Use of probiotic bacteria as growth promoters, anti-bacterial and their effects on physiological parameters of Oreochromis niloticus Yassir A. E.

Khattab 1, Adel M. E. Shalaby2 and Azza, Abdel-Rhman3 Fish Nutrition Department. 2- Fish Physiology and Hatchery Department. 3- Fish Disease Department. Central Laboratory for Aquaculture Research, Abbassa, Abo- Hammad, Sharkia. Key words: Nile tilapia, growth, FCR, PER, haematology, probiotics, antibacteria, Aeromonas hydrophila, Micrococcus luteus and Pseudomonas species. ABSTRACT Micrococcus luteus and Pseudomonas species were isolated from gonads and intestine of apparantly healthy Oreochrois niloticus respectively. M. luteus and Ps. species were safe for O niloticus and had antagonistic effect against the pathogenic Aeromonas hydrophila (the cause of motile aeromonas septicemia among freshwater fish) in vitro. The inhibition zone was 4 cm in diameter with M. luteus and 9 cm in diameter with Psuedomonas sp. The effect of supplemental dietary probiotic bacteria (M. luteus and Ps. species, 107 cells per gram food) used as anti-bacterial on some physiological parameters, growth performance and survival rate of O. niloticus was also studied. Nile tilapia (2.35 - 2.45 g) were assigned to four treatments, with three replicates each. Fish were fed frequently a diet of 30% crude protein (CP) at a rate of 3% of live body weight twice daily for six days a week for 90 days. The obtained results showed that the final weight, weight gain and specific growth rate of O. niloticus increased significantly in fish fed with diet containing M. luteus, while they were decreased significantly in fish fed a diet containing Psuedomonas sp, when compared to the control group. The highest growth performance was recorded with M. luteus. Fish survival rate was decreased in fish fed with diet containing P. species. Feed intake was similar except for the fish that were fed M. luteus. The best feed conversion ratio (FCR) was observed with the fish fed M . luteus. There was no significant differences in protein efficiency ratio (PER) except that containing Psuedomonas sp. Crude protein content in whole fish body was significantly increased in fish fed with a diet containing a mixture of the two bacteria species, while total lipids were decreased significantly in all treatments. Ash content in whole fish body was significantly increased in all treatments. Erythrocyte counts (RBCs) in fish fed diets containing Micrococcus luteus were significantly higher than that of the control, but the RBCs and haemoglobin content (Hb) were decreased significantly in fish fed with the diet containing Psuedomonas sp.. Haematocrit values (Hct) were decreased significantly in all treatments. Also, the plasma total protein, aspartate amiontransferase (AST). Alanine amionotransferase (ALT) and lactate dehydrogease (LDH) were significantly decreased in fish fed with all diets containing Psuedomonas sp. On the other hand, the glucose concentration increased in all treated groups. After the O. niloticus challenged by Aremonas hydrophila 0.3107 cells / ml i.p.route, the mortality rate was 25% with M. luteus while Ps. species 90 %. The mixed group had mortality rate 80% as the control one. M. luteus was safe to O. niloticus and had probiotic effect in vitro and in vivo, while Ps. species had probiotic effect invitro only.

INTRODUCTION Fish Diseases are major problem for the fish farming industry, which currently is the fastest growing food-protein producing sector with an annual increase of approximately 9%, among those, bacterial infections are considered as the major cause of mortality in fish hatcheries (Grisez and Ollevier, 1995). The motile aeromonas group, especially A. hydrophila, affects a wide variety of freshwater fish species and occasionally marine fish (Larsen and Jensen, 1977). The prophylactic and therapeutic control of the bacterial diseases is based on oral administration of antibiotics. However, such treatment may cause the development of resistant bacteria (Aoki et al., 1985), yeild residues in fish and introduce potential hazard to public health and to the environment. Furthermore, the normal microbial flora in the digestive tract, which is beneficial to fish, may also be killed or inhibited due to oral chemotherapy (Sugita et al., 1991). Although vaccines are being developed and marketed, they cannot be used alone as a universal disease control measure in aquaculture (Ambile-Cuevas et al., 1995). A new approach method, that is gaining acceptance within the industry, is the use of probiotic bacteria to control potential pathogens (Gomez-Gil et al., 2000, Robertson et al., 2000). In recent years, there is a great interest in the use of probiotic bacteria in aquaculture to improve disease resistance, water quality and/or growth of farmed fish (Verschuere et al., 2000). Probiotic bacteria have a posible competition for nutrients with pathogens in the digestive tract (Gatesovpe, 1997) or the hypothetical stimulation of the immune system, as the activation of macrophage (Perdign et al., 1990). The antibacterial effect of bacteria is generally due to either singly or in combination, production of antibiotics, bacteriocins, siderophores, lysozymes and proteases, and alteration of pH values by organic acids production (Sugita et al., 1998). The use of probiotics stimulates rainbow trout immunity by stimulating phagocytic activity, complement mediated bacterial killing and immunoglobulin (Ig) production (Nikoskelainen et al., 2003). Therefore, the present study was carried out to evaluate the role of M luteus and Psuedomonas sp. as growth promoters and antibacterials for Nile tilapia fry (O. niloticus) and their effect on some physiological parameters. MATERIALS AND METHODS Bacterial studies:Healthy O. niloticus (2.33- 2.45g) were randomly collected from the earthen pond at the Central Laboratory for Aquaculture Research (CLAR), Abbassa, Abou-Hammad, Sharkia Governorate, Egypt. Bacteriological examinations of the collected fish were done, where samples from the internal organs (liver, kidney, gonads, stomach and intestine) and gills were cultured on TSB and incubated at 30C for 24-48 hours. Purification and identification of the isolates were done using biochemical tests according to Bergey et al. (1984), Austin and Austin (1993) and API 20 E strip system (Bio Merieux). The isolated bacteria from the internal organs of the investigated fish (intestine, stomach and gonads) were examined for its inhibitory effects against the pathogenic A. hydrophila obtained from CLAR-Fish Diseases Department. The in vitro probiotic activity was done using agar diffusion method and the inhibition zoon determined as described by Ruiz et al., (1996).

