Abstract: This study aimed to characterize Bacillus sp. NP5 and to evaluate the effectiveness of its application as a probiotic on growth performances and health status of common carp (Cyprinus carpio ) before and after infected by Aeromonas hydrophila . Bacillus sp. NP5 cells were given to common carps through the feed for 30 days at doses of 106, 108 and 1010 CFU g–1 feed. On day 31, common carps were challenged with A. hydrophila , which was injected via intramuscular route. Result of phenotypic and genotypic identification of Bacillus sp. NP5 isolate showed that this isolate was Bacillus cereus with 99% of similarity index. Bacillus sp. NP5 was able to produce protease, amylase and lipase, it was also potential to inhibit A. hydrophila, Streptococcus agalactiae and Mycobacterium fortuitum. Supplementation of feed containing probiotic at a dose of 1010 CFU g–1 feed resulted a higher total bacterial count and a higher total probiotic count in the intestine, those followed with higher value of amylase, protease and lipase activity, along with the highest daily growth rate and the lowest feed conversion ratio (p<0.05). Total leukocytes and phagocytic activity in probiotic treatment with a dose of 1010 CFU g–1 feed was higher (p<0.05) compared to controls at the end of the rearing period over 30 days. Common carps fed probiotic showed survival rates after the challenge test with values ranging between 81-100%, while the survival rate of fish without probiotic supplementation was only 50%. The results of this study showed that probiotic supplementation on common carp could reduce the pathogenicity of disease caused by A. hydrophila infection, which indicated an improvement in cellular immune response.
INTRODUCTION
In the aquaculture industry of the world including Indonesia, common carp (Cyprinus carpio) is classified as the oldest aquaculture species (Balon, 2006) and it is one of the economically important species. As the increasing of animal protein source demands, aquaculture has emerged as one of the sustainable industries that can be relied to fulfill the nutritional requirements and to sustain the human food safety. However, most of the intensive farming centers face major obstacles caused by diseases outbreaks.
Motile Aeromonad Septicaemia (MAS) caused by Aeromonas hydrophila infection has become a common problem in the aquaculture industry, that attacks various fish species in the world. The MAS can spread very quickly, it generally causes mortality about 25%, but the mortality rate will be higher in high stocking density condition and poor water quality (Hoole et al., 2001). This pathogen has characteristics: Gram-negative, motile, short rod shape and it is commonly found in all aquatic environment types (Harikrishnan et al., 2003). The clinical symptoms of MAS include: Swelling tissues, dropsy, red spots on the body surface, necrosis, ulceration and haemorrhagic septicemia (Karunasagar et al., 1989). Some fish species infected by MAS include tilapia (Tellez-Banuelos et al., 2010), catfish (Ullal et al., 2008), goldfish (Harikrishnan et al., 2009), common carp (Jeney et al., 2009; Yin et al., 2009) and eel (Esteve et al., 1994). This pathogen generally acts as a secondary pathogen, but it is also proved as primary pathogen (Pridgeon and Klesius, 2011), it has acute mortality pattern that can cause a mortality less than 24 h.
Antibiotics and chemical drugs has commonly used to inhibit and to prevent pathogens growth because of growth promotion stimulation and chemotherapy (Jagoda et al., 2014). However, the use of those materials have high risk with the increasing of resistant bacterial strains, disruption of stability and balance of the intestinal microflora and the residue is accumulated in fish carcass and aquatic environment (Skjermo and Vadstein, 1999). The use of antibiotics also requires a long time for antibiotic residues withdrawal from fish body (Iregui et al., 2014), so now a days it has been prohibited to be used for fish diseases control. Probiotics are live or dead microbes or microbial components, which give some benefits to the host (Fuller, 1989; Lazado and Caipang, 2014). The use of probiotics is one of the eco-friendly alternative efforts to reduce the use of antibiotics, it improves fish appetite, it also maintains stability and the balance of intestinal microflora (Panigrahi et al., 2010). Probiotic has contribution in enzymatic digestion, because it can produce extracellular amylase, protease and lipase (Kuhlwein et al., 2014). Various probiotics which are commonly used are from Lactococcus genus (Sugita et al., 2009), Bacillus sp. (Kumar et al., 2008), Lactobacillus and Saccharomyces cerevisiae (Ramakrishnan et al., 2008). Probiotic derived from Bacillus sp. has several advantages, because it has spores, which are relatively stable to heat, some are able to survive at low pH (Barbosa et al., 2005) and its viability is relatively stable during long-term storage for the purpose of mass scale commercial feed production (Ringo et al., 2014).
