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 0.3×107 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.

where samples from the internal organs (liver. which currently is the fastest growing food-protein producing sector with an annual increase of approximately 9%.33.. The in vitro probiotic activity was done using agar diffusion method and the inhibition zoon determined as described by Ruiz et al. that is gaining acceptance within the industry. 1998). affects a wide variety of freshwater fish species and occasionally marine fish (Larsen and Jensen. there is a great interest in the use of probiotic bacteria in aquaculture to improve disease resistance..45g) were randomly collected from the earthen pond at the Central Laboratory for Aquaculture Research (CLAR).2. Robertson et al. niloticus (2. siderophores. Egypt. they cannot be used alone as a universal disease control measure in aquaculture (Amábile-Cuevas et al. such treatment may cause the development of resistant bacteria (Aoki et al. kidney. In recent years. Abou-Hammad. Austin and Austin (1993) and API 20 E strip system (Bio Merieux).. The use of probiotics stimulates rainbow trout immunity by stimulating phagocytic activity. 1977). niloticus) and their effect on some physiological parameters. 2000). 1990). MATERIALS AND METHODS Bacterial studies:Healthy O. 1995). as growth promoters and antibacterials for Nile tilapia fry (O. hydrophila obtained from CLAR-Fish Diseases Department.. the present study was carried out to evaluate the role of M luteus and Psuedomonas sp. 2000. 2000). the normal microbial flora in the digestive tract.. may also be killed or inhibited due to oral chemotherapy (Sugita et al. bacterial infections are considered as the major cause of mortality in fish hatcheries (Grisez and Ollevier. Abbassa. Sharkia Governorate.. A new approach method. (1984). and alteration of pH values by organic acids production (Sugita et al. The prophylactic and therapeutic control of the bacterial diseases is based on oral administration of antibiotics. The antibacterial effect of bacteria is generally due to either singly or in combination. yeild residues in fish and introduce potential hazard to public health and to the environment. bacteriocins. among those. The isolated bacteria from the internal organs of the investigated fish (intestine. (1996). Bacteriological examinations of the collected fish were done.. Therefore. 1991). The motile aeromonas group. stomach and intestine) and gills were cultured on TSB and incubated at 30°C for 24-48 hours. Furthermore. especially A. However. 1997) or the hypothetical stimulation of the immune system.. as the activation of macrophage (Perdigón et al. Probiotic bacteria have a posible competition for nutrients with pathogens in the digestive tract (Gatesovpe. stomach and gonads) were examined for its inhibitory effects against the pathogenic A. Purification and identification of the isolates were done using biochemical tests according to Bergey et al. hydrophila. Although vaccines are being developed and marketed.INTRODUCTION Fish Diseases are major problem for the fish farming industry.. lysozymes and proteases. 1985).. which is beneficial to fish. . 2003). 1995). is the use of probiotic bacteria to control potential pathogens (Gomez-Gil et al. production of antibiotics. gonads. complement mediated bacterial killing and immunoglobulin (Ig) production (Nikoskelainen et al. water quality and/or growth of farmed fish (Verschuere et al.