Safety of the two isolates probiotics was studied by using 90 apparently healthy fish that were acclimatized for two weeks in indoor tanks. The fish were divided into 3 equal groups (three replicates each). Fish of the 1st and 2nd groups were i.p. inoculated by 0.3 ml of saline containing 107 cells / ml of 24 hrs M. luteus or Pseudomonas sp., respectively. Fish of the 3rd group were I/P inoculated by 0.3 ml of saline as controls according to Irianto and Austin (2002). Preparation of probiotics diet:The probiotic bacteria were prepared by inoculating the bacterial isolates in TSB and incubated at 30C for 48 hrs, then centrifuged at 3000 rpm for 30 minutes. After centrifugation, the bacteria was washed twice with saline. The solution was added to the bacterial cells (till one ml saline contained 1010 bacterial cells). The saline containing the probiotic isolates was added to commercial sterilized food to give an initial number of 1010 bacterial cells/ g. Dry ingredients were sieve and ground to a small particle size in a Thoms-Wiley Laboratory Mill model 4, USA. Ingredients were mixed to formulated basic diet, which consisted of 13% hearring fish meal, 40% soybean meal, 5% wheat bran, 35.94% ground yellow corn, 2.5% fish and corn oil (1:1), 0.06% ascorbic acid, 1.5% vitamins and minerals mixture, 2% carboxymethyl cellulose as a binder. The experimental basic diet formulated to contain 30% crude protein and 421 k cal of gross energy/100g diet on a dry weight basis. Bacteria strains supplemented in base diet to conducted four treatments: base diet untreated (control); base diet mixed with M. luteus (T1), base diet mixed with Pseudomonas sp (T2) and a mixture from M luteu and Pseudomonas sp diets with equal amounts (T3). Experimental diet were passed with clean and sterile auger and diet of Spaghetti Machine, La Parmigiana, Model D45 LE, Italy and dried at room temperature. Diets transferred to black plastic bags and stored in a freezer (20 C) until immediately prior feeding. The number of probiotic bacterial cells in the prepared pellets were found to be 07 cells/g diet. Feeding culture system design: The feeding experiment was conducted in 12 glasses aquaria (754050 cm), at the Center Laboratory for Aquaculture Research, Abbassa, Abou-Hammad, Sharkia, Egypt. The aquaria were supplied with 100 ml well aerated tap water. Each aquarium was supplied by compressed air via-stone. Fresh tap water was stored in cylindrical fiberglass tanks for 24 hours under areation in order to dechlorenated the water. Each aquarium was cleaned daily by siphoning fish feces and remaining feed with 75% of total water volume, then refilled to fixed volume again. Water temperature range was (26- 28C). Nile tilapia fries, O. niloticus with an average weight (2.35 - 2.45 g / fish) were acclimatized in an indoor fiberglass for two weeks. The fish were distributed randomly at a rate of 20 fish / aquarium. Treatments in triplicate were arranged at random. The experimental fish were batch weighed at the beginning of the experimental period. Fish were fed twice daily, 6 days a week, at a fixed feeding rate of 3% body weight of fish per day. The feed given were adjusted at 2-weeks intervals after fish were weigh. The experiment ran for 90 days, after which all the experimental fish were collected , counted and weight. The growth parameters and feed utilization were calculated according to: Specific growth rate (SGR) = 100 (ln W2 ln W1) T -1

Where W1 and W2 are the initial and final weight, respectively, and T is the number of days in the feeding period. Feed conversion ratio (FCR) = FI (B2 + B dead B1)-1 Where FI, B1 and B2 are the feed intake, the biomass at the start and end, respectively, and B dead is the biomass of the dead fish. Protein efficiency ratio (PER) = (B2 B1) PI-1 Where B1 and B2 are the biomass at the start and the end of the experiment, and PI is the protein intake. . Physiological Analyses: At the end of experiment, blood samples were taken from the caudal vein of an anaesthetized fish by sterile syringe using EDTA solution used as an anticoagulant. The blood samples were used for determining erythrocyte count (Dacie and Lewis 1984) and hemoglobin content (Vankampen, 1961). Haematocrit value (Hct) were calculated according to the formulae mentioned by Britton (1963). Plasma was obtained by centrifugation of blood at 3000 rpm for 15 min and nonhaemolyzed plasma was stored in deep freezer for further biochemical analyses. Plasma glucose was determined using glucose kits supplied by Boehring Mannheium kit, according to Trinder (1969). Total protein content was determined colorimetrically according to Henry (1964). Activities of aspartate amninotransferase (AST) and alanine aminotransferase (ALT) were determined colorimetrically according to Reitman and Frankel (1957), while lactate dehydrogenases (LDH)) were measured by using Diamond diagnostics kits according to the method of Cobaud and Warblewski (1958). Challenge test:After 90 days of feeding, the fish of each group were divided into two subgroups, the first subgroup of each treatment was challenged I/P with pathogenic A. hydrophila (0.3 ml of 107 cells / ml) which was obtained from (CLAR). The second subgroup was injected i.p. by 0.3 ml of saline as control. Both subgroups kept under observation for 14 days to record the survival rate daily. Chemical analysis:The proximate chemical analysis of fish and diets were done according to the method of AOAC (1990). Dry matter was determined after drying at 105 C until constant weight and ash after heating of dry samples in muffle furnace at 580C for 6 hours. Crude protien was determined by the Kjeldahl procedure. Total lipid was determined by extraction by petroleum eather in soxhelt apparatus. Statistical analysis: The obtained results were analysed statistically using analysis of variance (ANOVA). Dauncans multiple range test (Dauncan, 1955) was used to evaluate the mean differences among different treatments at the 0.05 significant level. RESULTS Bacteriological examinations: In the present study, bacteriological examinations revealed that, the suspected probiotic bacterial isolates were identified as M. luteus and Psuedomonas sp. The physical and the biochemical characters of M. luteus revealed that it was Gram postive cocci, motile, oxidase and catalase were produced and oxidation/ fermintation were negative. It produced acid from arabinose, galactose and insitol. It did not produce acid

