- Email: [email protected]
Effects of potential probiotic Bacillus amyloliquifaciens FPTB16 on systemic and cutaneous mucosal immune responses and disease resistance of catla (Catla catla)
Fish & Shellfish Immunology 35 (2013) 1547e1553
Contents lists available at ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Full length article
Effects of potential probiotic Bacillus amyloliquifaciens FPTB16 on systemic and cutaneous mucosal immune responses and disease resistance of catla (Catla catla) Anushree Das a, Khriezhato Nakhro a, Supratim Chowdhury b, Dibyendu Kamilya a, * a
Department of Fish Health and Environment, College of Fisheries, Central Agricultural University, Lembucherra, Post Box No. 60, Agartala 799 001, Tripura, India Department of Fish Processing Technology, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, 5 e Budherhat Road, Chakgaria, P.O. e Panchasayar, Kolkata 700 094, West Bengal, India b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 June 2013 Received in revised form 12 August 2013 Accepted 27 August 2013 Available online 4 September 2013
Effects of dietary administration of Bacillus amyloliquefaciens FPTB16 on systemic and mucosal immunity and disease resistance of catla (Catla catla) against Edwardsiella tarda infection were evaluated in the present study. The laboratory maintained B. amyloliquefaciens was used to study antagonistic activity against fish pathogenic bacteria by agar well diffusion assay. Healthy catla were challenged by this bacterium for determination of its safety. For preparation of probiotic supplemented diet, the bacteria were added to the basal diet (control) at three different inclusion levels i.e., 1 109, 1 108 and 1 107 CFU/g diet. Fish (weight 25e30 g) were fed with these diets and various immune parameters and disease resistance study were conducted at 4 weeks and 8 weeks post-feeding. The bacterial antagonism study showed inhibition zone against E. tarda, Aeromonas hydrophila, Vibrio parahaemolyticus and V. harveyi. B. amyloliquefaciens was harmless to catla as neither mortalities nor morbidities were observed after the challenge. Study of different systemic and mucosal immunological parameters viz. superoxide anion production and nitric oxide production, myeloperoxidase content, lysozyme activity and total protein content showed significant enhancement (p < 0.05) in fish fed with 108 and 109 CFU/g B. amyloliquefaciens at both time points with the highest values observed in case of 109 CFU/g. For fish fed with 107 CFU/g B. amyloliquefaciens, all the parameters showed significant enhancement (p < 0.05) at both time points except the lysozyme activity of serum at 8 weeks. Diet containing 108 and 109 CFU/g B. amyloliquefaciens significantly enhanced (p < 0.05) the resistance of catla against bacterial challenge at both time points. These results collectively suggest that B. amyloliquefaciens is a potential probiotic species and can be used in aquaculture to improve health status and disease resistance with an optimal dietary supplementation of 109 CFU/g. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Bacillus amyloliquefaciens FPTB16 Catla (Catla catla) Probiotic Immune response Disease resistance
1. Introduction With the ever increasing demand for fish and aquatic food products, there has been a shift in aquaculture practices, moving from extensive systems towards the semi-intensive and intensive systems. With the increasing intensification and commercialization of aquaculture production, disease has become a major stumbling block in the fish farming industry [1]. The widespread use of broad-
* Corresponding author. Tel.: þ91 381 2865 264, þ91 381 2865 513; fax: þ91 381 2865 291, þ91 381 2917 048. E-mail addresses: [email protected], dibyendu_kamilya@ yahoo.co.in (D. Kamilya). 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.08.022
spectrum chemotherapeutics to combat such health related problems has led to the development and spread of drug resistant pathogens, environmental hazards and food security problems [2]. Use of probiotics has been gaining importance for diseases prevention in aquaculture as an eco-friendly alternative to antibiotics and other drugs [3,4]. Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host [5]. Probiotics exert beneficial effects on the host by providing nutrients and enzymatic contribution to digestion, improving water quality, enhancing growth, inhibiting pathogenic microorganisms and enhancing immune responses [6,7]. Apart from lactic acid bacteria, the most commonly used probiotics in fish farming industry belong to gram positive spore
1548
A. Das et al. / Fish & Shellfish Immunology 35 (2013) 1547e1553
forming Bacillus spp [3]. Bacillus preparations are resistant to the environment, have a long lasting shelf life and the beneficial roles of these bacterial species in the aquaculture field are well established [3,8]. Within the Bacillus genus, Bacillus amyloliquefaciens is a closely related species to Bacillus subtilis with potent biocontrol ability against a number of plant and post-harvest pathogens [9,10]. However, no information is available about B. amyloliquefaciens as a biocontrol agent for aquatic pathogens except the recent report where the potential antagonistic activity of B. amyloliquefaciens against eel-pathogenic Aeromonas hydrophila has been demonstrated [11]. In another study, the bacterium has also been found to improve the survival of Litopenaeus vannamei larvae with antagonistic activity against different Vibio spp [12]. However, to the best of our knowledge, no reports are available to describe its potential probiotic nature in finfish. Thus, the aim of the present study was to evaluate the efficiency of B. amyloliquefaciens to act as a potential probiotic, especially in terms of improving immunity and disease resistance in one of the commercially important Indian major carp species, catla (Catla catla).
