
B. abortus strain [RB.sub.51] is a rough strain that was derived after multiple passages of virulent strain 2308 on rifampin containing soy agar plate. Strain [RB.sub.51] is stable, attenuated rough variant that is essentially devoid of OPS, therefore, vaccination with [RB.sub.51] does not induce antibody to the OPS of Brucella species that are detected by the use of standard serodiagnostic tests for brucellosis.  Therefore, the elicited antibodies can be detected only using rough antigen derived from the same vaccinal strain.  The application of ELISA using rough antigen on serum of vaccinated animals revealed a detection of elevated rough antibodies titer which persisted up to 8th week postvaccination.
This result confirms previous studies [20,21] that used a dot blot or indirect ELISA techniques based on extraction of rough soluble antigen from [RB.sub.51] vaccinal strain. In these previous studies, the dot blot or indirect ELISA using rough antigen detected antibodies from [RB.sub.51] vaccinated mice for 8-week postvaccination.
The using of rough antigen in ELISA is considered by many workers to be the only tool to detect humoral immune responses induced by [RB.sub.51] vaccine. The antibody detected using rough [RB.sub.51] antigen in the current study was probably directed primarily to outer membrane proteins but not to the lipopolysaccharides (LPS) O antigen. [7,22]
Levamisole which has been used extensively as anthelmintic in man and domestic animals, recently attracted attention because it has immune-potentiating properties.  The mechanism by which levamisole could enhance serum antibody responses to the infective agent is not clearly known. 
In the present study, the most enhanced antibody responses could be seen in mice group treated with levamisole 7-day postvaccination (Group III) and mice treated with levamisole at 0 and 7 days postvaccination (Group IV) while mice group given levamisole simultaneously with vaccine showed the lowest have significantly enhanced antibody response. The immunomodulation is most likely due to the direct effects of levamisole on the immune system and to time at which it was administrated levamisole enhances macrophage and T lymphocytes function and reduces suppressor T cell function. 
Because antibody formation to most bacterial antigens is B lymphocyte dependent, the augmentation of the helper functions of T cells could enhance antibody production. The stimulatory effects of levamisole on macrophages may be particularly important because Brucella species tend to be an intracellular pathogen and macrophages are thought to be important in host defense in bovine brucellosis.  The positive effect of levamisole given on day 7 may be due to the fact that helper cells function is maximal at that time. DTH is in vivo assay of cell-mediated immune function and directly reflects the manifestation of function of the type T effector cell. 
DTH is a measure of antigen-specific T cell recall reaction at the site of rechallenging antigen in antigen-sensitized animals. 
In the present study, we evaluated the effect of administration of levamisole at different intervals post-[RB.sub.51] vaccination on the generation of DTH. The highest DTH response was observed in the group administrated levamisole 0 and 7 days postvaccination followed by group administrated with levamisole 7-day postvaccination. These results agree with  who suggested that levamisole being the most effective adjuvant modulates effectors T cells. Furthermore, Kimbell and Fisher  found that coinoculation of levamisole with plasmid not only preferentially increase the [IgG.sub.2] production but also prolongs the production. This may be due to its indirect effect on through activation of T lymphocytes to produce more interferon which in turn mediate for observed [Th.sub.1] type responses.  Suggested that levamisole could be a multifunctional regulator for both of [Th.sub.1] and [Th.sub.2] functions.
In the present study, following challenge with B. melitensis biovar 3, the bacteriological examination of sacrificed animal revealed that the challenge strain isolated from 30% of mice vaccinated with [RB.sub.51] alone (Group I), and the highest isolation rate was observed in spleen.
The level of protection offered by [RB.sub.51] vaccine may be due to the fact that this vaccine induces its immunity against infection mainly through cell-mediated immunity.  The immunity mediated by [RB.sub.51] against B. melitensis infections are primarily directed against outer membrane proteins which are prominently exposed on cell surface in the absence of LPS O side chain in [RB.sub.51].  Furthermore, Stevens and Olsen  proved by western immune-blot analysis that [RB.sub.51] vaccine induces small amount of IgG, but not IgM. The IgG had a role in protection of cattle and mice against virulent Brucella strains. It is clear from Table 3 that lower rate of isolation of B. melitensis biovar 3 was associated with mice groups vaccinated and treated with levamisole either 7 days or at 0 and 7 days postvaccination.
