New Approach to use Origanium Vulgare Extract as Immunostimulant to Increase Resistance to Pseudomonas aeruginosa and Pseudomonas flourscence

The aim of the present study to assess the use of ethanol extracts of Origanium vulgare as a growth and immunity promoter for Nile tilapia (Oreochromis niloticus L) fingerlings. Fish (Average 12.27 g) were randomly distributed into four treatments; three replicates each at a rate of 20 fish per 100– L aquarium. Fish were fed one of the tested diets containing similar crude protein (30%) and gross energy (4.40 kcal/g). In addition, 0.0, 0.5%, 1.0%, or 1.5% Origanium vulgare extract. Diets were given twice daily at a rate of 3% of live body weight, for six days a week during 10 weeks. After the feeding trial, fish of each treatment were challenged by pathogenic Pseudomonas aeruginosa and Pseudomonas flourscence, which was given by intraperitoneal (I/P) injection and they were kept under observation for 10 days to follow up any abnormal clinical signs and the daily mortality rate. The growth promoting influence of Origanium vulgare extract was observed on fish. The maximum growth was observed at 0.5 % Origanium vulgare extract as compared to the control. No significant differences in fish survival were reported among the experimental treatments, falling within the range of 93.3-100%. The control fish consumed less diet and gave a higher Feed conversion ratio (FCR), while fish fed diet supplemented with 0.5% Origanium vulgare extract demonstrated the highest protein efficiency ratio (PER), apparent protein utilization (APU), and energy utilization (EU). The supplementation of Origanium vulgare extract had no significant effect on the fish body composition (dry matter, crude protein, fat, and ash), mean which total protein, albumin, and globulin increased significantly to the highest values at 0.5% Origanium vulgare extract, as compared to the control. However, supplementation of Origanium vulgare extract did not significantly affect the albumin /globulin ratio (A/G). In conclusion, 0.5% Origanium vulgare extract in Nile tilapia diets increased the fish resistance to Pseudomonas aeruginosa and Pseudomonas flourscence, indicating the effective role of Origanium vulgare extract in disease prevention in tilapia culture. Moreover, the reduction in feed cost compared with the control diet showed 12.52% to produce one kg fish gain of treatment containing 0.5% extracted Origanum vulgare levels. *Corresponding author: El-Arabey AA, Pharmacology and Toxicology Department, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt, Tel: +00201006180922; E-mail: ph.amrcapa@gmail.com Received January 25, 2016; Accepted January 29, 2016; Published February 05, 2016 Citation: El Araby DA, El-Arabey AA (2016) New Approach to use Origanium Vulgare Extract as Immunostimulant to Increase Resistance to Pseudomonas aeruginosa and Pseudomonas flourscence. J Marine Sci Res Dev 6: 182. doi:10.4172/2155-9910.1000182 Copyright: © 2016 El Araby DA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
Tilapia species are widely distributed in many countries of the world. Their farming has grown extremely fast in the last decade, where they are cultured worldwide with annual growth rate of about 12.2% [1]. In Egypt, Nile tilapia (Oreochromis niloticus L) is a major species in aquaculture system and much appreciated by consumers. However, the success of intensive tilapia culture depends to large extent on supplemental feeding.
Medicinal plants as natural growth promoters have significant improvements of body weight, weight gain, and survival rate and feed conversion rate in broilers [2]. Particularly, aromatic plants are considered of massive interest for their flavors and for their medicinal properties, along with human consumption, animal foodstuff and ornamental uses; thus, they are especially appropriate for multifunctional sustainable crop models [3,4]. Several medicinal herbs are used in the medication of various diseases; like reducing high blood cholesterol, protection from cancer, chronic diseases, stimulating the immune system. Furthermore, these herbs contain also aromatic substances and essential oils used in food industries [5]. Antimicrobial substances are now widely used for the treatment of bacterial diseases for fish [6].
The genus origanum Bth. of the Labiatae occur in the Egyptian as fruticose shrubs or perennial herbs with ovate, entire leaves, and corymbose or paniculateds inflorescnces. This genus can is represented by two species, O. vulgare Linn and O. Maru Linn V. sinaicum Boiss. Origanum vulgare is a native to the Mediterranean, Euro-Siberian and Irano-Siberian regions [7]. Due to the variability in chemical and aromatic characteristics, Origanum plants belonging to different species and ecotypes are widely used in agriculture, pharmaceutical and cosmetic industries as a culinary herb, flavoring substance of food products, alcoholic beverages and perfumery for their spicy fragrance. The essential oil of Origanium vulgare involves more than 20 ingredients, most of which are phenolic antioxidants. Major components are thymol and carvacrol that constitute about 78 to 82% of the total oil. It has been demonstrated that the essential oil derived from oregano possess in vitro antimicrobial, antifungal, insecticidal and antioxidant properties. These properties are mainly attributed to thymol and carvcrol. Due beginning and at the end of experiment were analyzed according to the standard methods for moisture [10], protein, fat and ash. Moisture content was estimated by drying the samples at 85°C in an oven (GCA, model 18 EM, precision scientific group, Chicago, Illinois, USA) to constant weight and calculating weight loss. Nitrogen content was measured using a microkjeldahl apparatus Labconco (Labconco Corporation, Kansas, Missouri, and USA) and crude protein was estimated by multiplying nitrogen content by 6.25. Total lipids content was determined by ether extraction in the multi-unit extraction Soxhlet apparatus (Lab-Line Instruments, Inc., Melrose Park, Illinois, USA) for 16 hours and ash was determined by combusting samples at 550C for 6 hr in a muffle furnace (Thermolyne Corporation, Dubuque, Iowa, USA). Crude fiber was estimated according to method of Goering and Van Soest [11]. Gross energy was calculated according to NRC [9].