respectively. 2. Fish of the 1st and 2nd groups were i. Ingredients were mixed to formulated basic diet. Preparation of probiotics diet:The probiotic bacteria were prepared by inoculating the bacterial isolates in TSB and incubated at 30°C for 48 hrs.5% vitamins and minerals mixture. Abou-Hammad. Treatments in triplicate were arranged at random. The experimental fish were batch weighed at the beginning of the experimental period.3 ml of saline containing 107 cells / ml of 24 hrs M. at a fixed feeding rate of 3% body weight of fish per day. 1. Abbassa. Dry ingredients were sieve and ground to a small particle size in a Thoms-Wiley Laboratory Mill model 4. Each aquarium was cleaned daily by siphoning fish feces and remaining feed with 75% of total water volume. then centrifuged at 3000 rpm for 30 minutes. The solution was added to the bacterial cells (till one ml saline contained 1010 bacterial cells). The number of probiotic bacterial cells in the prepared pellets were found to be 07 cells/g diet. the bacteria was washed twice with saline. The experiment ran for 90 days. La Parmigiana.5% fish and corn oil (1:1). 0. The fish were distributed randomly at a rate of 20 fish / aquarium. The growth parameters and feed utilization were calculated according to: Specific growth rate (SGR) = 100 (ln W2 – ln W1) T -1 . The fish were divided into 3 equal groups (three replicates each). O. Sharkia. Model D45 LE. niloticus with an average weight (2. base diet mixed with Pseudomonas sp (T2) and a mixture from M luteu and Pseudomonas sp diets with equal amounts (T3). After centrifugation. after which all the experimental fish were collected . 35. The experimental basic diet formulated to contain 30% crude protein and 421 k cal of gross energy/100g diet on a dry weight basis.2. The feed given were adjusted at 2-weeks intervals after fish were weigh.. Each aquarium was supplied by compressed air via-stone.Safety of the two isolates probiotics was studied by using 90 apparently healthy fish that were acclimatized for two weeks in indoor tanks. Fish of the 3rd group were I/P inoculated by 0. Experimental diet were passed with clean and sterile auger and diet of Spaghetti Machine. Diets transferred to black plastic bags and stored in a freezer (–20 ºC) until immediately prior feeding. then refilled to fixed volume again. 6 days a week. base diet mixed with M. Water temperature range was (26. USA. inoculated by 0. 2% carboxymethyl cellulose as a binder. Italy” and dried at room temperature. 5% wheat bran. at the Center Laboratory for Aquaculture Research. luteus (T1).p. Egypt.3 ml of saline as controls according to Irianto and Austin (2002). The aquaria were supplied with 100 ml well aerated tap water. which consisted of 13% hearring fish meal. Feeding culture system design: The feeding experiment was conducted in 12 glasses aquaria (75×40×50 cm). The saline containing the probiotic isolates was added to commercial sterilized food to give an initial number of 1010 bacterial cells/ g. 40% soybean meal. counted and weight. Nile tilapia fries. Bacteria strains supplemented in base diet to conducted four treatments: base diet untreated (control). Fresh tap water was stored in cylindrical fiberglass tanks for 24 hours under areation in order to dechlorenated the water.94% ground yellow corn.45 g / fish) were acclimatized in an indoor fiberglass for two weeks.06% ascorbic acid. Fish were fed twice daily. luteus or Pseudomonas sp.28ºC).35 .

Both subgroups kept under observation for 14 days to record the survival rate daily. Dry matter was determined after drying at 105 ºC until constant weight and ash after heating of dry samples in muffle furnace at 580ºC for 6 hours. 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. Activities of aspartate amninotransferase (AST) and alanine aminotransferase (ALT) were determined colorimetrically according to Reitman and Frankel (1957). Statistical analysis: The obtained results were analysed statistically using analysis of variance (ANOVA). 1961). the first subgroup of each treatment was challenged I/P with pathogenic A. RESULTS Bacteriological examinations: In the present study. Challenge test:After 90 days of feeding.3 ml of 107 cells / ml) which was obtained from (CLAR). Physiological Analyses: At the end of experiment. hydrophila (0. blood samples were taken from the caudal vein of an anaesthetized fish by sterile syringe using EDTA solution used as an anticoagulant. luteus revealed that it was Gram postive cocci. Dauncan’s multiple range test (Dauncan. The second subgroup was injected i. while lactate dehydrogenases (LDH)) were measured by using Diamond diagnostics kits according to the method of Cobaud and Warblewski (1958). oxidase and catalase were produced and oxidation/ fermintation were negative. The physical and the biochemical characters of M. by 0. Chemical analysis:The proximate chemical analysis of fish and diets were done according to the method of AOAC (1990). Total lipid was determined by extraction by petroleum eather in soxhelt apparatus. the biomass at the start and end.3 ml of saline as control. Haematocrit value (Hct) were calculated according to the formulae mentioned by Britton (1963).Where W1 and W2 are the initial and final weight. respectively. according to Trinder (1969). respectively. 1955) was used to evaluate the mean differences among different treatments at the 0. and T is the number of days in the feeding period. B1 and B2 are the feed intake. Crude protien was determined by the Kjeldahl procedure. galactose and insitol. and PI is the protein intake. luteus and Psuedomonas sp. Feed conversion ratio (FCR) = FI (B2 + B dead – B1)-1 Where FI. Plasma glucose was determined using glucose kits supplied by Boehring Mannheium kit. It produced acid from arabinose. motile. the suspected probiotic bacterial isolates were identified as M. and B dead is the biomass of the dead fish. Total protein content was determined colorimetrically according to Henry (1964). It did not produce acid . bacteriological examinations revealed that.p. Protein efficiency ratio (PER) = (B2 – B1) PI-1 Where B1 and B2 are the biomass at the start and the end of the experiment.05 significant level. . the fish of each group were divided into two subgroups. The blood samples were used for determining erythrocyte count (Dacie and Lewis 1984) and hemoglobin content (Vankampen.