The previous increase of protection levels against infection with B. melitensis biovar 3 in levamisole-treated vaccinated groups is attributed to an augmentation of lymphocyte and macrophage proliferation, phagocytosis and increase of lysozyme enzymes released with increase of intracellular killing. 
Furthermore, levamisole stimulates cytotoxic T cells associated with enhancement of lymphokines production. The previous concept leads to an increase in the rate of clearance of infective agents. 
The results of the present investigation are in agreement with those obtained by  who found that the postvaccination injection of levamisole in guinea pigs caused a higher protection and survival rates than control animals.
Under the conditions of the present study, levamisole given 7 days or 0 and 7 days postvaccination caused enhanced humoral and cell-mediated immune responses to [RB.sub.51] strain. Whether this procedure could be used in the field to enhance resistance to bovine brucellosis, this requires further study.
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Ahmed M A Mansour
Department of Biotechnology, Faculty of Science, Taif University, Saudi Arabia
Correspondence to: Ahmed M A Mansour, E-mail: firstname.lastname@example.org
Received: November 05, 2017; Accepted: December 04, 2017
Table 1: Antibody profile of examined mice groups using ELISA Animal groups Weeks postvaccination 1 2 I 68[+ or -]6.11 96[+ or -]6.5 II 83[+ or -]6.63 132[+ or -]6.4 III 95 (**)[+ or -]6.90 192 (**)[+ or -]6.72 IV 160 (***)[+ or -]7.1 272 (***)[+ or -]6.31 V 0 0 IV 0 0 Animal groups 3 4 I 240[+ or -]5.9 544[+ or -]7.2 II 310[+ or -]7.4 715 (*)[+ or -]6.8 III 430 (**)[+ or -]6.3 960 (**)[+ or -]5.89 IV 640 (***)[+ or -]6.21 1216 (***)[+ or -]9.3 V 0 0 IV 0 0 Animal groups 5 6 I 704[+ or -]8.51 512[+ or -]8.53 II 952 (*)[+ or -]7.32 576 (*)[+ or -]6.1 III 983 (**)[+ or -]8.1 768 (**)[+ or -]6.01 IV 1088 (**)[+ or -]8.56 832 (**)[+ or -]5.3 V 0 0 IV 0 0 Animal groups 7 8 I 256[+ or -]6.8 208[+ or -]5.1 II 320 (*)[+ or -]7.1 230 (*)[+ or -]5.8 III 488 (**)[+ or -]7.4 320 (**)[+ or -]5.31 IV 510 (***)[+ or -]6.31 352 (***)[+ or -]6.11 V 0 0 IV 0 0 (*) Significant at P<0.05, (**) significant at P<0.01, (***) significant at P<0.001 Table 2: DTH elicited by examined mice groups Mice groups Elicitation (hours post soluble antigen inoculation) 14-day postimmunization 24 h 48 h I 6.15[+ or -]0.2 6.91[+ or -]0.3 II 6.91[+ or -]0.8 8.16 (*)[+ or -]0.7 III 10.22 (**)[+ or -]0.9 11.38 (**)[+ or -]0.6 IV 15.29 (***)[+ or -]0.6 16.94 (***)[+ or -]0.8 V 3.14 (*)[+ or -]0.3 3.65 (*)[+ or -]0.6 VI 2.15[+ or -]0.1 2.14[+ or -]0.2 Mice groups 72 h I 5.28[+ or -]0.2 II 5.51[+ or -]0.5 III 8.43 (**)[+ or -]0.4 IV 11.56 (***)[+ or -]0.6 V 2.08 (*)[+ or -]0.2 VI 2.09[+ or -]0.2 The mean represented of the footpad thickness (0.1 mm unit after BSA antigen inoculation). (*) Significant at P<0.05, (**) significant at P<0.01, (***) significant at P<0.001. DTH: Delayed-type hypersensitivity Table 3: Results of bacteriological examination of challenged mice Mice groups Organ subjected to bacteriological examination Spleen (%) Liver (%) Lung (%) Kidney (%) Positive mice (%) I 30 10 20 20 30 II 30 0 10 0 30 III 10 0 20 10 10 IV 10 0 0 0 10 V 100 70 40 60 100 VI 100 80 60 40 100
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|Title Annotation:||RESEARCH ARTICLE|
|Author:||Mansour, Ahmed M. A.|
|Publication:||National Journal of Physiology, Pharmacy and Pharmacology|
|Date:||May 1, 2018|
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