Growth parameters
Growth performance was determined and feed utilization was calculated as follows: Weight gain = W2 -W1; Specific growth rate (SGR) =100 (ln W2 -ln W1)/T; where W1 and W2 are the initial and final weights, respectively, and T is the number of days of the feeding period; especially to the in vitro antimicrobial property, the application of oregano essential oil (OEO) in poultry production would be expected to have both prophylactic and therapeutic results [7,8].
This study aimed to evaluate the effects of Origanum vulgare extract on the growth performance, feed utilization, whole body composition, and resistance of Nile tilapia to Pseudomonas aeruginosa and Pseudomonas flourscence infection.

Extraction of origanum vulgare
A known weight of air dried material of Origanum vulgare was subjected to exhaustive extraction with ethyl alcohol (95%) using soxhlet apparatus. The obtained extract was then cooled, filtered and evaporated under vacuum to a thick syrup, which was dissolved in Di-Methyl Sulf-Oxide (DMSO), the solution was mixed with petroleum ether b.P (40-60°C) and centrifuged at 5000rpm, a deep green powder was obtained, containing Turbines (an active principal which protecting Fresh water fishes from bacterial diseases).

Diet preparation
Four experimental diets were prepared containing 30% crude protein and 7% lipid in addition to different levels 0.0, 0.5, 1.0, 1.5% of Origanum vulgare extract (Table 1), Origanum vulgare were obtained from local market and extracted in Lab. The dry ingredients of each diet were thoroughly mixed, 100 mL of water per kg diet was added and all contents were blended using kitchen blender to make a paste. Pelleting of diets was carried out by passing the blended mixture through laboratory pellet machine with 1 mm diameter matrix. The pellets were dried in a drying oven model (Fisher oven 13-261-28A) for 24 hours at 85°C, stored in plastic bags and kept in a refrigerator at -2°C during the experimental period to avoid rancidity [9].