table (4) showed that the moisture content was approximately similar (27. niloticus. species were safe to fish in comparison to the control as show in table (1) which gives mortality rate 10%.68 g feed/fish except the fish group fed a diet containing M. It grows on nutrient agar contained 0. niloticus in the group which fed a diet containing mixed bacteria ( equal amounts of Psuedomonas sp and M. fractose. 6. sorbitol. These values decreased significantly in the fish group which fed a diet containing Psuedomonas sp (8. On the other hand.25%.28 g feed/fish. hydrophila in vitro. lutius and P. while control group and fish fed with diet containing M. niloticus increased significantly when fed a diet containing M.96. salicin.626 g/fish weight gain and 1.976 g/fish final weight.59 g/fish weight gain and 1..14 to 11. luteus was isolated from gonads of apparently healthy O.47 %/ day SGR) but less than control group (9. 10.69. respectively). P>0. xylose. 6. while the highest one was observed at control. xylose or manitol. Table (3) indicated that there was a significant increase in protein efficiency ratio (PER) with the diet containing M.58).536 %/day SGR). luteus and Psuedomonas sp were examined for a probiotic activity against the pathogenic A. ornithine and lycin decarboxylase were negative with it.99 g/fish weight gain and 1. 7. trehalose.34 %/day SGR). luteus were 100% (Table 2). sorbitol.0. Growth Performance: Results in Table (2) showed that the final weight.02 g/fish final weight. niloticus as no clinical signs or mortalities were noticed following challenge via I/P route. M. It did not hydrolysed starch or arginine. Table 3).156). 5.05.74 and 1. and 5% NaCl. 1. manitol or maltose. It isolated from intestine of apparently healthy O. niloticus.98. M. 3. Psuedomonas sp gave larger inhibition zone (9 cm) than M. . Crude protein content in whole fish body was approximately similar (53.026 and 1. It did not haemolyside blood. Safety of the isolated probiotics: Two isolates of the probiotics were harmless to O. The in. It produced acid from glucose and did not produce acid from sucrose. Protein efficiency ratio with the diet containing Psuedomonas sp showed the least PER (1. salicin. arabinose. oxidative.16g/fish final weight.16-55. the fish group that fed with Psuedomonas sp. hydrophila.39 g/fish final weight. and grow at 45ºC. luteus (4 cm). niloticus fed with diet contained P. weight gain and specific growth rate of O. respectively. Arginine hydrolysis. luteus only (P<0. lactose. The O. The control and mixed groups had protein efficiency ratio of 2. luteus in which the feed intake was slightly increased (12. species (95%) then mixed diet (97.773 g/fish weight gain and 1. It showed inhibitory effects against A. M. However. and that fed mixed bacteria with insignificant difference (1. single. galactose. the least feed conversion ratio (FCR) was observed with fish group fed diet contained M.87-31. luteus (1. motile.61 %/day SGR). luteus (2.from glucose. Survival rate was decreased in O. Feed intake was approximately similar and ranged from 11.58%). Concerning the proximate chemical analysis of whole fish body. bacilli.5%).72). facultative anaerobe and oxidase-postive. luteus) were slightly increased (8. Psuedomonas sp was gram negative.vitro probiotic activity: Two isolates.05).