from glucose, fractose, salicin, sorbitol, xylose, trehalose, manitol or maltose. It did not hydrolysed starch or arginine. It grows on nutrient agar contained 0.0, 3, and 5% NaCl, and grow at 45C. It did not haemolyside blood. M. luteus was isolated from gonads of apparently healthy O. niloticus. Psuedomonas sp was gram negative, bacilli, single, motile, oxidative, facultative anaerobe and oxidase-postive. It produced acid from glucose and did not produce acid from sucrose, salicin, arabinose, lactose, galactose, sorbitol, xylose or manitol. Arginine hydrolysis, ornithine and lycin decarboxylase were negative with it. It isolated from intestine of apparently healthy O. niloticus. The in- vitro probiotic activity: Two isolates, M. luteus and Psuedomonas sp were examined for a probiotic activity against the pathogenic A. hydrophila in vitro. It showed inhibitory effects against A. hydrophila. However, Psuedomonas sp gave larger inhibition zone (9 cm) than M. luteus (4 cm). Safety of the isolated probiotics: Two isolates of the probiotics were harmless to O. niloticus as no clinical signs or mortalities were noticed following challenge via I/P route. M. lutius and P. species were safe to fish in comparison to the control as show in table (1) which gives mortality rate 10%. Growth Performance: Results in Table (2) showed that the final weight, weight gain and specific growth rate of O. niloticus increased significantly when fed a diet containing M. luteus only (P<0.05., 10.16g/fish final weight, 7.773 g/fish weight gain and 1.61 %/day SGR). These values decreased significantly in the fish group which fed a diet containing Psuedomonas sp (8.02 g/fish final weight, 5.626 g/fish weight gain and 1.34 %/day SGR). The O. niloticus in the group which fed a diet containing mixed bacteria ( equal amounts of Psuedomonas sp and M. luteus) were slightly increased (8.976 g/fish final weight, 6.59 g/fish weight gain and 1.47 %/ day SGR) but less than control group (9.39 g/fish final weight, 6.99 g/fish weight gain and 1.536 %/day SGR). Survival rate was decreased in O. niloticus fed with diet contained P. species (95%) then mixed diet (97.5%), while control group and fish fed with diet containing M. luteus were 100% (Table 2). Feed intake was approximately similar and ranged from 11.14 to 11.68 g feed/fish except the fish group fed a diet containing M. luteus in which the feed intake was slightly increased (12.28 g feed/fish, Table 3). On the other hand, the least feed conversion ratio (FCR) was observed with fish group fed diet contained M. luteus (1.58), while the highest one was observed at control, the fish group that fed with Psuedomonas sp, and that fed mixed bacteria with insignificant difference (1.69, 1.74 and 1.98, respectively). Table (3) indicated that there was a significant increase in protein efficiency ratio (PER) with the diet containing M. luteus (2.156). Protein efficiency ratio with the diet containing Psuedomonas sp showed the least PER (1.72). The control and mixed groups had protein efficiency ratio of 2.026 and 1.96, respectively. Concerning the proximate chemical analysis of whole fish body, table (4) showed that the moisture content was approximately similar (27.87-31.25%; P>0.05). Crude protein content in whole fish body was approximately similar (53.16-55.58%).

