INTRODUCTION

Lactic acid bacteria are gram-positive, non-sporulating and catalase negative rods or cocci that ferment various carbohydrates mainly to lactate and acetate. Various amino acids, vitamins and minerals are essential for their growth (Kandler and Wise, 1986). Various authors have shown that lactic acid bacteria are also part of the normal intestinal flora of fish (Ringo and Gatesoupe, 1998). Most of the evidence comes from salmonid species like Arctic charr (Salvelinus alpinus) and Atlantic salmon (Salmo salar) (Ringo et al., 1995; Gonzalez et al., 2000). Few studies have described lactic acid bacteria in other freshwater fish (Kvasnikov et al., 1977; Cai et al., 1999). Kvasnikov et al. (1977) described the presence of lactic acid bacteria, including Lactobacillus in the intestines of various fish species at larval, fry and fingerling stages inhabiting ponds. However, it was discussed that some human activities like artificial feeding in ponds would have had an effect on the bacterial composition and load in some fish, like carp (Cyprinus carpio) which showed the highest content of lactic acid bacteria in the intestines.

Probiotics can be defined as a food (feed) or drug containing live microbes that, when ingested, is expected to give beneficial physiologic effects to the host animal through microbial actions (Ishibashi and Yamazaki, 2001). Most of the probiotics are marketed as foodstuffs or drug. Consideration of the safety of probiotics is therefore of extreme importance. The safety of the microbes that have been used traditionally in probiotics can be conformed through a long period of experience (Mayra-Makien and Bigert, 1993). Yasuds and Taga (1980) suggested that probiotic bacteria would be found to be useful not only as food but also as biological controllers of fish disease and activators of nutrient regeneration. In the biological control in aquaculture emerge and since then the research effort has continually increased. Bacillus sp. is often antagonistic against other fresh water fish pathogenic bacteria (Gatesoupe, 1999; Rengipipat et al., 2000). Generally bacteria play two major roles as beneficial bacteria and pathogenic forms, beneficial bacteria are helpful in nutrient recycling and organic matter degradation and thus clear the environment (Moriarty, 1997). Pathogenic bacteria are the causative agents of bad water quality, stress and diseases as they act as primary and secondary pathogens (Karunasagar et al., 1996). Among fish pathogenic Aeromonas hydrophila and direct cause the syptecimea diseases and indirectly affect the fish health through developing poor water quality, high cyclone and stress (Lightner et al., 1992).

In this present study investigated the inhibitory activity of Bacillus sp. isolated from fish digestive tract, identify and evaluate bacteria which could be used in disease prevention in Oreochromis mossambicus. To test bacterial isolates for antagonistic activity against putative fresh water pathogenic bacteria Aeromonas hydrophila.

MATERIALS AND METHODS

Lactic acid producing bacteria isolated from fish digestive tract:The tilapia Oreochromis mossambicus fish was collected from the Cauvery, fresh water river, Erode district, Tamilnadu, India. The fish were washed with sterile distilled to remove the unwanted particles. Then the animals were dissected to remove the digestive tracts by the sterilization condition. The digestive tracts were homogenized in the same sterile distilled water for centrifugation. After centrifugation the supernatant was taken and serially diluted in sterile distilled water in the test tubes to 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7 and 10^-8 dilution and were pour plated on nutrient agar plate and incubated for 24 h at room temperature. Individual colonies were taken and inoculated in Elliker`s broth. Then the broths were incubated for 18-24 h at 37°C. One hundred microliter of culture was inoculated in Bacillus selective medium Lactobacillus bulgaricus agar plate and different colonies were selected and subjected to confirmatory tests for (LAB).

Conformation of Bacillus sp.
Grams staining: Markedly available Gram`s staining kit purchased from Himedia, heat-fixed smears should be stained for 1 min with crystal violet, washed in tap water, covered with Grams iodine for 1 min re-washed, decolorized by a few seconds in acetone-alcohol and counterstained for 30 sec in safranin. The smears are washed thoroughly and gently air-dried and observed under the oil immersion objective.

Methyl Red test (MR): Inoculate the isolated Bacillus with buffered glucose broth and incubate at 37°C for 48 h. After incubation add a few drops of methyl red solution to the culture, read immediately.

Catalase test: A loopful of the culture was placed on a slide and few drops of 10% hydrogen peroxide were added. The slides were observed for effervescence.