2. Materials and methods 2.1. Experimental fish and rearing Healthy catla (weight 25e30 g) were collected from a local fish farmer. After collection they were brought immediately and acclimatized in 1000 L fibre-reinforced plastic (FRP) tanks. The fish were acclimatized at ambient temperature (26 1 C) with aeration and were fed twice daily with a basal pelleted diet @3% of body weight. The physicochemical parameters of water were maintained throughout the period of the experiment. Water exchange (upto 50%) was done to remove the waste feed and fecal matter, as and when required. 2.2. Bacteria B. amyloliquefaciens FPTB16 (GenBank Accession: KF319058), isolated from an indigenous fermented fish product ‘Shidal’, was used in all the experiments. This bacterium was obtained from the Department of Fish Processing Technology, West Bengal University of Animal and Fishery Sciences. The Edwardsiella tarda strain (ET-PG-29), known to be pathogenic to catla [13] is available in our laboratory. Other indicator strains such as A. hydrophila, Vibrio harveyi and V. parahaemolyticus were also obtained from Department of Fish Processing Technology, West Bengal University of Animal and Fishery Sciences. The bacteria were maintained in the laboratory by subculturing in nutrient agar (NA).
2.4. Safety of the probiotic strain The safety of B. amyloliquefaciens was evaluated by using a challenge study. Ninety healthy catla were distributed in six tanks equally (15 fish per tank) and were acclimatized for one week. The bacteria were grown in nutrient broth for 48 h at 30 C and the bacterial pellets were obtained by centrifugation at 3000 rpm for 30 min. Bacterial cells were washed twice with sterile saline solution (0.85% NaCl) and then resuspended in the same solution to obtain a bacterial suspension with the concentration of 109 CFU/ml. The fish from three tanks were intraperitoneally (i.p.) injected with 0.2 ml of B. amyloliquefaciens fresh culture suspension containing 109 bacteria ml1. Rest three tanks served as a control and i.p. injected with 0.2 ml sterile saline. All groups were fed on basal diet and kept under observation for 15 days and the mortality and morbidity were recorded. 2.5. Feed preparation and determination of the survival of the probiotics bacteria in feed The basal pelleted diet was prepared from locally available ingredients. Proximate analysis of the basal diet revealed 21.08% crude protein, 6.14% crude lipid, and 13.48% ash, 6.11% moisture, 14.28% crude fibre and 38.91% nitrogen free extract (digestible carbohydrate). The basal diet was used as control diet. Three probiotic supplemented diets, designated as D1, D2 and D3, were prepared with three levels of inclusion of B. amyloliquefaciens, i.e. 1 107, 1 108 and 1 109 CFU/g diet, respectively. The survival of the supplemented bacteria in the diet was assessed following storage at 4 C and at room temperature (26 C) on weekly basis for four weeks [15]. One gram of the diet was homogenized in 9.0 ml sterile saline solution, and serial dilutions down to 104 were prepared and 0.1 ml was spread onto triplicate plates of nutrient agar. The colonies were counted after incubation for 24 h at 30 C. Based on the survivability data feeds were prepared on weekly basis to ensure high probiotic levels in the diet. 2.6. Feeding schedule Three hundred catla fingerlings without the sign of any disease (gross and microscopic examination of different tissues from representative samples) and with no previous history of infection were divided into four equal groups. The groups were subdivided into 3 equal subgroups of 25 fish in each tank to determine the probiotic effect. The basal diet was fed to all fish during the acclimatization period. The fish were provided with aeration and water was renewed daily. Three groups of fish were fed with three probiotic supplemented diets (107, 108 and 109 CFU/g diet). The 4th group was given basal diet without probiotics (control). The fish were fed twice daily (at 9:00 h and 17:00 h) @3% of the body weight for 8 weeks. All the diets were prepared once a week and stored at 4 C.