On the other hand, content of the total lipids was low at the diet contained mixed bacteria (19.24%). The highest content of the total lipids was obtained in the control (23.5 %). Total lipid content in the fish groups which fed diet contained P. species and M. luteus were 20.93 and 22.31%, respectively. Ash content in whole fish body was significantly increased with diets containing P. species, mixed bacteria and M. luteus (25.90, 25.17 and 23.87%, respectively), while the least one was observed in the control (21.69 %). Physiological Parameters: Erythrocyte counts in fish fed diets containing M. luteus were significantly high (1.059 0.043 million/mm3), while the decreased significantly in fish fed with diet containing Psuedomonas sp (0.626 0.034 million/mm3). The erythrocyte count in fish fed diets containing mixed bacteria and control group were 0.821 0.034 and 0.997 0.067 million/mm3, respectively. Hemoglobin content was slightly increased in fish fed diet containing M. luteus (5.12 g/100 ml ). Also, the hemoglobin content decreased in fish fed diet containing Psuedomonas sp (3.29 g/100 ml). Hemoglobin content in fish fed diets containing mixed bacteria and control group were 4.115 and 4.57 g/100 ml, respectively. Haematocrit value (Hct) decreased at fish fed diets containing P. species (9.0%), while other treatments were approximately similar and ranged from 14.75% to 18.5% (Table 5). The plasma glucose concentration of the fish control group was 85.72 3.45 (mg%). Data presented in Table (6) indicated that fed of O niloticus with pro biotic induced an increase in the plasma glucose as compared to the control group. It was significantly increase in fish fed with diet containing Psuedomonas sp and mixture of M. luteus and Psuedomonas sp. On the other hand, as shown in table (6) the plasma total proteins were decreased significantly in fish fed with diet containing Psuedomonas sp and mixture of M. luteus and Psuedomonas sp (2.66 0.136 and 2.75 0.170 g/L, respectively) where the control group was 3.17 0.118 (g/L). Also, the AST and ALT activities in plasma of O niloticus were significantly decreased in fish fed with diet containing Psuedomonas sp and mixture of M. luteus and Psuedomonas sp (Table 6) when compared to treatment fish fed with diet containing M. luteus only and the fish in control group. Same as the trend, the average level of plasma LDH activity in fish control group was ( 189.90 8.16 IU/L). As shown in Table (6), the lactate dehydrogease was significantly decreased in fish fed with diet containing Psuedomonas sp and mixture of M. luteus and Psuedomonas sp. Challenge test results: Table (7) showed that M. luteus had a probiotic effect with fish. The mortality rate was 25% of O. nilotecus fed on a diet containing M. luteus for three months and challenged i.p. by pathogenic A. hydrophila (0.3 ml 0f 107 cells/ml) . The mortality rate of O. niloticus which fed on a diet containing Psuedomonas sp was 90% , this ratio was more than control. The fish which fed on diet contained Psuedomonas sp had external sings including ulceration and hemorrahges, and the fish becam sensitive to any infection. The mixed and the control groups had the same mortality rate (80%).

DISCUSSION Probiotics, which are micro-organisms or their products with health benefit to the host, have been used in aquaculture as a means of diseases control, supplemnting or even in some cases replacing the use of antimicrobial compounds. In the present study, The physiological and the biochemical characters of suspected probiotic bacterial isolates were identified as Micrococcus luteus and Psuedomonas sp. as identified by Bergey et al. (1984) and Austin and Austin (1993). A wide range of Gram-positive (Bacillus, Carnobacterium, Enterococcus, Lactococcus, Lactobacillus, Micrococcus and Streptococcus) and Gram-negative bacteria (Aeromonas, Alteromonas, Photorhodobacterium, Pseudomonas and Vibrio) has been evaluated as probiotics in aquaculture (Irianto and Austin 2002a). M. luteus and Psuedomonas sp. showed inhibitory effects in vitro against A hydrophila. However, Psuedomonas sp. gave larger inhibition zone (9 cm) than M luteus (4cm). M. luteus and Psuedomonas sp. were isolated from gonads and intestine, respectively, of apparently healthy O. niloticus, but Irianto and Austin (2002b) isolated M. luteus A1-6 from digestive tract of Oncorhychus mykiss. Sugita et al. (1998) isolated M. luteus. and Psuedomonas sp from the intestine of seven fish species and recorded that the bacteria had inhibitory effect against Vibrio vulnificus. They isolated Alteromonas haloplanktis from gonads of Argopecten purpuratus broadstock. Riquelme et al. (1996) noticed that Alteromonas haloplanktis had inhibitory activity against Vibrio sp and A. hydrophila. Lewus et al. (1991) reported that the bacteriocins which produced by lactic acid bacteria had inhibitory effect against A. hydrophila. Also, they isolated Pseudomonas (C30, 217) from broadstock. Spanggaard et al. (2001) isolated Psuedomonas sp. from O. mykiss and used as probiotics in O. mykiss mixed in water. Smith and Davey (1993) reported that P. flurescence reduced diseases caused by A. salmonicida. From the present study M. luteus had a probiotic effect in vitro and in vivo against A. hydrophila, while Psuedomonas sp had a probiotic effect in vitro only and in vivo it changed to pathogenic bacteria. Our results agree with Chang and Liu (2002b) who indicated that Bacillus toyoi suppressed the growth of Edwarsilla tarda in vitro, but did not reduce mortalities in eels due to edwardsillosis in vivo. Irianto and Austin (2002) used M. luteus with feed as a potential combating A. salmonicida infection in rainbow trout (O. mykiss). M. luteus gave mortality rate 25% at 107 cells / g of feed among O. niloticus challenged by A. hydrophila. Lactobacillus rhamnosus was administered at a dose of 109 and 1012 cells / g of feed to rainbow trout for 51 days and reduced mortalities from 52.6 to 18.9 % (109 cells / g feed) and to 46.3% (1012 cells / g of food) following challenge with A. salmonicida (Nikoskelainen et al., 2001). The mortality rate between O. niloticus which fed on a diet containing Psuedomonas sp was 90% which did not agree with Gram et al. (1999) who reported that P. fluorescens strain (AH2) reduced the mortality of 40 g rainbow trout infected with a pathogenic V. anguillarum. The mortality rate at the treated fish was 32% compeared to control 47%. The mode of action of the probiotics is rarely investigated, but possibilities include competitive exclusion, i.e. the probiotics actively inhibit the colonization of potential pathogens in the digestive tract by antibiosis or by competition for nutrients and

