Department of  Brucella,

Animal Health Research Institute, Dokki Giza.

Monitoring of immune response against Brucella melitensis Rev- 1 vaccine following administration

of some antibiotics

(With 4 Tables and 4 Figures)

By

Hoda M. Zaki; E.M. EL-Bauomy

 and R.M. Khoudair

 (Received at 27/12/2005)

قياس رد الفعل المناعى ضد لقاح البروسيلا مليتنسز ريف-1

عقب استخدام بعض المضادات الحيوية

هدى زکى ، عصام البيومى ، رمضان خضير

الأوکسى تتراسيکلين والاستربتوميسين والانروفلوکساسين اکثر المضادات الحيوية شيوعاً واستعمالاً لعلاج کثير من أمراض الحيوان. لذا فقد اجريت هذه الدراسة بهدف معرفة تأثير هذه المضادات الحيوية على الحالة المناعية للحيوانات المحصنة واستخدمت خنازير غينيا المحصنة بلقاح ريف-1 کمثال لذلک. تم تحصين عدد 350 حيوان من خنازير غينيا بلقاح الريف-1 ثم تقسيمهم إلى 7 مجموعات کل مجموعة تحتوى على 50 حيوان. المجموعة الأولى بعد تحصينها تم ترکها بدون أى معاملات أخرى واستخدمت کضابط للتجربة. أما المجموعة الثانية والثالثة والرابعة فقد أعطيت الأوکسى تتراسيکلين والاستربتومايسين والانروفلوکساسين على الترتيب بالتوافق مع التحصين بجرعات محددة لمدة 3 أيام فى حين أن المجموعة الخامسة والسادسة والسابعة قد تم معالجتها بنفس رجيم العلاج ولکن عند بلوغ رد الفعل السائلى قمته (4 أسابيع بعد التحصين). تم ذبح 5 حيوانات من کل مجموعة أسبوعياً لمدة 8 أسابيع هى فترة التجربة. تم تجميع عينتا دم من کل حيوان مذبوح أحدهما على مانع للتجلط (هيبارين) بهدف قياس رد الفعل المناعى السائلى والخلوى لهذه الحيوانات. أظهرت النتائج أن الأوکسى تتراسيکلين أکثر المضادات الحيوية تأثيراً ضاراً على رد الفعل المناعى کذلک الاستربتومايسين ولکنه أقل تثبيطاً من الأوکسى تتراسيکلين فى حين أن الانروفلوکساسين ليس له مثل هذه التأثير على رد الفعل المناعى سواءاً الخلوى أو السائل. وعليه فقد خلصت هذه الدراسة إلى استنتاج مفادة أن الحيوانات المريضة المحصنة بلقاحات البروسيلا يحظر علاجها بالأوکسى تتراسيکلين أو الاستربتومايسين وأن الأنروفلوکساسين هو العلاج الأمثل لمثل هذه الحالات.

Summary

The humoral and cellular immune response in guinea pigs vaccinated with Rev-1 vaccine and simultaneously treated with antibiotics (oxytetracycline, streptomycin or enrofloxacin) or 4 weeks post-vaccination were evaluated in the present study. Groups vaccinated and simultaneously treated with oxytetracycline or 4 weeks post-vaccination showed delayed or abrupt reduction respectively in antibody production, lymphocyte transformation, phagocytic percent and intra-cellular killing one week post administration of this antibiotic while groups vaccinated and simultaneously treated with streptomycin or 4 weeks post-vaccination showed abrupt reduction in antibody titer and moderate reduction in phagocytic percent, intra-cellular killing and lymphocyte transformation. Treatment by enrofloxacin simultaneously with Brucella melitensis Rev-1 vaccine or at peak of vaccination (4 weeks post-vaccination) cause non-significant variation in both humoral and cellular immune response. Therefore oxytetracycline and streptomycin should not be recommended for animal treatment during exposure to Brucella melitensis Rev-1 vaccine. In contrast enrofloxacin exhibited a satisfactory margin of safety upon the humoral and cellular immune responses to Brucella melitensis Rev-1 vaccine.