Some probiotics generally show their dominance in the fish gastrointestinal tract only in treatment or administration period. Therefore, the main challenge to be achieved in the probiotics administration is to create bacterial colonization in the fish intestine in long term period, it is especially subjected to probiotic, which is sourced from different fish species. This research used Bacillus sp. NP5 isolated from the digestive tract of tilapia (Putra and Widanarni, 2015) and it has been proven being able to control streptococcosis on tilapia (Widanarni and Tanbiyaskur, 2015). One of the factors that affect probiotic performance is dose (Nayak, 2010), because probiotic which is given in sufficient quantities will produce the beneficial effect for growth and health on fish, so the dose of probiotic, which is given to the host must be carefully determined to avoid the overdose that can give unexpected side effects and lost in production cost (Dash et al., 2014). Nikoskelainen et al. (2001) stated that low doses of probiotic may fail to stimulate the fish immune system, but high doses of probiotic can cause interference in the host. Probiotic in the very high dose will cause imbalance of the microbiota in the digestive tract and interfere immune response that can cause the lost of energy, which used for the growth (Li et al., 2012; Ramos et al., 2013). To produce maximum therapeutic effect is required minimum concentration of 106 CFU mL–1 (Kurman and Rasic, 1991), the range of 107-108 CFU mL–1 or in higher number (Jayamanne and Adams, 2006). The administration of Bacillus subtilis and Lactobacillus acidophilus at a concentration of 107 CFU g–1 feed for 1 and 2 months in tilapia could increase the activity of lysozyme and bactericidal serum against Aeromonas hydrophila with post-challenge test survival of 43.52% (Aly et al., 2008).
Before it is applied to aquatic organisms, probiotic bacteria need to be characterized and identified to differentiate with other species, which are pathogenic potential species. Moreover, the characterization and identification of probiotic bacteria are also important for quality control and the patent purpose (Gomez-Gil et al., 2000; Romero and Navarrete, 2006). Based on the above cases, this study aimed to characterize Bacillus sp. NP5 and to evaluate the effectiveness of its application on the growth performances and health status of common carp (Cyprinus carpio) before and after infected by Aeromonas hydrophila.
MATERIALS AND METHODS
Identification of Bacillus sp. NP5: Phenotypic character of Bacillus sp. NP5 was evaluated using api® 50 CHB V4.0 kit, bioMérieux, Marcy-I'Etoile, France. Genotypic character was evaluated through amplification of 16S rRNA encoding gene with 63 forward primer (5’-CAG GCC TAA CACATG CAA GTC-3’) and 1387 reverse primer (5’-GGG CGG WGT GTA CAA GGC-3’) (Marchesi et al., 1998). The composition of master mix PCR in each tube consisted of 25 μL GoTaq (Promega), 4 μL 63 forward primer, 4 μL 1387 reverse primer, 17 μL ddH2O and DNA template, which was taken directly from Bacillus sp. NP5 isolate using a toothpick. The PCR conditions were as follow: Predenaturation at 95°C for 5 min, denaturation at 95°C for 1 min, primer annealing at 55°C for 1 min, elongation at 72°C for 1 min and primer extension at 72°C for 5 min. Amplification process on PCR machine consisted of 30 cycles. Termination reaction was carried out by temperature droping to 4°C. The PCR products were run for electrophoresis on agarose gel 1% in TAE buffer 1x at 80 V for 45 min, followed by visualization using UV transiluminator. Sequencing was done by sending amplified-DNA to sequencing service provider company. Sequence results were aligned with database in Gene Bank using BLAST-N online software program (www.ncbi.nlm.nih.gov).