/ or space, alteration of microbial metabolism, and/or by the stimulation of host immunity (Irianto and Austin, 2002a). Numerous trials were conducted with microorganisms known as probiotics to improve culturability of food species and to improve human health and welfare. Appropriate probiotic applications were shown to improve intestinal microbial balance, thus leading to improve food absorption (Parker, 1974; Fuller, 1989), and reduced pathgogenic problems in the gastrointestinal tract (Lloyd et al., 1977; Goren et al., 1984). The final weight, weight gain, specific growth rate, survival rate feed intake and protein efficiency ratio were increased among O. niloticus fed a diet containing M. luteus, so it may be considered as a growth promoter in fish aquaculture. These results agree with Rengpipat et al. (1998) and Prabhu et al. (1999) were reported that the probiotic treated group enhancing growth rate of shrimps and maintaining water quality parameters. Survival of shrimps was significantly greater in treated group compared with the control group. Lactic acid bacteria had an effect as growth promotor on the growth rate in juvenile carp but not in sea bass (Noh et al., 1994). Also, Emterococcus. faecium had also been used to improve growth when fed to sheat fish, Silurtes glanis L (Bogut et al., 2000). Probiotics may stimulate appetite and improve nutrition by the production of vitamins, detoxification of compounds in the diet, and by the breakdown of indigestible components(Irianto and Austin, 2002a). Streptococcus faecium improved the growth and feed efficiency of Israeli carp (Noh et al., 1994 and Bogut et al., 1998). A several probiotic species were used including Lactobacillus sp. (Jonsoon, 1986) and mixes cultures (Lessard and Brisson 1987). The use of probiotics can improve the nutrition level of aquacultural animals and improve immunity of cultured animals to pathogenic microorganisms. In addition, the use of antibiotics can be reduced and frequent outbreaks of diseases can be prevented. Riquelme et al. (1997) studied the naturally occurring bacteria which are able to promote the growth and survival of Argopecten purpuratus larvae by inhibiting the activity of other bacteria that flourish in hatchery cultures. Tovar-Ramrez et al. (2004) notced that the growth of larvae of sea bass fed 1.1% live yeast as a probiotic was increased than control group. Also, survival of larvae was significantly higher than the control. Kennedy et al. (1998) showed that the addition of a gram-positive probiotic bacterium increased survival, size uniformity, and growth rate of marine fish larvae (snook, red drum, spotted seatrout and stripped mullet). Much less work has been directed at the immunological enhancement of defence mechanisms of fish by probiotic bacteria or the protective mechanisms of probiotic bacteria in fish (Nikoskelainen et al., 2003). Also less work has been directed at the blood parameters. Results of the present investigation showed that Psuedomonas sp in diet of O. niloticus caused a decrease of blood parameters (RBSC, HB and Hct). These results are in agreement with those of Palikova et al (2004) who observed pathmorphological findings (haemorrhages in the skin, eyes, hepatopancreas and in swim bladder) in the common carp after exposure to Cyanobacteria extract. Ishikawa (1998) showed that the spleen, liver and kidney of unhealthy fish held fairly severe infection , suggesting that haematopoiesis was also severely affected and this affected the peripheral blood by decreasing erythrocyte

volume. Also Ranzani-Paiva et al (2004) showed that the decrease in erythrocytes count and haematocrit of Nile tilapia inoculated with Mycobacterium marinum may lead to a tendency to develop hypochromic, microcytic anaemia. M. luteus had a good effect on erthrocytes count, heamoglobin content and haematocrit value was increasing than control group. These results agree with Irianto and Austin (2002), who recorded an increase in erthrocyte count in fish fed on probiotic bacteria than control group. The production of immunoglobulin A (IgA) is also stimulated and protecting the mice against Salmonella typhimurium (Perdigon et al., 1990). Nikoskelainen et al. (2003) reported that the selected probiotic bacteria may have an impact on the spasific and innate immunity of fish. They recorded that (Ig) level was significantly increased with the probiotic feed group. The plsma glucose concentration was significantly increased in fish fed with diet containing Psuedomonas sp and mixture of M. luteus and Psuedomonas sp. These the results are in agreement with those of Peters et al (1988) who found elevated of glucose levels in plasma of Salmo gairdneri when injected with Aermonas hydrophila or add to water of aquarium. The present study indicated that Psuedomonas sp may cause stress of fish. On the other hand, the plasma total proteins showed decreased significance in fish fed with diet containing Psuedomonas sp and mixture of M. luteus and Psuedomonas sp. These results agree with those of Cruz et al. (1989) who found lower total protein in plasma of Salmo gairdneri when injected with Vibrio anguillarum extracellular products intramuscularly. The present study indicated that the AST, ALT and LDH activities in plasma of O niloticus was significantly decreased in fish fed with diets containing Psuedomonas sp. and mixture of M. luteus and Psuedomonas sp. The present results agree with Palikova et al (2004) who indicated decrease of enzyme activities (AST, ALT and LDH ) in Cyprinus carpio after exposure to extract of Cyanobacteria. The decrease of these parameter may be due the severe damage of some organs liver, spleen, muscle and kidney (Cruz et al .,1989). At the same time, results of the present investigation proved that Psuedomonas sp exhibited contrary results in the same studied parameters and hence can not be considered as O. niloticus culture. However, a mixture of both bacterial species improved the protein content of fish. In conclusion, Micrococcus luteus isolate was clearly beneficial for cultured O. niloticus when administered as a food additive. It is argued that such probiotic has a role in disease control strategies, growth promotion and it improves the blood picture and biochemical parameters among O. niloticus in aquaculture.