2.3. Antagonism study Agar well diffusion test was conducted to study antagonistic activity of B. amyloliquefaciens following a previously described method [14] with some modification. The antagonistic activity was examined against indicator pathogenic bacteria like E. tarda, A. hydrophila, V. harveyi and V. parahaemolyticus. Briefly, a culture of the indicator bacteria was independently spread on nutrient agar plates, and then wells of 4e5 mm diameter were made on solidified agar. The base of the wells was sealed with one drop of molten agar. Wells were then filled with 100 ml of overnight bacterial culture. The plates were incubated at 30 C and zones of inhibition around the wells were observed and recorded after 24e48 h.
2.7. Measurement of systemic and cutaneous mucosal immune responses Different systemic and cutaneous mucosal immune responses were measured separately for probiotic treated and control fish after 4 weeks and 8 weeks of feeding. 2.7.1. Sampling, serum collection, mucus collection and isolation of head kidney (HK) leucocytes At each time point, 3 fish were randomly sampled from each tank, thus a total of 9 fish were sampled for each treatment. Before the separation of the HK, anaesthetized fish (using clove oil
A. Das et al. / Fish & Shellfish Immunology 35 (2013) 1547e1553
@100 ml L1) were bled directly from the heart/caudal vein and allowed to clot at room temperature. Serum was collected from the clotted blood and stored at 20 C in sterilized vial until further use. Mucus was sampled by gentle scraping from the fish body. An equal volume of phosphate buffered saline (PBS, pH 7.2) containing 0$02% azide was added to collected mucus, vortexed for 1 min and stored at 4 C overnight. Mucus was then centrifuged at 4000 rpm for 10 min and supernatant collected. The processed mucus samples were stored at 20 C until further use. Isolation of HK leukocytes was done following the method described previously [16]. Following blood collection, HK was removed aseptically from the treatment and control fish and the cell suspension was obtained in complete RPMI-1640 medium. The cell suspension was washed by the centrifugation for 10 min at 1000 rpm and the pellet was resuspended in RPMI. It was then carefully layered on the top of the lymphocyte isolation medium, HiSep (Hi-media) and centrifuged at 1500 rpm for 30 min. Cells at the medium-Hisep interface were transferred into the clean tubes and washed twice by the centrifugation at 1000 rpm for 10 min. Purified leucocytes were counted using a haemocytometer and the cell viability was determined by the trypan blue exclusion test.
1549
control tanks and distributed into another 12 tanks for challenge study. Challenge was performed by injecting each fish intramuscularly with 0.2 ml of E. tarda (1 107 CFU ml1). The same challenge test was repeated 8 weeks later using same number of fish. The challenged fish were kept under observation for 15 days and the dead fish were removed from the tank. The cause of death and pathological changes were verified by re-isolation of bacteria from dead/infected samples. The mortalities were recorded and the survival percentage was determined. 2.9. Statistical analysis Statistical analysis of data was performed using SPSS-15.0 for windows software (SPSS Inc., Chicago, IL, USA). Results are presented as mean standard deviation (SD). Data were analyzed by the one-way analysis of variance (ANOVA) and Tukey’s range test. Probability levels of 0.05 were used to find out the significance in all cases. 3. Results 3.1. Antagonism study and safety of B. amyloliquefaciens
2.7.2. Assessment of systemic and mucosal immunological parameters Production of intracellular superoxide anion by the HK leukocytes was evaluated by nitroblue tetrazolium reduction assay [16]. Production of nitric oxide (NO) was assessed by measuring absorbance of nitrite from the leukocyte culture supernatant using Griess reaction [17]. The total myeloperoxidase content of serum and mucus was determined as described by Mohanty and Sahoo [18]. The lyzozyme activity of serum and mucus was measured following a turbidimetric method, modified to a microtitre plate assay [19]. Lysozyme activity was expressed as units ml1 where one unit was defined as the reduction in absorbance of 0.001 min1. The protein content of serum and mucus was estimated following the biuret method [20].