Semi-quantitative test of amylolytic, proteolytic and lipolytic activity: This test aimed to measured amylolytic, proteolytic and lipolytic activity of probiotic bacteria through hydrolysis test of starch, skim milk and olive oil. Hydrolysis ability was characterized by clear zone around the bacterial colony. Amylolytic, proteolytic and lipolytic activity were evaluated by Amylolytic Index (AI), Proteolytic Index (PI) and Lipolytic Index (LI) that can be measured by the equation according to Lim et al. (1987) as follows:
Quantitative test of amylolytic, proteolytic and lipolytic activity: Enzymes were obtained from centrifugation of bacterial culture at a speed of 5000x g for 30 min. Amylase activity was measured according to Bernfeld (1955). Amylase activity of supernatant was measured quantitatively with a spectrophotometer at a wavelength of 550 nm. One unit of amylase activity is defined as the amount of enzyme that produces reductor sugar (maltose) as much as 1 μmol min–1 in test condition. Protease activity was measured according to Walter (1984). Absorbance was read at a wavelength of 578 nm with a standard used was L-tyrosine 5 mM. One unit of protease activity is defined as the amount of enzyme that is able to produce 1 μmol of tyrosine per minute. Lipase activity was measured according to Kwon and Rhee (1986). Measurement of lipase activity was performed using margarat acid (heptadecanoat acid) standard curve, which was measured its absorbance at 715 nm. One unit of lipase activity is defined as the amount of enzyme that liberates 1 μmol mL–1 min–1 of fatty acid at 60°C.
Inhibition test of Bacillus sp. NP5 against pathogenic bacteria: Inhbitory activity was evaluated by Kirby-Bauer method and competition test between Bacillus sp. NP5 with pathogenic bacteria (Aeromonas hydrophila, Streptococcus agalactiae and Mycobacterium fortuitum). Inhibition test with Kirby-Bauer method was performed using paper disc by measuring the inhibition zones, while competition method was performed by growing Bacillus sp. NP5 with each pathogen isolate, which has been given antibiotic resistant marker in Trypticase Soy Broth (TSB) medium, it was followed by enumerating the No. of pathogenic bacteria cells (CFU mL–1) which grew on Trypticase Soy Agar (TSA) medium, which was supplemented with antibiotic marker (Widanarni et al., 2003). Result of growth inhibition of pathogenic bacteria in competition test was measured by the equation as follows:
Preparation of probiotic: Probiotic bacteria used as feed supplement in this study, was Bacillus sp. NP5 which was previously given antibiotic rifampicin marker (Bacillus sp. NP5 RfR) (Widanarni et al., 2003). Probiotic cells were cultured in TSB medium and incubated in a water bath shaker at a temperature of 29-30°C, 160 rpm for 24 h. The suspension of bacterial cells was then centrifuged and the bacterial cells pellet was rinsed twice using Phosphate Buffer Saline (PBS) solution.
Preparation of test feed: Preparation of test feed using commercial feed with a protein content of 31% was carried out by adding three probiotic doses (106, 108 and 1010 CFU g–1 feed) into the feed. Mixing feed and probiotic was done by adding 2% egg white as a binder, while the control feed was only added 2% egg white.
Rearing condition and experimental design: This study was conducted over 45 days, including 30 days of rearing period with feeding trial using test feed, one day of rest period before the challenge test and 14 days of the challenge test. Experimental animals used in this study were common carps with an average weight of 4.81±0.25 g, which have previously been adapted to the experimental medium for 2 weeks. Fish were stocked randomly in each treatment aquarium measuring 65×40×40 cm3 with a density of 15 fish per aquarium. This study, used a completely randomized design, consisting of five treatments with three replicates, control (-) (feed without probiotic and without pathogen infection), control (+) (feed without probiotic and with pathogen infection), A (feed suplementation with probiotic at a dose of 106 CFU g–1 feed and with pathogen infection), B (feed supplementation with probiotic at a dose of 108 CFU g–1 feed and with pathogen infection) and C (feed supplementation with probiotic at a dose of 1010 CFU g–1 feed and with pathogen infection). Feeding was done by at satiation method, in which fish were fed three times a day (08.00, 12.00 and 16.00 Western Indonesia Time) for 30 days. Water quality was maintained by removing feces and water replacement in the rearing medium as much as 20% every day. Water quality was monitored during rearing period with the parameters and ranges were: Temperature at 28.0-28.5°C, DO at 7.2-8.1 mg L–1, pH at 7.4-8.04 and TAN at 0.0032-0.0065 mg L–1.
Observation of bacterial population in the intestine of common carp: The intestine of fish was weighed as much as 0.1 g and was homogenized in 0.9 mL PBS. Serial dilutions were performed in PBS, then each dilution (0.05 mL) was spread on TSA medium for total bacterial count and TSA with rifampicin suplementation for total Bacillus sp. NP5 RfR count. The No. of bacteria in the sample could be determined by enumerating the No. of colonies that grew on the medium multiplied by the dilution factor in CFU g–1unit (Madigan et al., 2003).