References
Ambile-Cuevas, C.F.; Grdenas-Garci, M. and Ludgar, M. (1995): Antibiotic resistance. Am. Sci., 83: 320-329. AOAC (1990): Official Methods of Analyses of the Association of Official Analytical Chemists International. "15th edition, Association of Official Analytical Chemists, Arlington, VA, USA. Aoki, T.; Kanazawa, T. and Kitao, T. (1985): Epidemiological surveillance of drugresistant Vibrio anguillarum strains. Fish Patho., 20: 199-208.

Austin, B. and Austin, D.A. (1993): "Bacterial Fish Pathogens: Diseases in Farmed and Wild Fish. 2nd ed. Ellis Horwood Ltd., Chichester, England . Bergey, D.; Sneath, P. and John, H. (1984) Bergeys Manual of Systematic Bacteriology. Williams & Wilkins, Baltimore. Vol. I section 5. Bogut, I.; Milakovic, Z.; Brkic, S.; Novoselic, D. and Bukvic, Z. (2000): Effects of Enterococcus faecium on the growth rate and content of intestinal microflora in sheat fish , (Silurtes glanis L). Vet. Med. 45: 107-109. Bogut, I.; Milakovic, Z.; Brkic, S. and Zimmer, R. (1998): Influence of probiotic (Streptococcus faecium M74) on growth and content of intestinal microflora in carp (Cyprinus carpio). Czech J. Anim. Sci., 43: 231-235. Britton, C.J. (1963): "Disorders of the Blood",9th ed. I. A. Churchill, Ld. London. United Kingdom. Chang, C.I. and Liu, W.Y. (2002): An evaluation of two probiotic bacterial strains, Enterococcus faecium, SF68 B. toyoi, for reducing edwardsiellosis in cultured European eel, Anguilla anguilla L. J. Fish Dis., 25: 311-315. Cobaud, P. A. and Warblewski. T(1958):Colorimeteric determination of lactic acid dehydrogenase of body fluids. Am. J. Path,10: 234- 236. Cruz, M. C.; De- La. And Mroga, K. (1989): The effect of Vibrio anguillarum extracellular products on Japanese eels. Aquaculture, 80 (3-4): 2010210. Dacie, J. V. and Lewis, S. M. (1984): Practical Haematology., Churchill Living Stone. London Duncan, D. B. (1955): Multiple range and multiple (F) test. Biometrics, 11: 1- 42. Fuller, R. (1989): Probiotic in man and animals. J. Appl. Bacteriol., 66: 365-378. Gatesovpe, F.J. (1997): Siderophor production and probiotic effect of Vibrio sp. Associated with turboe larvae, Scophthalmus maximus. Aquatic Living Resour., 10: 239-246. Goren, E.; De Jong, W. A.; Doornenbal, P.; Koopman, J.P. and Kennis, H. M. (1984) Protection of chicks against salmonella infection induced by spray application of intestinal microflora in the hatchery. Veterinary Quarterly., 6: 73-79. Gomez-Gil, B.; Roque, A. and Turnbull, J.F. (2000): The use and selection of probiotic bacteria for use in the culture of larval aquatic organisms. Aquac.191: 259-270. Gram, L.; Melehiorsen, J.; Spanggaard, B.; Huber, L. and Nielsen, T.F. (1999): Inhibition of Vibrio anguillarum by Pseudomonas fluorescens AH2, a possible probiotic treatment of fish. Appl. Environ. Microbiol., 65: 969-973. Grisez, L. and Ollevier, F. (1995): Vibrio (Listonella) anguillarum infection in marine fish larviculture. In:Lavens, P., Jaspers, E., Roelande, l. (Eds.), Larvi 91-fish and crustacean larviculture symposium. Eur. Aquac. Soc. Gent. p. 497, Special publication no, 24. Henry, R. J. (1964): Colorimetric determination of total protein. In: Clinical Chemistry. Harper and Row Publ., New York, pp 181. Irianto A. and Austin B. (2002a): Probiotics in aquaculture (Review). J. Fish. Diseases, 25: 633-642. Irianto A. and Austin B. (2002b): Use of dead probiotics to control furunculosis in rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish. Diseases, 25: 333342.