Detection of antagonistic activity: Isolated probiotic Bacillus sp. was assayed by the agar well diffusion method according to Benkerroum et al. (1993). The plates were examined for lysis around the wells at different time intervals for a total of 24 h. The agar well disc diffusion assay was used by two different isolated bacteriocin such as extra cellular protein (ECP) and intra cellular protein (ICP), the zone of inhibition was observed against common fish pathogen A. hydrophila.

Probiotic study-Maintenance of fish: The fish employed in this study were common fish Oreochromis mossambicus with weight range between 20±2 g fish was collected from the Cauvery fresh water river, Erode district, Tamilnadu, India. The major physical and chemical factors affect the growth of fish in a closed system. The fish were feed with commercially available artificial diet twice a day. The parameters were maintained, temperature (26-30°C), salinity (1-2 ppm), dissolved oxygen (6-7 mg L^-1), pH (6.5-8) and light.

Immersion study of Bacillus sp. against Aeromonas hydrophila: Tilapias (Oreochromis mossambicus) had not been exposed to fish diseases and were deemed specific pathogen free. The tilapias were acclimated for one week in four tanks to laboratory conditions before the start of the trial. After the acclimation period the average weight of the fish was 22±2g and the fish were randomly divided into twenty 500 L capacity polypropylene tanks with three treatments and three replicates per treatment, each containing 50 tilapia fish. The water temperature was held at 26-30°C during the whole trial. One treatment served as the control and was fed with regular diet (without the probiotics) during the entire trial period.

In order to obtain the bacterial suspensions, the probiotic strains were grown in MRS broth in a shaking incubated at 30°C overnight. After incubation, the cells were harvested by centrifugation (2000 rpm), washed twice with PBS buffer and re-suspended in the same buffer. The absorbance at 600nm was adjusted to standardize the number of bacteria (10⁵-10⁶CFU mL^-1). Commercial artificial feed was used as the basal diet for the supplementation of probiotic strains. Probiotic diets were prepared with cells resuspended in 5mL of PBS to 10⁶ CFU mL^-1 and mixed. Tilapia was fed two times daily at 5% body weight per day with a 50% water change every day, during 25 day feeding trials and survival was estimated visually each morning. A. hydrophila was used as an infectious agent in the experiment. The strain was grown for 12 h at 30°C in nutrient broth. Directly immersion of the tank 3 and 4, treatments and control were exposed to pathogenic A. hydrophila at a level of 10⁵-10⁶ CFU mL^-1 at 28°C by adding the bacteria to the water. Dead fish were collected and recorded daily.

Statistical analysis: The results were analyzed using one-way analysis of variance (ANOVA). Fisher`s range test was used to determine differences (p<0.05) between tested groups. All statistics were performed using SPSS 10 for Windows.

Fig. 1:	Antagonistic activity against Aeromonas hydrophila, A: Antibiotic, B: Bacteriocin (Extra cellular protein), I: Intra cellular protein, C: Control

Fig. 2:	Accumulated mortality of Bacillus sp. infected with 105-106 CFU mL-1 of pathogenic A. hydrophila for 25 days with probiotic treatment

The Bacillus sp. colonies were obtained from the Oreochromis mossambicus fish gut by the 10^-6 and 10^-7 dilutions; these were conformed as the initial screening for grams staining. Lactic acid bacteria are cocci or bacilli forms, non-motile, gram positive, Methyl red test positive and Catalase negative. Inhibition zone obtained the fish pathogen by antagonistic activity assay.

The inhibition zone 8 mm was observed on the plates inoculated with Aeromonas hydrophila by Bacillus sp. indicates the production of secondary metabolite of antimicrobial substances of bacteriocin against A. hydrophila (Fig. 1) by agar well diffusion method. Immersion study was observed in the four different groups of tanks contain O. mossambicus Values are given as mean±SD for three tanks in each group, Significant at (p<0.05), NS: Non significant, ¹Tank 1. Control, ²Tank 2. Bacillus sp. (10⁵-10⁶CFU mL^-1), ³Tank 3. Aeromonas hydrophila (10⁵-10⁶CFU mL^-1), ⁴Tank 4. Aeromonas hydrophila (10⁵-10⁶ CFU mL^-1) + Bacillus sp. (10⁵-10⁶CFU mL^-1) with 10⁵-10⁶ CFU mL^-1 cultures of Bacillus sp. and A. hydrophila and mortality was observed (Table 1).

Table 1:	Immersion study of A. hydrophila by Bacillus sp.

Isolated bacillus strains were inoculated second tank 87.5% of mortality rate was observed by the 25 days experiment. At the same mass culture of common fish pathogen A. hydrophila (10⁵-10⁶CFU mL^-1) were inoculated third tank was observed zero % of mortality rate. Both two stains were immersed fourth tanks survival rates were increased compare to the third tank. 65% of mortality rates were observed by probiotic stains using fourth tank (Fig. 2).

