INTRODUCTION

The increase of productivity in fish culture has been accompanied by stressful conditions and problems related to fish diseases. Enhancement of the immune system seems to be the most promising method for preventing fish diseases. This enhancement can be achieved with immunostimulants. Some immunostimulants that have been used in aquaculture are: synthetic substances (like levamisole), biological substances (like bacterial derivatives), nutritional factors, animal and plant compounds (Sakai, 1999). Unlike chemical substances used in aquaculture, plant immunostimulants have no residuals in the environment and do not cause drug resistance. On the other hand, in traditional medicine plants have been used in order to prevent and treat a lot of diseases in human and animals. Recently, these plants have received more attention for their immune stimulating functions in fish culture too (Ardo et al., 2008; Choi et al., 2008; Dügenci et al., 2003; Jian and Wu, 2003, 2004; Rao and Chakrabarti, 2005; Yin et al., 2006). Zataria multiflora is one of the medicine plants that is a member of Labiatae family. Main compositions in the Zataria are Thymol, Carvacrol, Para-cymene, Terpinene and β-caryophyllene that provide 37.59, 33.65, 7.72, 3.88 and 2.06%, respectively in this essential oil (Sharififar et al., 2007). In traditional medicine Z. multiflora is used for intestinal and liver inflation, regulating the menstruation, removing different parasites, bloating and even disinfecting the environment. There are few studies on effect of this plant in aquaculture. Sharifrohani (2004) showed that Zataria multiflora is effective against fungal diseases in rainbow trout eggs till eyed egg stage and significant differences were observed particularly at 50-100 ppm compared with control group.

In spite of positive effects of this plant in controlling some microbial diseases and its immunostimulation effect in human and some animal models (Basti et al., 2005; Dakhili et al., 2006; Fazeli et al., 2007; Khosravi et al., 2007; Khosravim et al., 2008; Sharififar et al., 2007; Shokri et al., 2006; Vigo et al., 2004) no information is available concerning its effect on the fish immunity. Therefore, the aim of present study was to investigate the effect of this plant on some non-specific and specific immune responses of common carp (Cyprinus carpio) as one of the main worldwide farmed species. Particularly the aim was to study effect of the essential oil of this plant on non-specific immune system of common carp under lower water temperature similar to winter condition that fish are faced in the ponds.

MATERIALS AND METHODS

Experimental Conditions
Healthy common carp, Cyprinus carpio, weighing 30-35 g were obtained from a fish farm in Gilan provine, Iran. The fish were acclimated with essential oil free feed in out door 300 L tanks for one month. Water quality parameters consisting of water temperature, dissolved oxygen, ammonia, nitrite, hardness were 16-17°C, >6, <0.01, 0.1, 180 mg L^-1 and 7.8, respectively through out the experiment. This study was conducted in 18 months from April 2007 to December 2008.

Administration of Z. multifora
Fish were divided into 4 groups (20 fish/group) and fed diets containing different doses of 30, 60 and 120 ppm essential oil of Z. multiflora (Barij essence company, Iran) for a period of 8 continuous days. Fish were fed at the rate of 1% body weight. A control group was included without any treatment.

Blood Sampling
Four fish per treatment group were bled from caudal vein 1, 2, 8, 15 and 22 days post-treatment. A proportion of blood sample was put into an Eppendorf tube to prepare serum for assaying lysozyme, serum bactericidal activity, total protein, globulin and albumin and the remaining blood samples were taken into another tube containing 10% EDTA to measure white blood cell count.

Serum Lysozyme Activity
The turbidimetric assay for lysozyme content was carried out to measure according to Kumari (2006). A suspension of 150 μL of Micrococcus lysodeikticus (0.2 mg mL^-1 in 0.02 M sodium acetate buffer, pH 5.5) was added to 96-well U-bottom microtitre plates containing a 15 μL test serum in each well. Initial O.D was then obtained at 450 nm immediately after adding the substrate and final O.D was obtained after 1 h incubation at 24°C. Lyophilized hen egg white lysozyme, HEWL (Sigma), was used to develop a standard curve. Serum lysozyme values were expressed as μg mL^-1 equivalent to hen egg white lysozyme activity.