Ishikawa, C. M(1998): Oreochromis niloticus (Tilapia do Nilo) inoculados experimentalmente com Mycobacterium marimum ATCC 927. M. Sc. Thesis . University of Sao Paulo. SaoPaula, Brazil. Jonsoon, E. (1986) Persistence of lactobacillus strain in the gut of sucking piglets and its influence on performance and health. Swed. J. Agric. Res. 16, 43-60. Kennedy, S.B.; Tucker, J.W.; Thoresen, M. and Sennett, D.G.(1998): Current methodology for the use of probiotic bacteria in the culture of marine fish larvae. Aquaculture 98, World Aquaculture Society. Baton Rouge, P. 286. Larsen, J.L. and Jensen, N.J. (1977): An Aeromonas species implicated in ulcer-disease of the cod (Gadus morhua). Nord Vet. Med., 29: 199-211. Lessard, M. and Brisson G.J. (1987) Effect of a lactobacillus fermentation product on growth, immune response and fecal enzyme activity in weaned pigs. Can. J. Anim. Sci. 67, 509-516. Lewus , C.B.; Kaiser, A. and Montville, T.J. (1991): Inhibition of food-born bacterial pathogens by bacteriocins from lactic acid bacteria isolated from meat Appl. Environ. Microbiol. 57: 1683-1688. Lloyd, A. B.; Coming, R.B. and Kent, R.D. (1977) Prevention of Salmonella typhimurium infection in poultry by pretreatment of chickens and poultry with intestinal extracts. Australian Veterinary Journal 53, 82-87. Nikoskelainen, S.; Ouwehand, A.C.;Bylund, G.; Salminen, S. and Lilius, E. (2003): Immune enhancement in rainbow trout (Oncorhynchus mykiss) by potential probiotic bacteria (Lactobacillus rhamnosus). Fish and Fish Immun. 15: 443452. Nikoskelainen, S.; Ouwehand, A.; Salminen, S. and Bylund, G. (2001) Protection of rainbow trout (Oncorhynchus mykiss) from furunculosis by Lactobacillus rhamnosus. Aquaculture 198, 229-236. Noh, S.H.; Han, K.; Won, T.H. and Choi, Y.J. (1994): Effect of antibiotics, enzyms, yeast culture and probiotics on the growth performance of Israeli carp. Korean J. Anim. Sci., 36: 480-486. Palikova, M.; Navratil, S.; Krejcf, R.; Sterba, F.; Tichy, F. and Kubala, L(2004): Outcomes of repeated exposure of carp (Cyprinus carpio L) to Cyanobacteria extract. Acta. Vet. Brno., 73: 259- 265. Parker, R. B. (1974) Probiotics, the other half of the antibiotic story. Anim. Nutr. Health 29, 4-8. Perdign, G.; Alvarez, S.; Nader de Macas, M.E.; Roux, M.E. and Ruiz Holgado, A.P. (1990): The oral administration of lactic acid bacteria increase the mucosal intestinal immunity in response of enteropathogens. J. food Protec. 53: 404-410. Peters, G.; Faisal, M.; Lang, T. and Ahmed, I (1988): Stress caused by social interaction and its effect on susceptibility to Aeromonas hydrophilia infection in rainbow trout Salmo gairdneri. Aquat. Org., 4 (2):889. Prabhu, N.M.; Nazar, A.R.; Rajagopal, S. and Ajmal-Khan, S. (1999): Use of probiotics in water quality management during shrimp culture. J. Aquac. Tropics. 14 (3): 227-236. Ranzani- Paiva, M. T.; Ishikawa, C. M.; Eiras, A. C. and Silveira, V. R (2004): Effects of an experimental challenge with Mycobacterium marinum on the bood

parameters of Nile tilapia , Oreochromis nitoticus (Linnaeus, 1757). Brazilian. Archives Biology Technology., 47 (6):945- 953. Reitman, S. and Frankel, S. (1957): Colorimetric determination of glutamic oxaloacetic .and glutamic pyruvic transaminases. Amer. J. Clin. Pathol., 28: 53-56 Rengpipat, S.; Phianphak, W.; Piyatiratitivorakul, S. and Menasveta, P. (1998): Effects of a probiotic bacterium on black tiger shrimp Penaeus monidon survival and growth. Aquac. 167: 301-313. Riquelme, C.; Araya, R.; Vergara, N.; Rojas, A.; Guaita, M. and Candia, M. (1997): Potential probiotic strains in the culture of the Chilean scallop Argopecten Purpuratus (Lamarck, 1819). Aquacult., 154: 17-26. Riquelme, C.; Hayashida, G.; Araya, R.; Uchida, A.; Satomi, M. and Ishida, Y. (1996): Isolation of native bacterial strain the scallop Argopecten Purpuratus with inhibitory effects against pathogenic vibrios. J. Shelfish Res. 15 (2): 369-374. Robertson, P. A. W.; ODowd, C.; Burrells, C.; Williams, P. and Austin, B. (2000): Use carnobacterium sp. as probiotic for Atlantic salmon (Salmo salar L) and rainbow trout (Oncorhynchus mykiss, Walbaum). Aquaculture., 185: 235-243. Ruiz, C.M.; Roman, G. and Snchez, J.L. (1996): A marine bacterial strain effective in producing antagonisms of other bacteria. Aquac. Intern., 4: 289-291. Smith, P. and Davey, S. (1993): Evidence for the competitive exclusion of Aeromonas salmonicida from fish with stress inducible furunculosis by fluorescent pseudomonad. J. fish Dis. 16 (6): 521-524. Spanggaard, B.; Huber, I.; Nielsen, E. B.; Pipper, C. B.; Martinussen, T.; Slierendrecht W. J. and Gram, L. (2001): The probiotic potential against vibriosis of the indigenous microflora of rainbow trout. Environmental Microbiology., 3: 755765. Sugita, H.; Hirose, Y.; Matsuo, N. and Deguchi, Y. (1998): Production of the antibacterial substance by Bacillus species strain NM12, an intestinal bacterium of Japanese coastal fish. Aquacult., 165: 269-280. Sugita, H.; Miyajima, C. and Deguchi, Y., (1991): The vitamin B12-producing ability of the intestinal microflora of freshwater fish. Aquacult., 92: 267-276. Tovar-Ramrez, D.; Zambonino-Infante, J.; Cahu, C.; Gatesoupe, F.J. and VzqueJurwz, R. (2004): Influence of dietary live yeast on European sea bass (Dicentrarchus labrax) larval development. Aquacult. 234: 415-427. Trinder, P. (1969): Determination of glucose concentration in the blood. Ann. Clin. Biochem., 6:24. Vankampen, E. J. (1961): Determination of haemoglobin. Clin. Chem. Acta, 5: 719720. Verschuere, L.; Rombaut, G.; Sorgeloos and Verstraete, W. (2000): Probiotic bacteria as biological agents in aquaculture. Micro. And Mole. Biol. Rev., 64 (4): 655-671.