Challenge test: After receiving test feed for 30 days, common carps from each treatment (except negative control) were challenged by Aeromonas hydrophila. The aim of the challenge test was to evaluate the probiotic performance in improving the resistance of common carp against A. hydrophila infection. Common carps were infected by A. hydrophila at a concentration of 107 CFU mL–1 (0.1 mL individual–1). Infection was done by injection using a syringe via intramuscular route and negative control fish were injected with PBS. Clinical symptoms and mortality of common carp were observed for 14 days after the injection.
Experimental parameters: Experimental parameters observed consisted of bacterial population in the intestine, digestive enzymes activity included amylase, protease (Drapeau, 1974) and lipase (Debnath et al., 2007), growth performances, immune responses and the survival rate of common carp after the challenge test that described fish resistance to infection. Growth performance parameters observed were Daily Growth Rate (DGR) and Feed Conversion Ratio (FCR). The bacterial population in the intestine included total bacterial count and total Bacillus sp. NP5 RfR count. Growth performances and bacterial population in the intestine were evaluated immediately after 30 days of probiotic treatment. Immune responses parameters observed included total leukocytes, phagocytic activity (Balcazar et al., 2006), differential leukocytes (lymphocytes, monocytes and neutrophils), hematocrit, hemoglobin and total erythrocytes. Immune responses and survival rate of fish were measured twice: After 30 days of feeding trial (before the challenge test) and 14 days after the challenge test.
Statistical analysis: Data obtained were statistically analyzed using SPSS Statistics 17.0 software and the test was continued with Duncan test for significant difference test (p<0.05).
RESULTS
Identification of 16S rRNA: Result of identification of phenotypic character of Bacillus sp. NP5 using api® 50 CHB V4.0 kit showed that the species of Bacillus sp. NP5 was Bacillus cereus strain EM5. The identification based on the result of partial sequencing of 16S rRNA gene and BLAST-N analysis with 1250 bp alignment also showed that Bacillus sp. NP5 had closeness with Bacillus cereus strain EM5 on similarity index of 99%.
Amylolytic, proteolytic and lipolytic activity: The activity of amylase, protease and lipase of Bacillus sp. NP5 obtained the range of Amylolytic Index (AI), Proteolytic Index (PI) and Lipolytic Index (LI) were 0.50, 0.20 and 1.33, respectively (Table 1). The highest activity of amylase was achieved at 48 h of observation (0.044 U mg–1 protein), protease at 96 h (0.0030 U mg–1 protein) and lipase was observed only up to 24 h (0.062 U mg–1 protein) (Table 2).
Inhibitory ability of Bacillus sp. NP5 against pathogenic bacteria: The result of inhibition test of Bacillus sp., NP5 against pathogenic bacteria; A. hydrophila, S. agalactiae and M. fortuitum showed that Bacillus sp. NP5 had inhibitory activity against pathogenic bacteria with the inhibition index values were 0.44, 0.70 and 0.15, respectively (Table 3) and it was able to reduce those pathogens in competition test with the percentage values were 66.1, 98.81 and 30%, respectively (Table 4).
Bacterial population in the intestine of common carp: Probiotic supplementation through the feed on common carp for 30 days had significantly effect (p<0.05) on the bacterial population in the intestine of common carp. The highest value of total bacterial count in the fish intestine was found in C that was not significantly different (p>0.05) with B, but it was significantly different (p<0.05) with control and A.
| Table 1: | Potential index of amylolytic, proteolytic and lipolytic activity of Bacillus sp. NP5 |
| Table 2: | Amylase, protease dan lipase activity produced by Bacillus sp. NP5 on different time |
| na: Not analyzed | |
| Table 3: | Inhibitory activity of Bacillus sp. NP5 against pathogenic bacteria evaluated by Kirby-Bauer method |
| Table 4: | Inhibitory activity of Bacillus sp. NP5 against pathogenic bacteria evaluated by competition test |
In addition, the highest total Bacillus sp. NP5 count in the fish intestine was also found in C that was significantly different (p<0.05) with all treatments (Table 5).
Growth performances of common carp: The digestive enzymes (amylase, protease and lipase) activity on probiotic treatment with a dose of 1010 CFU g–1 feed was higher (p<0.05) compared to control (Table 6). There was significant difference in daily growth rate between C with control and A (p<0.05), but it was not significantly different (p>0.05) with B. The value of FCR in the probiotic treatments were lower (p<0.05) compared to control, but there was no difference (p>0.05) between probiotic treatmeants (Table 7).