17 ± 0. luteus and Psuedomonas sp (2.45 (mg%).75% to 18.043 million/mm3).16 IU/L).90. while the least one was observed in the control (21. As shown in Table (6). hydrophila (0.24%).93 and 22. The mixed and the control groups had the same mortality rate (80%). species. The erythrocyte count in fish fed diets containing mixed bacteria and control group were 0. respectively.87%.067 million/mm3. 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.059 ± 0. species (9. luteus (25. The highest content of the total lipids was obtained in the control (23. luteus (5. Same as the trend.90 ± 8. luteus had a probiotic effect with fish. luteus were significantly high (1. Hemoglobin content was slightly increased in fish fed diet containing M. 25. Physiological Parameters: Erythrocyte counts in fish fed diets containing M. while other treatments were approximately similar and ranged from 14.On the other hand. respectively) where the control group was 3.626 ± 0. by pathogenic A. while the decreased significantly in fish fed with diet containing Psuedomonas sp (0.3 ml 0f 107 cells/ml) . the average level of plasma LDH activity in fish control group was ( 189. Also.118 (g/L). The plasma glucose concentration of the fish control group was 85.72 ± 3. luteus only and the fish in control group. Also.0%).69 %).170 g/L.136 and 2.57 g/100 ml.821 ± 0. niloticus which fed on a diet containing Psuedomonas sp was 90% .p. respectively.75 ± 0.5% (Table 5).997 ± 0. It was significantly increase in fish fed with diet containing Psuedomonas sp and mixture of M. the lactate dehydrogease was significantly decreased in fish fed with diet containing Psuedomonas sp and mixture of M. luteus for three months and challenged i. Haematocrit value (Hct) decreased at fish fed diets containing P. luteus and Psuedomonas sp (Table 6) when compared to treatment fish fed with diet containing M.29 g/100 ml).115 and 4. and the fish becam sensitive to any infection.31%. The mortality rate was 25% of O.034 and 0. The mortality rate of O. respectively). The fish which fed on diet contained Psuedomonas sp had external sings including ulceration and hemorrahges.5 %). Hemoglobin content in fish fed diets containing mixed bacteria and control group were 4. species and M. . respectively. On the other hand. Challenge test results: Table (7) showed that M.12 g/100 ml ). this ratio was more than control.034 million/mm3). 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. Total lipid content in the fish groups which fed diet contained P.17 and 23.66 ± 0. the hemoglobin content decreased in fish fed diet containing Psuedomonas sp (3. mixed bacteria and M. nilotecus fed on a diet containing M. 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. Ash content in whole fish body was significantly increased with diets containing P. content of the total lipids was low at the diet contained mixed bacteria (19. luteus and Psuedomonas sp. luteus were 20.

(1984) and Austin and Austin (1993). (2001) isolated Psuedomonas sp. In the present study. Our results agree with Chang and Liu (2002b) who indicated that Bacillus toyoi suppressed the growth of Edwarsilla tarda in vitro.DISCUSSION Probiotics. luteus had a probiotic effect in vitro and in vivo against A. Lewus et al. Photorhodobacterium. niloticus which fed on a diet containing Psuedomonas sp was 90% which did not agree with Gram et al. of apparently healthy O. niloticus challenged by A. luteus and Psuedomonas sp. but Irianto and Austin (2002b) isolated M. salmonicida. salmonicida infection in rainbow trout (O. hydrophila. (1991) reported that the bacteriocins which produced by lactic acid bacteria had inhibitory effect against A. Enterococcus. which are micro-organisms or their products with health benefit to the host. (1998) isolated M. the probiotics actively inhibit the colonization of potential pathogens in the digestive tract by antibiosis or by competition for nutrients and . anguillarum. hydrophila. as identified by Bergey et al. from O. Psuedomonas sp. Spanggaard et al. 2001). 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. and Psuedomonas sp from the intestine of seven fish species and recorded that the bacteria had inhibitory effect against Vibrio vulnificus. but did not reduce mortalities in eels due to edwardsillosis in vivo. However. hydrophila. hydrophila. The mode of action of the probiotics is rarely investigated. The mortality rate at the treated fish was 32% compeared to control 47%. luteus gave mortality rate 25% at 107 cells / g of feed among O. luteus A1-6 from digestive tract of Oncorhychus mykiss. (1996) noticed that Alteromonas haloplanktis had inhibitory activity against Vibrio sp and A. flurescence reduced diseases caused by A. mykiss). i. luteus and Psuedomonas sp. M. mykiss and used as probiotics in