Table (1) challenge test to the probiotc bacterial isolates among O. niloticus Micrococcus Pseudomonas Isolates control luteus species .i.p .i.p i.p. Route of injection dose 0.3ml of saline (0.3ml (107 cells/ml (0.3ml (107 cells/ml Mortality 10 10 a 0a 0a Morbidity a 10 10 0a 6.66 6.66 a Healthy 10a 80 100 a 3.33a 93.33 . Mean S.E. having the same letter in the same row and the same rout of injection are not significantly different at P<0.05.
Table(2):Growth performance of Nile tilapia (O. niloticus) fed diet containing probiotic bacteria. Items Initial weight (g/fish) Final weight (g/fish) Weight gain (g/fish) Weight gain (%) Control 2.406 a 0.01 9.39 b 0.465 6.99 b 0.474 290.84 b 3.25 1.536 ab 0.044 100 Micrococcus luteu 2.386 a 0.009 10.16 a 0.040 7.773 a 0.049 325.76 a 3.32 1.61 a 0.007 100

Pseudomon as species
2.38 a 0.009 8.02 d 0.063 5.626 c 0.056 234.41 d 1.64 1.34 c 0.005 95

Mix 2.40 a 0.007 8.976 ca 0.319 6.59 b 0.319 276.7 c 6.25 1.47 b 0.004 97.5

S G R (%/day) Survival (%)

The same letter in the same row is not significantly different at P<0.05. Table (3): Dry feed intake, food conversion ratio (FCR) and protein efficiency ratio (PER) of Nile tilapia (O. niloticus) fed diet containing probiotic bacteria. Pseudomonas Items Control Micrococcus Mix species luteu DFI 11.68 ab 12.28 a 11.14 b 11.403 b 0.343 0.013 0.090 0.233 FCR 1.693 a 1.58 b 1.743 a 1.98 a 0.067 0.01 0.049 0.006 2.026 ab 2.156 a 1.72 d 0.076 0.014 0.006 The same letter in the same row is not significantly different at P<0.05. PER 1.96 ac 0.056

Table (4): Proximate chemical analysis (%; on dry matter basis) of whole

body of Nile tilapia (O.niloticus) fed diet containing probiotic bacteria. Items Moisture Control

Micrococcus luteu

Pseudomonas species

Mix

31.25a 27.87 b 28.80 b 29.35 b 0.071 0.231 0.51 0.111 b a a Crude Protein 54.80 54.84 53.16 55.58 a 0.532 0.489 0.938 0.668 a b b Total lipid 23.50 22.31 20.93 19.24 c 0.524 0.11 0.606 0.128 b b c Ash 21.69 23.87 25.90 25.17 cb 0.225 0.837 0.273 0.072 The same letter in the same row is not significantly different at P<0.05.

Table (5): Changes in erythrocyte counts ((RBCs), hemoglobin content ((HB) and haematocrit value (Hct) in the blood of Nile tilapia (O.niloticus) fed diet containing probiotic bacteria. Items Control Micrococcus Pseudomonas Mix species luteu a a Erythrocyte count 0.997 1.059 0.626 b 0.821 b 6 3 10 /mm 0.067 0.043 0.034 0.034 a a b Hemoglobin 4.57 5.12 3.29 4.115 a (g/100ml) 0.222 0.151 0.236 0.272 a a b haematocrit value 18.5 17.0 9.0 14.75 c (%) 0.866 0.816 0.365 0.478 The same letter in the same row is not significantly different at P<0.05.

Table (6): Changes in glucose levels , total proteins, aspartate aminotransferase (AST), alanine amino transferase (ALT) and lactated dehydrogease (LDH) activities in plasma of Nile tilapia (O. niloticus) fed diet containing probiotic bacteria. Pseudomonas Items Control Micrococcus Mix species luteu Glucose 85.72 a 91.73 ab 121.8 c 101.12 b (mg/L) 3.45 5.59 7.10 4.45 Total protein 3.17 a 3.28 a 2.66 b 2.75 a (g/L) 0.118 0.205 0.136 0.170 AST(IU/L) 79.8 a 78.56 a 54.34 b 61.53 c 3.29 2.58 1.38 2.75 ALT (IU/L) 26.31 a 26.64 a 14.81 b 19.75 b 1.61 1.05 2.05 1.78 LDH (IU/L) 189.90 a 185.1 a 113.2 b 147.5 c 8.16 8.66 7.36 4.78 The same letter in the same row is not significantly different at P<0.05.

Table (7). Mortality rate of O. niloticus, fed diet containing probiotic bacteria for 90 days and challenged with A. hydrophila.
Items No. of injected fish Dose of bacteria Rout of injection Mortality rate after two weeks of injection Control Micrococcus luteu Pseudomonas species Mix

20
0.3 ml of 10 cells/ml
7 7

20
0.3 ml of 10 cells/ml
7

20
0.3 ml of 10 cells/ml

20
0.3 ml of 107cells/ml

I/P 80

I/P 25

I/P 90

I/P 80