The in-vitro antimicrobial assay by agar well diffusion technique showed that the B. amyloliquefaciens induced an inhibition zone against E. tarda, A. hydrophila, V. harveyi and V. parahaemolyticus of 8.9 mm, 9.1 mm, 9.3 mm and 6.6 mm, respectively. The intraperitoneal injection of B. amyloliquefaciens was harmless to the catla as neither mortalities nor morbidities were observed during the 15 days of post-challenge observation. 3.2. Survivability of the probiotics bacteria in feed The storage of feed supplemented by B. amyloliquefaciens showed an average 44.11% reduction in viable cells when stored at room temperature (28 C) for 4 weeks. However, the average reduction was 18.61% when feed was stored at 4 C for 4 weeks.
2.8. Challenge test
3.3. Immune responses
Four weeks after the start of the feeding experiment, 10 fish were collected from each of the probiotic supplemented and
3.3.1. Superoxide anion (O2) production The superoxide anion production increased significantly (p < 0.05) in all the diet groups when compared with that of the control (Fig. 1). These increments were evident for all the groups after 4 weeks and 8 weeks of probiotic feeding. The maximum values were obtained for the highest level of probiotic inclusion to the diet (D3, 1 109 CFU/g diet), whereas the lowest values were obtained for lowest probiotic dose (D1, 1 107 CFU/g diet). The values were higher for all the dietary groups after 8 weeks of feeding than the 4 weeks of feeding.
Fig. 1. Superoxide anion production by catla head kidney leucocytes. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
3.3.2. NO production The NO production showed similar trend with that of the superoxide anion production. The increments were statistically significant (p < 0.05) for all the probiotic supplemented groups after 4 weeks and 8 weeks of feeding (Fig. 2). The maximum values were obtained in the highest level of probiotic inclusion to the diet (D3, 109 CFU/g diet), whereas the lowest values were obtained for lowest probiotic dose (D1, 107 CFU/g diet). Similar to superoxide anion production the values were higher for all the dietary groups after 8 weeks of feeding than the 4 weeks of feeding. 3.3.3. Myeloperoxidase content of serum and mucus The total myeloperoxidase content of both serum and mucus increased significantly (p < 0.05) in all the dietary groups. The
1550
A. Das et al. / Fish & Shellfish Immunology 35 (2013) 1547e1553
Fig. 2. Nitric oxide production by catla head kidney leucocytes. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
myeloperoxidase content of serum was higher in the group D3 (109 CFU/g diet) after 4 weeks of feeding than the 8 weeks of feeding (Fig. 3). In contrary, the myeloperoxidase content of mucus for all the dietary treatment groups was higher after 8 weeks of feeding than the 4 weeks of feeding (Fig. 4). At both point of time, an increase in serum and mucosal myeloperoxidase content was observed with the increasing supplementation level of B. amyloliquefaciens. 3.3.4. Lysozyme activity of serum and mucus The lysozyme activity of catla serum increased significantly (p < 0.05) after 4 weeks of feeding with different probiotic supplemented diets when compared to that of the control (Fig. 5). Statistically significant increase (p < 0.05) of serum lysozyme activity was also observed after 8 weeks of feeding with 108 and 109 CFU/g B. amyloliquefaciens. However, the increment was not statistically significant (p > 0.05) for the dietary group D1 (107 CFU/ g diet). The lysozyme activity of catla mucus increased significantly (p < 0.05) after 4 and 8 weeks of probiotic feeding for all the treatment groups (Fig. 6). The values of lysozyme activity of mucus were higher for all the dietary groups after 8 weeks of feeding than the 4 weeks of feeding.
Fig. 3. Myeloperoxidase activity of catla serum. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
fed control diet exhibited lowest survival rate (i.e. 20%) after 8 weeks of feeding.
4. Discussion Use of probiotics in aquaculture is now increasingly being considered as an eco-friendly approach to mitigate health related problems. Disease preventing abilities of probiotics are achieved through enhancement of immunity and exclusion of pathogens. Among probiotics, Bacillus strains are gaining more and more importance and are widely used in aquaculture. The present study describes the effects of a little explored potential probiont B. amyloliquefaciens in ameliorating the health of catla in an 8weeks feeding trial. A common way to select probiotics is to perform in vitro antagonism tests, in which pathogens are exposed to the candidate probiotics or their extracellular products [21]. In the present work B. amyloliquefaciens showed antagonistic activity against the pathogenic indicator bacteria like E. tarda, A. hydrophila, V. harveyi and V. parahemolyticus The inhibitory activity of
3.3.5. Total protein content of serum and mucus After both 4 and 8 weeks of feeding, the total protein content of serum and mucus from 3 treatment groups were significantly higher (p < 0.05) than that of the control (Figs. 7 and 8). Fish fed with 109 CFU/g B. amyloliquefaciens showed the highest level of protein content of serum and mucus at both the time points. The protein contents of serum from all the dietary groups were always higher than that of mucus at all the time points.