O. M.e. Irianto and Austin (2002) used M. Riquelme et al. Alteromonas. Carnobacterium. fluorescens strain (AH2) reduced the mortality of 40 g rainbow trout infected with a pathogenic V. 217) from broadstock. Smith and Davey (1993) reported that P. Micrococcus and Streptococcus) and Gram-negative bacteria (Aeromonas. Lactococcus. The mortality rate between O. were isolated from gonads and intestine. Also. (1999) who reported that P.. Lactobacillus.9 % (109 cells / g feed) and to 46. From the present study M.6 to 18. luteus with feed as a potential combating A. luteus. while Psuedomonas sp had a probiotic effect in vitro only and in vivo it changed to pathogenic bacteria. they isolated Pseudomonas (C30. niloticus.3% (1012 cells / g of food) following challenge with A. have been used in aquaculture as a means of diseases control. gave larger inhibition zone (9 cm) than M luteus (4cm). The physiological and the biochemical characters of suspected probiotic bacterial isolates were identified as Micrococcus luteus and Psuedomonas sp. salmonicida (Nikoskelainen et al. They isolated Alteromonas haloplanktis from gonads of Argopecten purpuratus broadstock. respectively. showed inhibitory effects in vitro against A hydrophila. A wide range of Gram-positive (Bacillus. supplemnting or even in some cases replacing the use of antimicrobial compounds. but possibilities include competitive exclusion. Sugita et al. M. mykiss mixed in water. Pseudomonas and Vibrio) has been evaluated as probiotics in aquaculture (Irianto and Austin 2002a).

Survival of shrimps was significantly greater in treated group compared with the control group. and reduced pathgogenic problems in the gastrointestinal tract (Lloyd et al. Results of the present investigation showed that Psuedomonas sp in diet of O. Also. Emterococcus./ or space. (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. survival rate feed intake and protein efficiency ratio were increased among O. 2002a). HB and Hct). 2002a). (2004) notced that the growth of larvae of sea bass fed 1. Goren et al. (1998) showed that the addition of a gram-positive probiotic bacterium increased survival. and growth rate of marine fish larvae (snook. hepatopancreas and in swim bladder) in the common carp after exposure to Cyanobacteria extract.. faecium had also been used to improve growth when fed to sheat fish. weight gain. Also less work has been directed at the blood parameters. red drum. Fuller.. (1999) were reported that the probiotic treated group enhancing growth rate of shrimps and maintaining water quality parameters. 1994). and by the breakdown of indigestible components(Irianto and Austin. These results agree with Rengpipat et al. A several probiotic species were used including Lactobacillus sp. (Jonsoon. Ishikawa (1998) showed that the spleen. These results are in agreement with those of Palikova et al (2004) who observed pathmorphological findings (haemorrhages in the skin. size uniformity. Silurtes glanis L (Bogut et al. 1994 and Bogut et al. Streptococcus faecium improved the growth and feed efficiency of Israeli carp (Noh et al. eyes. luteus. 1977. specific growth rate. (1998) and Prabhu et al. Probiotics may stimulate appetite and improve nutrition by the production of vitamins. The use of probiotics can improve the nutrition level of aquacultural animals and improve immunity of cultured animals to pathogenic microorganisms. 2003).. niloticus fed a diet containing M. Kennedy et al. Lactic acid bacteria had an effect as growth promotor on the growth rate in juvenile carp but not in sea bass (Noh et al. thus leading to improve food absorption (Parker. Also. niloticus caused a decrease of blood parameters (RBSC..1% live yeast as a probiotic was increased than control group. 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. the use of antibiotics can be reduced and frequent outbreaks of diseases can be prevented. 1989). so it may be considered as a growth promoter in fish aquaculture. Numerous trials were conducted with microorganisms known as probiotics to improve culturability of food species and to improve human health and welfare. spotted seatrout and stripped mullet). 1974.. alteration of microbial metabolism. and/or by the stimulation of host immunity (Irianto and Austin.. detoxification of compounds in the diet. suggesting that haematopoiesis was also severely affected and this affected the peripheral blood by decreasing erythrocyte . In addition. Appropriate probiotic applications were shown to improve intestinal microbial balance. Riquelme et al. The final weight. 1986) and mixes cultures (Lessard and Brisson 1987). 1998). 1984). liver and kidney of unhealthy fish held fairly severe infection . survival of larvae was significantly higher than the control.. Tovar-Ramírez et al. 2000).