Immune responses: The value of total leukocytes, phagocytic activity, hemoglobin and total erythrocytes before the challenge test in C was higher (p<0.05) compared to A, B and controls. The value of total leukocytes after the challenge test in B and C was higher (p<0.05) compared to A and controls. Hemoglobin and total erythrocytes of fish in all treatments, which were injected with pathogen decreased at the end of the challenge test (Table 8 and 9).
Survival rate: The survival rate of fish before the challenge test ranged from 80-100%.
| Table 5: | Total bacterial count and total Bacillus sp. NP5 count in the intestine of common carp (Cyprinus carpio) after 30 days of rearing period |
| nd: Not detected, different letters in the same row indicate significant differences (Duncan, p<0.05), the values shown are means and standard deviations | |
| Table 6: | Digestive enzymes activity (U mg–1 protein) of common carp (Cyprinus carpio) fed probiotic at a dose of 1% (1010 CFU g–1 feed) |
Different letters in the same row indicate significant differences (Duncan, p<0.05). The values shown are means and standard deviations | |
| Table 7: | Survival Rate (SR), Daily Growth Rate (DGR) and Feed Conversion Ratio (FCR) of common carp (Cyprinus carpio) after 30 days of feeding trial |
| Different letters in the same row indicate significant differences (Duncan, p<0.05). The values shown are means and standard deviations | |
| Table 8: | Immunity parameters of common carp (Cyprinus carpio) after 30 days of feeding trial |
| Different letters in the same row indicate significant differences (Duncan, p<0.05). The values shown are means and standard deviations | |
| Table 9: | Survival Rate (SR) and immunity parameters of common carp (Cyprinus carpio) after the challenge test with Aeromonas hydrophila |
| Different letters in the same row indicate significant differences (Duncan, p<0.05). The values shown are means and standard deviations | |
However, the survival rate of fish after the challenge test in all probiotic treatments and negative control were higher (p<0.05) compared to positive control. The survival rate obtained in negative control, A, B and C were 94.00, 81.33, 87.67 and 100.00%, respectively, while positive control only reached 50.00% (Table 9).
DISCUSSION
Identification of 16S rRNA gene is a molecular approach that was conducted to determine the bacteria species based on similarity of gene sequences with the data in Gene Bank. Based on the distribution of BLAST-N result, Bacillus sp. NP5 had a homology value of 99% with Bacillus cereus. Homology showed that those sequences having evolutionary relationship (Pertsemlidis and Fondon III, 2002). Bacillus sp. NP5 is used as probiotic associated with its ability to produce digestive enzymes such as amylase, protease and lipase. In addition, this probiotic also has inhibitory activity against several aquaculture pathogenic bacteria such as A. hydrophila, S. agalactiae and M. fortuitum.
Introduction of every types of bacteria into the intestine has a particular requirement, in which the bacteria must be able to compete with the resident microflora, which have been formed with the ecological niche (nutrients and attachment) in order to survive. Probiotic strains must be able to stick to the intestinal mucosa layer and utilize mucus as nutrients source for colonizing, it must be persistent and must be able to proliferate in the digestive tract of fish (Merrifield et al., 2010). The ability of Bacillus sp. NP5 to survive in the intestine of common carp was shown by the data of total probiotic count in the fish intestine, total probiotic count values were higher on the highest dose of probiotic treatment. Probiotic is an intestinal microflora, which plays a role in the mechanism of host resistance against stress conditions and pathogens; through immune response stimulation, production of specific antimicrobial substance (bacteriocin), release of metabolic product such as Short Chain Fatty Acid (SCFA) which causes the pH of intestinal fluid decreasing to a level under the optimum conditions for the pathogens growth, improvement of intestinal epithelial cells and action mechanism of imunity cells.