DISCUSSION

The concept of biological control for health maintenance has received widespread attention during the last few years, driven in large part by consumers and the lay press. The first report of the existence in freshwater of bacteria with an inhibitory effect against a Aeromonas sp. has been attributed to Ochoa and Olmos (2006) Subsequently, Rosenfeld and ZoBell (1947) described a study of antibiotic-producing marine microorganisms and since then research has begun to develop biological control strategies based on the application of these bacteria.

The isolated LAB species conformed by the methyl red and catalyzed negative test by Jack et al. (1995). Gatesoupe et al. (1999) observed that L. lactis showed an inhibitory activity against fish pathogen. This observation was confirmed by an inhibition zone of 8 mm as observed in this study. The culture media (supernatant) ECP were used, which showed highest inhibition against A. hydrophila. The inhibitory effects of Bacillus sp. may be due to production of antibiotics, bacteriocins, lysozymes, proteases and/or hydrogen peroxide and the alteration of pH values by the production of organic acids (Verschuere et al., 2000). All the LAB species produced by secondary metabolite of bacteriocin compounds against A. hydrophila (Santos et al., 1996). Adolfo (2004) was reported to the isolated Lactobacillus sp., both homofermenters and heterofermenters, were able to inhibit the human and fish pathogens by acid production when using a high glucose concentration. A few strains also inhibited both gram-positive and gram-negative fish and human pathogens with low (0.2%) concentration of glucose in the medium. Inhibitory activities of these strains have been usually detected against related species such as Staphylococcus aureus, Clostridium and other fish pathogenic bacteria (Schillinger and Lucke, 1989). But, only few Bacillus growing at low glucose concentration have been reported to inhibit a broader range of microorganisms, including gram negative foodborne and human pathogens. Some of these are currently used as probiotics (Jacobsen et al., 1999).

Bacteriocins are bactericidal or bacteriostatic peptides that are mostly active against bacteria closely related to the producer (Klaenhammer, 1988). Immersion application against pathogenic bacteria, O. mossambicus fish mortality rate was increased by the above experiments. A. hydrophila produces extracelluar hemolysis compounds after 24 h 10⁵-10⁶ CFU mL^-1 culture inoculations. In vivo condition A. hydrophila hemolytic toxins were produced at the same time as Bacillus sp. had produced by the secondary metabolite of extracelluar bacteriocin compounds. The isolated Bacillus sp. produces hydrogen peroxidase, other organic acids and bacteriocin (Klaenhammer, 1988; Daeschel, 1989). A growing concern about the high consumption of antibiotics in aquaculture has initiated a search for alternative methods of disease control (Gildberg et al., 1997) and growth promotion (Byun et al., 1997). The isolated Bacillus sp. was checked the disc diffusion assay and to improved resistance against infectious diseases can be achieved by the use of probiotics (Gildberg et al., 1997). Probiotic are living preparations of microbial cells that, when ingested in high enough concentration, beneficially affect the host`s health and growth by improving the intestinal microbial balance (Fuller, 1989; Havenaar et al., 1992). Selection of probiotic strains is achieved by screening procedures for several characteristics in vitro, such as inhibitory activities against several fish pathogens and gastric and intestinal secretions (Byun et al., 1997; Joborn et al., 1997). Cai et al. (1998) was reported to the similar experiments have shown that the inoculation of some probiotic strains, mainly lactic acid bacteria, increase fish survival after being challenged with fish pathogens while lactic acid bacteria populations in the gut increase and in one study lactic acid bacteria inoculation was related with an increase of fish growth rate.

The present study suggests that it is possible to maintain artificially the gut isolated Bacillus sp. treatment of fish by the immersion techniques proved successful as against fish pathogen of A. hydrophila. It is apparent that lactic acid producing bacteria may be used to control the A. hydrophila infection in fresh water fishes. Statistical analysis displayed significant differences (p<0.05) in the mortality of the tilapia between the treatment and control groups. Mortality in probiotic treatments and control were observed starting on the third day of contact with the pathogen, but the probiotic treatments were significantly different from the control when the mortality stabilized ending on the 25th day (Fig. 2). The high dose of the Bacillus sp. did not observe any negative side effects and this dose was associated with some improvements in the specific growth rate. However, further studies in which dosages of Bacillus sp. are repetitively fed to fish and to evaluate the biological effects of such treatments.