Key words: Brucellosis, Brucella melitensis, vaccination, antibiotics.

Introduction

Brucellosis is a zoonotic disease that causes heavy economic losses and human suffering. Under most conditions vaccination and serological identification and culling of infected animals are the only practical means to achieve its eradication (Monreal et al., 2003). Until now the best vaccine available for sheep has been the smooth (S) Brucella melitensis Rev-1 strain (Marine et al., 1999). Several authors have concluded that vaccination with Brucella melitensis Rev-1 vaccine is effective immunizing agent against Brucella organisms irrespective of challenge strain (El-Bauomy 1993 and EL-Gibaly et al., 1995). Protection against the facultative intra-cellular Brucella organism depends mainly on cellular response following vaccination, whereas humoral response is not indicative of good immunization (Nicoletti, 1990).

Therefore any exogenous agents which result in crucial depression of cellular response to the given Brucella vaccine will consequently affect the overall process of immunization against infection and leave non immune host. Since the use of chemotherapeutic agents was widely introduced in veterinary field many author have studied their influence on the immune response against animal disease. Immune suppression still represent the worst indirect adverse reaction that may be induced by chemotherapies, such agents have the capability to suppress the immune system even at therapeutic levels (Nagwa et al., 2005). Immunosuppressive properties of some antibiotics are effective in inhibition of both cellular and humoral immune responses to a variety of vaccines (Shalaby, 1989).

When initiating medication you must select not only the most biologically efficient therapeutic agent but also selecting an efficacious and safe drug which does not induce direct or indirect adverse reaction (Morris, 1995 and Harkness and Wanger, 1995). So the present study was designed to clarify the effect of medication with some antibiotics extensively used in veterinary medicine for treatment of several diseases namely oxytetracycline, streptomycin and enrofloxacin on both humoral and cellular immune response of guinea pigs vaccinated with Brucella melitensis Rev-1 vaccine.

The effect of medication was studied during vaccination and shortly after vaccination. As treatment of animals may become obligatory in certain instances therefore this study was also done to determine which of the used antibiotics is less harmful to the immune status of the animals and consequently can be used safely in concomitant with the vaccination process.

Materials and Methods

Vaccine: Brucella melitensis Rev-1 was obtained from Rhone Merieux Lyone, France.

Experimental animals:

A total of 350 guinea pigs (weighing 200 to 300g) free from any clinical signs were strictly selected and purchased from a trustable well established lab. animals unite of a well known institute. They were allocated randomly into 7 groups (50 animals/ each). Animals of all groups were vaccinated with Brucella melitensis Rev-1vaccine in a dose of 2×10⁸ CFU. The 1^st group received the vaccine alone and served as control group. The 2^nd group was injected I/M with oxytetracycline in a therapeutic dose 5 mg daily for 3 consecutive days simultaneous with the vaccination process. The 3^rd group injected with streptomycin in a dose of 10 mg daily for 3 consecutive days simultaneous with the vaccination process. The 4^th group was injected with enrofloxacin in dose of 10 mg for 3 consecutive days simultaneous with the vaccination process.

The 5^th group was inoculated with oxytetracycline with the same dose used for group 2 for 3 successive days at the 4^th week post vaccination. The 6^th group was inoculated with streptomycin with the same dose used for group 3 for 3 successive days at the 4^th week post vaccination. Whereas the last 7^th group was inoculated with enrofloxacin with the same regime used for group 4 at the 4^th week post vaccination .

Blood samples: 5 guinea pigs from each group were sacrificed  prior vaccination and weekly during 8 weeks post vaccination. Two samples (serum and heparinized blood) were collected from each sacrificed animal.