Fish culture and feeding regime
Fingerlings of Nile tilapia, O. niloticus L. were obtained from fish hatchery, at the Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharkia. Fish were kept in indoor fiberglass tanks for 2 weeks for acclimation to the laboratory condition and they were fed a commercial diet, containing 30% crude protein. Fifteen fish were frozen at -20°C for proximate analysis at initial. Four groups of Nile tilapia ranged from 11 to 13 g were randomly distributed into four groups, each contains 15 fish per 100-L aqurium (triplicate each treatment). Each aquarium was supplied with compressed air via airstones using aquarium air pumps. Settled fish wastes were cleaned daily by siphonation with a three quarets of aquarium's water, which was replaced by aerated water from the storage tank. Water temperature ranged between 27°C -29°C. The feeding rate was 3% of live body weight, fish fed twice daily at 9.00 and 13.00 hours; six days a week for a period of 10 weeks. Fish in each aquarium were group-weighed every 2 weeks and the amounts of feed given were readjusted accordingly. Dead fish was daily recorded and removed.

Chemical analysis of diets and fish
The tested diets and whole-fish body from each treatment at the   Energy utilization (EU; %) =100 [Energy gain in fish (g) / energy intake in diet (g)].

Challenge test
The experimental fish were divided into two groups. The first group comprised fish fed control diet, and challenged I/P with pathogenic Ps. aeruginosa and Ps. Flourscence (0.2 ml of 4 x 10 6 CFU). The second group, comprised fish fed diets containing different levels of Origanum vulgare extract (0.5, 1.0 and 1.5) and challenged I/P with the same pathogen (0.2 ml of 4 x 10 6 CFU). Both groups kept under observation for 10 days to record the daily mortality rate. Isolation and identification of pathogens were determined according to method of Schäperclaus et al. [12].

Physiological measurement
At the end of the experiment feeding trial, fish were fasted for the 24 hour immediately prior to sampling and five fish per aquaria were randomly chosen and anesthetized with tricaine methane sulfate (20 mg/L). The blood samples were put in micro centrifuge tubes, without anticoagulant, left to clot at 4°C and centrifuged at 5000 rpm for 5 minutes at room temperature. The collected serum was stored at -20°C for further assays. Total protein content in plasma was determined colorimetrically according to Henry method [13], Albumin and globulin in plasma according to method of Wotton and Freeman [14].

Economical evaluation
The cost of feed required to produce a unit of fish biomass was estimated using a simple economic analysis. The estimation was based on the local retail sale market price of all the dietary ingredients during the time of this study. These prices (in LE/kg) were as follows: herring fish meal, 10; soybean meal, 2.0; corn meal, 1.50; starch, 3.0; Wheat bran, 1.25; fish oil, 7.0; corn oil, 5.0; vitamin premix, 7.0; mineral mixture, 3.0; extracted Origanum 30.

Statistical analysis
The obtained data were analyzed by one-way ANOVA to evaluate the effect of Origanum vulgare extract. Differences between means were tested at the 5% probability level using Duncan Multiple Range test. All the statistical analyses were done using SPSS program version 10 (SPSS, Richmond, USA) as described by Dytham [15].