. . niloticus culture. 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. However. M.. ALT and LDH ) in Cyprinus carpio after exposure to extract of Cyanobacteria. The plsma glucose concentration was significantly increased in fish fed with diet containing Psuedomonas sp and mixture of M. niloticus in aquaculture. The present study indicated that the AST. Association of Official Analytical Chemists. Sci. It is argued that such probiotic has a role in disease control strategies. luteus and Psuedomonas sp. luteus and Psuedomonas sp. (2003) reported that the selected probiotic bacteria may have an impact on the spasific and innate immunity of fish. growth promotion and it improves the blood picture and biochemical parameters among O. niloticus when administered as a food additive. 1990). ALT and LDH activities in plasma of O niloticus was significantly decreased in fish fed with diets containing Psuedomonas sp. 20: 199-208. who recorded an increase in erthrocyte count in fish fed on probiotic bacteria than control group. These results agree with Irianto and Austin (2002). They recorded that (Ig) level was significantly increased with the probiotic feed group. M. the plasma total proteins showed decreased significance in fish fed with diet containing Psuedomonas sp and mixture of M. T.1989). In conclusion.volume. Am. AOAC (1990): Official Methods of Analyses of the Association of Official Analytical Chemists International. VA. (1989) who found lower total protein in plasma of Salmo gairdneri when injected with Vibrio anguillarum extracellular products intramuscularly. M.. and Ludgar. Gárdenas-Garciá. At the same time. luteus and Psuedomonas sp. and mixture of M.F. muscle and kidney (Cruz et al .. Arlington. 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. 83: 320-329. Nikoskelainen et al. (1985): Epidemiological surveillance of drugresistant Vibrio anguillarum strains. Fish Patho. T. Kanazawa. microcytic anaemia. C. and Kitao. On the other hand. These results agree with those of Cruz et al. References Amábile-Cuevas. heamoglobin content and haematocrit value was increasing than control group. The present study indicated that Psuedomonas sp may cause stress of fish. Aoki. Micrococcus luteus isolate was clearly beneficial for cultured O. The production of immunoglobulin A (IgA) is also stimulated and protecting the mice against Salmonella typhimurium (Perdigon et al. "15th edition. (1995): Antibiotic resistance. luteus had a good effect on erthrocytes count.. a mixture of both bacterial species improved the protein content of fish. spleen. T. 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. USA.. The decrease of these parameter may be due the severe damage of some organs liver. The present results agree with Palikova et al (2004) who indicated decrease of enzyme activities (AST.

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33± .34 c ± 0.626 c ± 0. PER 1.47 b ± 0.58 b 1.406 a ± 0.007 8. food conversion ratio (FCR) and protein efficiency ratio (PER) of Nile tilapia (O. Mean ± S.014 ± 0.01 ± 0.25 1.026 ab 2.76 a ± 3.343 ± 0.p.693 a 1.009 10. Route of injection dose 0.66± 6.05.25 1.233 FCR 1.05.38 a ± 0.33a 93. niloticus) fed diet containing probiotic bacteria.090 ± 0.3ml (107 cells/ml (0.474 290.076 ± 0.40 a ± 0. Items Initial weight (g/fish) Final weight (g/fish) Weight gain (g/fish) Weight gain (%) Control 2.773 a ± 0.16 a ± 0.007 100 Pseudomon as species 2.319 6.465 6.41 d ± 1.Table (1) challenge test to the probiotc bacterial isolates among O.p .536 ab ± 0.049 325.976 ca ± 0.61 a ± 0.006 2.01 9.14 b 11.59 b ± 0.66 a Healthy 10a ± 80 100 a 3.3ml (107 cells/ml Mortality 10 ± 10 a 0a 0a Morbidity a 10 ± 10 0a 6.32 1.049 ± 0.004 97. Table(2):Growth performance of Nile tilapia (O.156 a 1. niloticus) fed diet containing probiotic bacteria.02 d ± 0.05.743 a 1.39 b ± 0.96 ac ± 0.7 c ± 6.68 ab 12.98 a ± 0.84 b ± 3.i.72 d ± 0.E.056 Table (4): Proximate chemical analysis (%. Pseudomonas Items Control Micrococcus Mix species luteu DFI 11.64 1.5 S G R (%/day) Survival (%) The same letter in the same row is not significantly different at P<0.040 7.p i. having the same letter in the same row and the same rout of injection are not significantly different at P<0.056 234.99 b ± 0. on dry matter basis) of whole .013 ± 0. Table (3): Dry feed intake.i.009 8.044 100 Micrococcus luteu 2.067 ± 0.063 5.28 a 11.386 a ± 0.005 95 Mix 2.403 b ± 0. niloticus Micrococcus Pseudomonas Isolates control luteus species .3ml of saline (0.319 276.006 The same letter in the same row is not significantly different at P<0.