Serum Bactericidal Activity
Serum bactericidal activity was estimated following the method of Barnes et al. (2003) with slight modification. Aeromonas hydrophila isolate was cultured overnight in blood agar and the grown colonies were collected in sterile PBS and centrifuged at 2000 rpm (1500x g) for 15 min. The pelleted bacterial cells were then washed and resuspended in sterile PBS to an OD (540 nm) equivalent to 5.32x10⁷ cfu mL. This was diluted 50-fold in PBS and a volume of 25 μL was incubated with an equal volume of heat-inactivated (at 44°C for 20 min) serum sample for 1 h at room temperature. Complement activity was provided by the addition of 200 μL fresh normal serum. Following incubation at room temperature for 1.5 h, bacterial suspensions were serially diluted in PBS and spotted (10 μL) onto TSA plates, incubated at 25°C for 24 h and plates containing 30-300 viable colony forming unit were then counted.

Leukocyte Population Size
Leukocyte count was determined using a haemocytometer with Neubauer counting chamber.

Serum Total Protein, Albumin and Globulin
Serum protein was estimated by Biuret method and serum albumin by bromocresol green method using Pars Azmun kits (Iran) and Auto analyzer (Ependorf). Serum globulin was estimated by subtracting these two amounts.

Serum Antibody Titer
The remaining fish were intraperitoneally injected with formalin-killed whole cells of Aeromonas hydrophila (6x10⁸ cells mL^-1) (Khoshbavar-Rostami et al., 2007) and blood samples were obtained 3 weeks later after anesthetizing fish with clove oil (100 mg L^-1). Blood sera were then separated after overnight keeping at 4°C.

Micro agglutination titer was estimated by method described by Robertson (1990). A 25 μL of PBS was added to all wells of round- bottomed plates (except the first well) followed by adding 25 μL of sample serum to first and second plates. Serum of test and control fish were serially diluted from second to twelve plates and then 25 μL of Aeromonas hydrophila suspension prepared in PBS (9x10⁸ cells mL^-1) was added to all plates, kept overnight at room temperature and the last well with positive agglutination was considered as the antibody titer in each treatment.

Statistical Analysis
All data were statistically analyzed by one-way ANOVA, Kruscal-Wallis and Mann-Whitney using SPSS version16. The level of significance was p<0.05.

RESULTS

The obtained data are shown in Fig. 1-6. No significant difference was observed between control fish and treatment groups (p>0.05) in lysozyme content. Significant increase of serum bactericidal activity was just measured in groups treated with 60 ppm of Z. multiflora on day 15 and 22, respectively (Fig. 1). Mean while, white blood cell count was significantly higher in groups fed 30 and 60 ppm of Z. multiflora (P<0.05) (Fig. 2) after 15 days post-treatment.

Fig. 1:	Serum bactericidal activity in common carp fed diets containing different doses of Zataria multiflora. Data are expressed as the Mean±Standard error of mean

Fig. 2:	White blood cells in common carp fed diets containing different doses of Zataria multiflora. Data are expressed as the Mean±Standard error of mean

During this study, there were no significant changes in plasma protein during the whole experiment (p>0.05) (Fig. 3). Globulin content did not show any significant differences too (p>0.05) (Fig. 4). No significant difference was also observed between control fish and treatment groups (p>0.05) in albumin content.

Fig. 3:	Serum total protein in common carp fed diets containing different doses of Zataria multiflora. Data are expressed as the Mean±Standard error of mean

Fig. 4:	Serum albumin in common carp fed diets containing different doses of Zataria multiflora. Data are expressed as the Mean±Standard error of mean

A significant increase in antibody titer was seen in the fish treated with 30 and 60 ppm of Z. multiflora 3 weeks post-immunization with A. hydrophila whole cell antigens (p<0.05) (Fig. 6).