Probiotic produced significant effect on the digestive enzymes activity of common carp, because probiotic is able to improve digestibility of protein, carbohydrate and fat. This is further clarified by the better value of daily growth rate and FCR. Results of this study, showed that Bacillus sp. NP5 having an ability to assist the digestive process, it has been proven by the better growth of the fish, because it produces various extracellular enzymes, such as amylase, protease and lipase to facilitate the absorption and utilization of nutrients becoming more efficient. This is in line with the result of the study by Bairagi et al. (2002) which stated that Bacillus sp. isolated from the gut of C. carpio had high amount of extracellular amylolytic, proteolytic and lipolytic activity. The effects of probiotic supplementation on the digestibility improvement have also been observed in various fish species (Tovar-Ramirez et al., 2004). Protease secreted by probiotic serves to break down peptide bonds in protein stucture, then break down into protein core elements as protein monomers and free amino acids, which are very useful for the fish nutritional status improvement. Bacillus sp. NP5 is also able to secrete lipase, which triggers the production and assimilation of essential fatty acids, resulting in higher growth and immunity of common carp. Essential fatty acids are not only become a booster for the immune system, but also promotes the growth (Sharma et al., 2010). Bacterial enzymatic hydrolysis promotes the growth of common carp that is supported by a lower value of FCR, it shows the increasing of protein and fat bioavailability. Amylase and lipase are the major enzymes associated with the carbohydrate and fat break down. The results showed that the amylase, protease and lipase activity were higher in fish fed probiotic compared to control. The activity of amylase, protease and lipase in the probiotic treatment were largely the result of probiotic stimulation, there by encouraging exoenzymes to synthesize digestive endoenzymes, which ultimately synergize to improve nutrient digestibility and growth performances.
Dose of probiotic is a limiting factor to achieve the optimum beneficial effects (Minelli and Benini, 2008). Dose of probiotic in aquaculture generally ranges from 106-1010 CFU g–1 feed with optimum dose varies depending on fish species and immunity parameters observed. The optimum concentration is not only indicated by the bacterial colonization and proliferation in the gut, but also by the growth, immune responses and protection of the host. Effective dose of Bacillus sp. is 2×108 CFU g–1 feed for Oncorhynchus mykiss resulting in a low mortality percentage after the challenge test with pathogenic bacteria (Brunt et al., 2007). Phagocytic activity increased in higher value on O. mykiss given Lactobacillus rhamnosus at a dose of 1011 CFU g–1 feed for 30 days, but decreased at a dose of 109 CFU g–1 feed (Panigrahi et al., 2004). Best dose of Lactobacillus plantarum to increase growth, immune response and protection of grouper (Epinephelus coioides) is 108 CFU kg–1 feed, this dose showed better results than 106 and 1010 CFU kg–1 feed (Son et al., 2009). The administration of 0.5% dried Bacillus NP5 showed better results than higher doses (1 and 2%), this dose resulted the best growth performances in tilapia and was effective to control streptococcosis in tilapia with higher post-challenge test survival rate, better hematological parameter values and could inhibit S. agalactiae growth in the host target organs (Utami et al., 2015a, b). Low doses are not sufficient to stimulate maximum growth performance and cellular immune system related to the lack of colonization capacity, but high doses can cause high mortality, as happened on O. mykiss given L. rhamnosus at a dose of 1012 CFU g–1 feed, but it did not occur at a dose of 109 CFU g–1 feed (Nikoskelainen et al., 2001).
The addition of probiotic (106-1010 CFU g–1 feed) could reduce the amount of feed, which was required for the growth of common carp, resulting in reduction of production costs. The administration of Lactobacillus casei at a dose of 5×107 CFU g–1 for 30 days and 5×108 CFU g–1 for 60 days significantly improved the growth performances (daily growth rate and FCR) of Barbus gryprus (Vand et al., 2014). Lactobacillus plantarum at a dose of 108 and 1010 CFU g–1 feed given to Labeo rohita could improve daily body weight gain and FCR, it also showed an improvement in SGR (Giri et al., 2013). Similar results were also found in Epinephelus coioides (Son et al., 2009), tilapia nilotica (Oreochromis niloticus) (Aly et al., 2008), gilthead sea bream (Sparus aurata) (Suzer et al., 2008), Clarias gariepinus (Al-Dohail et al., 2009) and Macrobrachium rosenbergii (Venkat et al., 2004). Suplementation of commercial probiotic (Streptococcus faecium) was able to improve growth and feed efficiency on Israeli carp than antibiotics and yeast (Saccharomyces cerevisiae) (Bogut et al., 1998), it also occurred in terrestrial mammals, especially on pig (Bertin et al., 1997). The increasing doses of probiotic also showed the influence on the presence of bacteria in the intestine of common carp; the higher doses showed higher values on total bacterial count and total probiotic count. The high survival of Bacillus in intestinal mucosa cells can be caused by the competitive elimination mechanism against other bacteria, especially against pathogenic bacteria that has been demonstrated on the results of inhibition test (in vitro) of probiotic against some potential pathogens. This probiotic has antagonistic activity against A. hydrophila which is a resident pathogen of freshwater fish (Gonzalez et al., 1999), through the inhibitor compounds production. The increasing of probiotic population can produce some fermentative products such as lactic and acetic which cause a reduction in the pH of the intestinal fluid under optimum conditions for pathogens. Therefore, supplementation of Bacillus sp. NP5 could increase protection and resistance of common carp against a pathogen, which was showed by high survival rates after the challenge test. This is in line with the results of study by Tamamdusturi et al. (2016), which reported that the oral supplementation of microencapsulated probiotic Bacillus sp. NP5 showed higher survival rate of catfish (Pangasianodon hypophthalmus) after the challenge test with A. hydrophila compared to positive control. The oral supplementation of microencapsulated probiotic Bacillus sp. NP5 also showed higher survival rate of tilapia after the challenge test with Streptococcus agalactiae compared to positive control (Agung et al., 2015).