3.4. Challenge test Diet containing 108 and 109 CFU/g B. amyloliquefaciens significantly enhanced (p < 0.05) the resistance of catla against E. tarda challenge at both time points (Fig. 9). However, diet containing 107 CFU/g B. amyloliquefaciens increased the survival percentage of catla than that of the control, but it was not statistically significant (p > 0.05). The highest post-challenge survival rate (p < 0.05) was observed in fish group fed diet containing 109 CFU/g B. amyloliquefaciens (i.e. 76.67%) after 8 weeks of feeding. The fish
Fig. 4. Myeloperoxidase activity of catla mucus. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
A. Das et al. / Fish & Shellfish Immunology 35 (2013) 1547e1553
Fig. 5. Lysozyme activity of catla serum. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
1551
Fig. 7. Total protein content of catla serum. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
B. amyloliquefaciens has also been demonstrated against A. hydrophila [11] and against V. harveyi and V. parahemolyticus [12]. The result indicated that the probiont has potential antagonistic activity against a diverse group of pathogenic bacteria. The inhibitory activity of B. amyloliquefaciens may be attributed to its ability to produce a vast array of secondary metabolites with antimicrobial activity [22]. Another criterion to select probiotics is that the probiont should not be pathogenic to the hosts and this should be confirmed prior to acceptance [21]. The probiont used in the present study was not non-pathogenic in catla as mortalities and/or morbidities were not observed during the 15 days post-challenge. The present finding agreed with Austin et al. [23] and Aly et al. [24] who showed the safety of probiotics via intramuscular and intraperitoneal injection in Atlantic salmon and tilapia, respectively. The storage of the probiotic-supplemented diet, under cold temperature demonstrated the durability of B. amyloliquefaciens in the feed. There were a greater number of viable cells in the probiotic supplemented diet stored in the refrigerator at 4 C than that stored at room temperature (26 C). Such decline in viability of the bacteria is common is probiotic supplemented diet. Therefore, fresh diets were prepared at weekly interval to ensure high probiotic
levels in the diets. Similar way of preparing diet has also been reported from other works [24,25]. Probiotics interact with different immune cells to enhance immune responses. Respiratory burst activity of HK leukocytes is one of the important innate immune parameters which is widely used as an indicator of immunocompetence, especially when provoked by stimulators [26]. Increased superoxide anion production in the present study indicated inducement of one of the innate immunity components following probiotic administration. The respiratory burst activities by fish leukocytes following probiotics treatment are often contradictory. Some studies indicate that probiotics do not have significant impact on this innate defense mechanism of fish [27,28], while several other studies showed significant increase in respiratory burst activity in fish by various probiotics [25,29,30]. Apart from superoxide anion, NO is another effector molecule associated with activated fish granulocytes, and is involved in the immune response [31]. In the present study, the trend of nitric oxide production was similar to that of superoxide production following probiotic supplemented diet administration. However, studies on NO production following probiotic application are
Fig. 6. Lysozyme activity of catla mucus. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
Fig. 8. Total protein content of catla mucus. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
1552
A. Das et al. / Fish & Shellfish Immunology 35 (2013) 1547e1553
Fig. 9. Effects of dietary administration of B. amyloliquefaciens on the post-challenge survival of catla after infection with Edwardsiella tarda. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
limited. In one study, increased NO production following lactobacillus lactis admiministration was observed in turbot (Scophthalmus maximus) [32]. In another study, sea cucumbers fed with B. subtilis at 109 CFU/g diet also showed significantly higher NO production than the control diet [33]. The probiotic administration enhanced the synthesis of reactive oxygen and nitrogen species in activated leukocytes which were persistent throughout the feeding period. This may be attributed to the weekly addition of young culture of B. amyloliquefaciens to the diets thereby maintaining a high level of probionts in the diet. The myeloperoxidase is an important enzyme that utilizes oxidative radicals to produce hypochlorous acid to kill pathogens. During oxidative respiratory burst, it is mostly released by the azurophilic granules of neutrophils [34]. In the present study myeloperoxidase content of serum as well as mucus was significantly higher after 4 and 8 weeks of feeding with B. amyloliquefaciens