1- Humoral immune response:-

A - Conventional serological test:

Antibody titers in collected serum samples were measured using Serum agglutination (SAT), Mercapto-ethanol (ME) and Rivanol (Riv.T) tests. Serum agglutination and mercapto-ethanol tests were done according to the methods adopted by Alton et al. 1975 whereas Rivanol test was carried out according to the procedures of the National Vet Services Laboratories, Ames, Iowa, USA 1984 (Alton et al.1988)

B - ELISA:

ELISA technique was performed by the method of Bodwen et al.1999. Briefly, polystyrene plates sensitized by 5µg of Brucella melitensis biovar 3 lipopolysaccharide prepared by phenol extraction of killed bacteria (Grain Bastuj et al. 1990) and blocked with 3% skim milk in phosphate buffer saline (PBS). The plates were washed with PBS 0.05% Tween 20 and the sera under study were added (diluted 1: 100) in PBS- 0.05% Tween 20 containing 1% skim milk. After incubation, the plates were washed and incubated with 100 µl of 1: 10.000 diluted horse radish peroxidase conjugated goat anti guinea pig IgG (Htt; zymed, S. Sanfrancisco, Calif.) the reaction was developed with o-phenylenediamine (2µg/ml) and 0.03 H₂O₂ in 0.1 M citrate phosphate buffer (ph 5) and was stopped with 4 N H₂SO₄. The resulting colour was read at 492 nm O.D Negative and positive controls were assayed in the same run.

2- The cell mediated immune response:

A- Phagocytic activity: Polymorph nuclear (PMN) cells were isolated from blood by the method described by Rouse et al. 1980. The mixture of PMN and bacteria (Staphylococcus aureus) were incubated at 37ºC for 2 hour with regular stirring and then the mixture was centrifuged at 20 X g for 5 minutes at 4ºC. The supernatant was used to estimate the percentage of bacteria phagocytised. The mixture of bacteria and PMN were treated with one cycle of freezing and thawing and the percentage of bacteria killed was estimated according to the formula described by Woldehiwet and Rowan 1990.

B- Lymphocyte transformation: Lymphocyte proliferation was measured by a blastogenesis assay using 3-(4, 5 dimethylthiazol 1,2) -2,5 diphenyl tetrazolum bromide (MTT) (Sigma USA) as a tetrazolium dye. MTT is reduced to a blue Formazan compound by succinate dehydrogenase Mitochondrial enzyme produced by liver cells. MTT blastogenesis microassay was conducted as described by Denizot and Lange, 1991 with some modifications. Maslak and reynlods 1995. Two hundred µl culture medium containing mitogen phytohaemagglutinine (PHA) at 25 µg/ml or no mitogen (control well) was dispensed into each 96 well flat bottom tissue culture plate. Ten µl of cell suspension containing 10⁵ lymphocyte was added to each well. Plates were incubated at 37ºC for 72 hours in humid CO₂ incubator. Two hours before the termination of incubation 20 µl MTT 10 µg/ml was added to each well. At the end of incubation period 10% SDS in PBS was used to lysis the cells (50µl/ well). Then absorbance value was detected in an automated microtitre plate reader at wave length of 550 nm. The difference in reading between treated and control wells were subjected to statistical analysis.

Results

Animals of the 1^st group vaccinated with Rev-1 and not treated gave positive results to the current serological tests used (SAT,ME and Riv.T) and ELISA starting at 1^st week post vaccination reached their peak at the 5^th week then started to decline gradually till the end of the experiment (8 weeks post vaccination). Whereas animals vaccinated and simultaneously treated with oxytetracycline or streptomycin (Groups 2 and 3 respectively) showed significant reduction in antibody production as measured by conventional tests and ELISA.

Animals of groups 5&6 which treated at the expected peak of immunization titer with oxytetracycline or streptomycin showed abrupt decline in antibody titers. These decline in antibody titers occurred one week post administration of antibiotics while vaccinated animals simultaneously treated with enrofloxacin or 4 weeks post-vaccination (Groups 4&7) showed no significant differences in antibody titers in comparison to vaccinated non treated group (Tables 1& 2 and Figure 1).