Probiotic supplementation could improve the cellular immune responses of common carp to facilitate the elimination of potential pathogens in the intesinal tissue. A positive result from the administration of probiotic on hemoglobin, hematocrit and total erythrocytes also occurred in Catla catla (Hamilton), which were supplemented with Lactobacillus acidophilus in diet and it was related toprobiotic ability to improve hematological parameter values as a result of haemopoetic stimulation (Renuka et al., 2014). Cytotoxicity of A. hydrophila and accumulation of its extracellular products (α and β hemolysin, aerolisin, enterotoxin ACT, ALT and AST, protease and RNase) cause erythrocytes necrotic, hemolysis of erythrocytes and iron ions (Rey et al., 2009), thus causing a decreasing in total red blood cells of experimental fish. This is in line with the results of this study, total erythrocytes of infected common carp decreased from total erythrocytes of common carp before getting an infection. Probiotic interacts with mononuclear phagocytic cells (monocytes and macrophages) and polymorphonuclear leukocytes (neutrophils) and natural killer cells. Probiotic can act as an effective trigger for phagocytic cells, thus increasing phagocytic activity as happened on tilapia (Oreochromis niloticus) given feed containing L. rhamnosus for 2 weeks (Pirarat et al., 2006). An increasing in phagocytic activity also occurred in gilthead sea bream (Sparus aurata L.) given a diet containing Lactobacillus delbrueckii spp. lactis (CECT 287) at a dose of 107 CFU g–1 for 14 days (Salinas et al., 2005). Phagocytosis is responsible for initial activation of inflammatory response before antibodies production occured and it acts as mediator of phagocytic cells (neutrophils, monocytes and macrophages). In vivo activation of phagocytic cells by immunomodulator also causes secretion of a large number of active biological molecules such as inhibitor enzymes, cationic peptides, complement components, production of oxygen and nitrogen reactive (ROS and NOS), which are entirely involved in bactericidal activity (Kwak et al., 2003). Bacillus sp. NP5 could increase active phagocytic cells and total leukocytes. Stressors cause stress response in leukocytes on all vertebrates including fish (Davis et al., 2008). An increasing in total leukocytes could be caused by the experimental fish were in stress condition due to daily consumption of feed containing probiotic, which was recognized as foreign material. Along with the increasing of total leukocytes, fish mortality in probiotic treatment (in a dose of 1010 CFU g–1) showed a lower value after the challenge test with pathogen compared to positive control, which indicated the improvement on health status of the fish, which consumed feed containing probiotic.
CONCLUSION
Result of phenotypic and genotipic identification of Bacillus sp. NP5 isolate showed that this isolate was Bacillus cereus with 99% of similarity index. Supplementation of feed containing probiotic at a dose of 1010 CFU g–1 feed resulted a higher total bacterial count and a higher total probiotic count in the intestine, those followed with higher value of amylase, protease and lipase activity, along with the highest daily growth rate and the lowest feed conversion ratio (p<0.05). This dose also showed higher value of total leukocytes and phagocytic activity (p<0.05) compared to controls at the end of the rearing period. Common carps fed probiotic showed survival rates after the challenge test with values ranging between 81-100%, while the survival rate of fish without probiotic supplementation was only 50%. This showed, that probiotic supplementation on common carp could reduce the pathogenicity of disease caused by Aeromonas hydrophila infection, which indicated an improvement in cellular immune response.