Fig. 5:	Serum globulin in common carp fed diets containing different doses of Zataria multiflora. Data are expressed as the Mean±Standard error of mean

Fig. 6:	Antibody titer estimated by bacterial micro agglutination test in common carp fed diets containing different doses of Zataria multiflora. Data are expressed as the Mean± Standard error of mean

DISCUSSION

In aquaculture industry fishes are inevitably subjected to different stresses during the period of culture thus use of immunostimulant can act as an alternative way for the prevention and control of disease outbreaks. Diet supplemented with Zataria multiflora essential oil enhanced common carp immunity to some extent even though fish can not express their potential maximum immunity during low temperature. These lower immune responses may in part due to the lower water temperature used in this study (Bagni et al., 2005; Tort et al., 2004). In some regions such as Iran growing of carp encounters a sever stress during the cold weather in Winter because of lower water temperature of about 12-17°C. Thus enhancing the fish immune response may improve the fish health condition to some pathogenic microorganisms.

Several innate immune responses of the common carp were examined after oral administration of different doses of Zataria multiflora during low water temperature. Essential oil supplementation in common carp had no effects on serum lysozyme activity for the duration of the experiment. On the other hand, WBC significantly increased in 30 ppm and 60 ppm fed groups on day 15 with slight increase on day 2 in 30 ppm groups. Elevation in serum bactericidal activity was just noted on day 15 and 22 in 60 ppm fed groups when compared to control and a slight but not significant increase was observed on day 2 in this group too. In our experiment, total protein, albumin and globulin were measured, but neither of these parameters was affected by Zataria essential oil but antibody titer measured showed a significant increase in lower doses, 30 and 60 ppm, 3 weeks after injecting formalin killed Aeromonas hydrophila. It is note worthy to mention that even in blood smears prepared from different treatments in lower doses higher amount of lymphocytes were observed that is in agreement with the results in antibody titers (Data not shown).

Only few studies have been focused on the innate immune responses of Z. multiflora in some species of terrestrial animal models but till now there were no studies to determine the effects of this plant on fish immune system. The BALB/C mice respiratory burst activity was significantly influenced by intraperitoneally injection of Z. multiflora 4 and 7 days after treatment (Shokri et al., 2006). In rabbit, intra coutaneous injection of this essential oil affected antibody titer against Candida albicans, neutrophil phagocytosis plus significant difference in lymphocyte transformation (Khosravi et al., 2007).

Diet supplemented with this essential oil to some extent enhanced common carp immunity even though fish can not express their maximum immunity potential during low temperature. This conclusion is supported by the out come of the challenge experiments in sea bass (Dicentrachus labrax) and gilthead sea bream (Sparus aurata) that reduction in total WBC, complement and lysozyme activities was observed during reduction of water temperature (Bagni et al., 2005; Tort et al., 2004).

Dose optimizing of an immunostimulant and its administration duration are also other parameters need to be considered. Li et al. (2006) showed that using 100 mg kg^-1 of levamisole, the best immunity responses was achieved while 1000 mg kg^-1 of levamisole caused chronic toxicity and higher FCR so further studies using even longer administration period for Zataria should be considered. Also, the essential oil of Zataria multiflora used in this study contains several ingredients such as Thymol, Carvacorol and Para-cymene. Therefore, the fish immune responses to a single component of this herbal oil need further studies.

In conclusion, this study showed that Z. multiflora essential oil had the potential for enhancing innate immune system of carp under temperature stress but further studies are required to evaluate the immunostimulatory effect of Z. multiflora essence in carp in different situations during the period of culture.

ACKNOWLEDGMENT

The authors thank staff of Department of Qquatic Animal Health, Central Lab of Veterinary Faculty of Tehran University and Barij Essence Company. This study was funded by the research council of university of Tehran.