Animals vaccinated and simultaneously treated with oxytetracycline or 4 weeks post-vaccination (Groups 2&5) displayed significant reduction in phagocytic percent and intra-cellular killing. Also animals vaccinated and simultaneously treated with streptomycin or 4 weeks post-vaccination (Groups 3&6) showed reduction in phagocytic and intra-cellular killing percent, but less than that occurred in oxytetracycline treated groups. This reduction observed one week after administration of antibiotics. In contrast enrofloxacin simultaneously treated group or 4 weeks post-vaccination disclosed no reduction in phagocytic activity comparing to vaccinated non treated group (Table 3 and Figures 2&3).

Simultaneously oxytetracycline treated group with vaccine or 4 weeks post-vaccination showed significant reduction in lymphocyte transformation after administration of antibiotics by one week. While streptomycin treatment cause moderate reduction (among treated groups) in lymphocyte transformation. Whereas treatment with enrofloxacin simultaneously with the vaccine or 4 weeks post vaccination showed no effect on lymphocyte transformation (Table 4 and Figure 4).

Table 1: Serological profiles of Rev-1 vaccinated and vaccinated treated animals with oxytetracycline, streptomycin or enrofloxacin.

* Mean antibody titre.

1- Vaccinated non treated (control).

2- Vaccinated simultaneously treated with oxytetracycline.

3- Vaccinated simultaneously treated with streptomycin.

4- Vaccinated simultaneously treated with enrofloxacin.

5- Vaccinated and treated at peak (4 weeks post immunization) with oxytetracycline.

6- Vaccinated and treated at peak (4 weeks post immunization) with streptomycin.

7- Vaccinated and treated at peak (4 weeks post immunization) with enrofloxacin.

Table 2: Evaluation of humoral immune response using ELISA test in Rev-1 vaccinated and vaccinated treated animals with oxytetracycline, streptomycin or enrofloxacin.

1- Vaccinated non treated (control).                                                                     

2- Vaccinated simultaneously treated with oxytetracycline.

3- Vaccinated simultaneously treated with streptomycin.                            

4- Vaccinated simultaneously treated with enrofloxacin.

5- Vaccinated and treated at peak (4 weeks post immunization) with oxytetracycline.                       

6- Vaccinated and treated at peak (4 weeks post immunization) with streptomycin.

7- Vaccinated and treated at peak (4 weeks post immunization) with enrofloxacin.

* Significant at P < 0.01 using t-student test comparing with control (G1)

** Significant at P < 0.001 using t-student test comparing with control (G1)

Table 3: Phagocyctic activity in Rev-1, vaccinated and vaccinated treated animals with oxytetracycline, streptomycin or enrofloxacin.

Ph % = phagocytic percent.            K% = intra-cellular killing percent.

1 - Vaccinated non treated (control).                                                       

2 - Vaccinated simultaneously treated with oxytetracycline.

3 - Vaccinated simultaneously treated with streptomycin.                                       

4 - Vaccinated simultaneously treated with enrofloxacin.

5 - Vaccinated and treated at peak (4 weeks post immunization) with oxytetracycline.           

6- Vaccinated and treated at peak (4 weeks post immunization) with streptomycin.

2 - Vaccinated and treated at peak (4 weeks post immunization) with enrofloxacin.

* Significant at P < 0.01 using t-student test comparing with control (G1)

** Significant at P < 0.001 using t-student test comparing with control (G1)

Table 4: Lymphocyt transformation using MTT in Rev-1 vaccinated and vaccinated treated animals with oxytetracycline, streptomycin or enrofloxacin.

1 - Vaccinated non treated (control).                                                                              

2 - Vaccinated simultaneously treated with oxytetracycline.

3 - Vaccinated simultaneously treated with streptomycin.                        

4 - Vaccinated simultaneously treated with enrofloxacin.

5 - Vaccinated and treated at peak (4 weeks post immunization) with oxytetracycline.

6- Vaccinated and treated at peak (4 weeks post immunization) with streptomycin.

7- Vaccinated and treated at peak (4 weeks post immunization) with enrofloxacin.

* Significant at P < 0.01 using t-student test comparing with control (G1)

** Significant at P < 0.001 using t-student test comparing with control (G1)