Results and Discussion
In the present study, the experimental diets contained 30% crude protein and 4.4 kcal/g diet (Table 1) are similar to that used by [16]. Changes in body weight (g) of Nile tilapia fed different levels of Origanum vulgare extract during experiment are illustrated in Figure 1. Initial body weight at all experimental groups did not differ significantly ( Table 2). The fish actively and efficiently grew without any external signs of nutritional deficiency. Growth performance (final weight, weight gain, weight gain % and specific growth rate) increased significantly (P<0.05) with the experienced diets ( Table 3). The highest growth was obtained with diet of 0.5% Origanum vulgare extract, as compared to the control diet. No significant differences were reported in fish survival rate among different treatments (P>0.05), since it ranged from 93.3 to 100%. This may indicate that Origanum vulgare extract enhance fish growth, feed utilization and immunity. The improvement of live body weight, body weight gain, weight gain %, SGR and survival rate may be due to the content of essential oil and extracts of Origanum species containing (turbines) antimicrobial, antioxidant and other biological activities [7,8]. Furthermore a better growth performance was observed in Nile tilapia fed on diet contained 0.5% cinnamon and 1% marjoram as a feed additive to tilapia feeds were recommended by Abd El-Maksoud et al., [17].
As compared to control fish fed diet supplemented with 0.5% Origanum vulgare extract produced the highest protein efficiency ratio (PER), apparent protein utilization (APU) and energy utilization (EU), (Table 4). Moreover, fish fed diet supplemented with 0.5% Origanum vulgare extract improves fish growth and feeding efficiency; this improvement may be contributed to improve feed intake and nutrient digestibility. In addition, Origanum vulgare extract contains several nutrients, especially essential oils, vitamins and minerals that may help in fish growth promotion. These results are in agreement with those of Abdel-Maaksoud et al., [17] and Shalaby et al., [18].
The extract of Origanum vulgare had no significant (P>0.05) effect on the whole body composition (dry matter, crude protein, fat and ash) ( Table 2). Similar finding were given by Abdel-Maksoud et al. [17] and Shalaby et al. [18].
Results of fish challenging against of Ps. aeruginosa and Ps. flourscence for ten days were shown no mortalities in all diets containing different levels of Origanum vulgare extract. The highest overall fish mortality rates were observed in the control group (90% and 100%, respectively). This enhanced immune response may be induced by the essential oils content and extracts of Origanum species which contains (turbines) antimicrobial, antioxidant and other biological activities [7,8]. Furthermore, study by Sahin et al. [19] prove the antibacterial effect of oils and extracts of Origanum species on many bacterial species like Escherichia coli, Enterobacter sp., Bacillus sp., Salmonella sp., Staphylococcus aureus, Klebsiella pneumoniae, Listeria monocytogenes and Campylobacter jejuni. Total protein, albumin, and globulin values increased significantly (P ≤ 0.5) with Origanum vulgare extract, reaching the highest value at 0.5% of the diet, as compared to the control (Table  5). On the other hand, Origanum vulgare Extract showed negligible insignificantly effect on the albumin/globulin (A/G) ratio (Table 5). Glutamic Oxaloacetic Transaminase (GOT) and Glutamic Pyruvate Transaminase (GPT) values decreased with increasing origanum vulgare extract and the highest values was obtained at the control diet ( Table 5). The results indicate that the improvement of fish health was took place when fed diet with Origanum vulgare extract. Moreover, the measurement of total protein, albumin and globulin in serum or plasma are of considerable diagnostic value in fish, as it affects the general nutritional status as well as the integrity of the vascular system and liver function [12].
The economical evaluation of the experimental diets contained different extracted Origanum vulgare levels 0.0, 0.5%, 1% and 1.5% are shown in (Table 6). The highest reduction in feed cost compared with the control diet showed to produce one kg fish gain of treatment supplemented with 0.5% extracted Origanum vulgare. The reduction in feed cost compared with the control diet showed 12.52% to produce one kg fish gain of treatment supplemented with 0.5% extracted Origanum vulgare levels. Previous studies showed that the use of spices in small amounts gave lower incidence cost and higher profit index of fish species [18].

Conclusion
It could be concluded that Origanum vulgare extract improved growth performance and feed efficiency of Nile tilapia and strengthen its resistance to Ps. auroginsa and Ps. fluorescence infection. Thus, the feeding of Origanum vulgare extract at level of 0.5% in diets for Nile tilapia is recommended as an immunostimulant.   Feed intake, feed conversion ratio (FCR), protein efficiency ratio (PER), apparent protein utilization (APU), apparent fat utilization (AFU) and energy utilization (EU) of Nile tilapia fed diets containing different levels of Origanum vulgare extract. Means the same letter (a/b/c) in the same row is not significantly different at P<0.05. Means the same letter (a/b/c) in the same row is not significantly different at P<0.05.