16 ± 8.38 ± 2.0 9.87 b 28.87 25.034 ± 0.837 ± 0. aspartate aminotransferase (AST).34 b 61.56 a 54.80 54.1 a 113.80 b 29.73 ab 121. Items Moisture Control Micrococcus luteu Pseudomonas species Mix 31.938 ± 0.24 c ± 0.11 ± 0.17 cb ± 0. alanine amino transferase (ALT) and lactated dehydrogease (LDH) activities in plasma of Nile tilapia (O.05.118 ± 0.05.64 a 14.90 a 185.222 ± 0.5 c ± 8.78 The same letter in the same row is not significantly different at P<0.115 a (g/100ml) ± 0.body of Nile tilapia (O.59 ± 7.29 ± 2.816 ± 0.205 ± 0. Pseudomonas Items Control Micrococcus Mix species luteu Glucose 85.78 LDH (IU/L) 189. total proteins.8 a 78.272 a a b haematocrit value 18.17 a 3. niloticus) fed diet containing probiotic bacteria.866 ± 0.2 b 147. .626 b 0.034 a a b Hemoglobin 4.606 ± 0.36 ± 4.51 ± 0.25a 27.69 23.273 ± 0.75 a (g/L) ± 0.75 c (%) ± 0.niloticus) fed diet containing probiotic bacteria.111 b a a Crude Protein 54.231 ± 0.niloticus) fed diet containing probiotic bacteria.75 b ± 1.58 a ± 0.12 3.05.478 The same letter in the same row is not significantly different at P<0.50 22.365 ± 0.05 ± 2. Items Control Micrococcus Pseudomonas Mix species luteu a a Erythrocyte count 0.0 14.75 ALT (IU/L) 26.57 5.31 20.071 ± 0.29 4.72 a 91.81 b 19.12 b (mg/L) ± 3. hemoglobin content ((HB) and haematocrit value (Hct) in the blood of Nile tilapia (O.236 ± 0.072 The same letter in the same row is not significantly different at P<0.668 a b b Total lipid 23.067 ± 0.136 ± 0.128 b b c Ash 21.28 a 2.61 ± 1.53 c ± 3.997 1. Table (6): Changes in glucose levels .16 55.66 b 2.90 25.151 ± 0. Table (5): Changes in erythrocyte counts ((RBCs).31 a 26.059 0.5 17.93 19.05 ± 1.489 ± 0.170 AST(IU/L) 79.66 ± 7.45 Total protein 3.043 ± 0.45 ± 5.821 b 6 3 10 /mm ± 0.84 53.532 ± 0.524 ± 0.8 c 101.225 ± 0.58 ± 1.10 ± 4.35 b ± 0.

hydrophila. fed diet containing probiotic bacteria for 90 days and challenged with A.Table (7). 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 20 0. Mortality rate of O.3 ml of 10 cells/ml 20 0. niloticus.3 ml of 107cells/ml I/P 80 I/P 25 I/P 90 I/P 80 .3 ml of 10 cells/ml 7 7 20 0.

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