supplemented diet. Similar result of elevated myeloperoxidase level in serum was observed for B. subtilis in rainbow trout (Oncorhynchus mykiss) [35], B. subtilis in gilthead seabream (Sparus aurata L.) [36] and Lactobacillus plantarum in grouper (Epinephelus coioides) [37]. However, to our knowledge, no reports are available regarding the myeloperoxidase content of cutaneous mucus of fish fed with probiotic diet. Lysozyme is an important humoral component of the systemic as well as mucosal immune system and act as defensive factor against invasive microorganisms in vertebrates [19]. In the present study, enhanced serum and mucosal lysozyme activity was recorded in all the treatment groups throughout the study period. Different probiotic species have been reported to trigger the lysozyme level in teleosts. Elevated serum lysozyme activity were observed by probiotics in rainbow trout [38], in brown trout, Salmo trutta [14] and in grouper [37]. Apart from serum, probiotics have also been reported to enhance the lysozyme level in skin mucosa of fish [39,40]. After 4 and 8 weeks of feeding, the total protein content of serum and mucus from treatment groups were significantly higher than that of the control. Similar increment of the serum protein content was observed in rohu (Labeo rohita) fed with B. subtilis supplemented diet [41]. The rise in total protein level may be a joint contribution form group of serum and mucosal proteins like
agglutinins, lectins, lysozyme, immunoglobulins etc. which are important defense molecules. Increase in total protein level in both serum and mucus corroborates the fact that these defense molecules were synthesized in ample quantity as a result of the stimulation of the systemic as well as mucosal immunity following dietary administration of B. amyloliquefaciens. In aquaculture, probiotics help in achieving natural resistance and high survivability of larvae and post larvae of fishes [42]. The effectiveness of probiotics in terms of protection against infectious pathogens is often attributed to the elevate immunity. In the present study dietary administration of B. amyloliquefaciens significantly increased the survival rates of catla challenged with Edwardsiella tarda. In a previous study protection was achieved in eel (Anguilla anguilla) against Aeromonas hydrophila infection after the fish were fed with diet supplemented with B. amyloliquefaciens @3 109 CFU/g [11]. Protection against edwardsiellosis [40,41], enteric red mouth disease [43], furunculosis [15], lactococossi and streptococcosis [44] and aeromoniasis [35] have successfully been accomplished through probiotics feeding. The results indicate the involvement of an activated and prolonged immune status of catla to resist E. tarda infection. As a final note, the present study delineates the efficacy of B. amyloliquefaciens as a potential probiotic for use in aquaculture. This probiont with an optimal dietary supplementation of 109 CFU/ g was effective in maintaining an elevated immunity level persistently, thereby conferring resistance against edwardsiellosis. We suggest long term feeding with frequent addition of fresh preparation of probiotics in the diet to maintain the persistent activation of immune cells throughout the feeding period. However, more studies are required to elucidate other mode of action of this probiont, especially its effect on growth, colonization in gut etc. to further characterize its efficacy.
Acknowledgment The authors are thankful to AICRP on Post Harvest Technology (ICAR), Kolkata Centre, Department of Fish Processing Technology, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, India for providing the probiotic strain. The authors are also thankful to Vice Chancellor, Central Agricultural University, Imphal, India and Dean, College of Fisheries, CAU, Lembucherra, Tripura, India for providing necessary research facilities.
References [1] Bondad-Reantaso MG, Subasinghe RP, Arthur JR, Ogawa K, Chinabut S, Adlard R, et al. Disease and health management in Asian aquaculture. Vet Parasitol 2005;132:249e72. [2] Ai Q, Xu H, Mai K, Xu W, Wang J, Zhang W. Effects of dietary supplementation of Bacillus subtilis and fructooligosaccharide on the growth performance, survival, non-specific immune response and disease resistance of juvenile large yellow croaker Larimichthys crocea. Aquaculture 2011;317:155e61. [3] Wang YB, Li JR, Lin J. Probiotics in aquaculture: challenges and outlook. Aquaculture 2008;281:1e4. [4] Nayak SK. Probiotics and immunity: a fish perspective. Fish Shellfish Immunol 2010;29:2e14. [5] Food and Agriculture Organization of the United Nations (FAO). Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. In: The joint FAO/WHO expert consultation report on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria (October 2001) 2001. [6] Verschuere L, Rombaut G, Sorgeloos P, Verstraete W. Probiotic bacteria as biological control agents in aquaculture. Microbiol Mol Biol Rev 2000;64:655e 71. [7] Balcazar JL, de Blas I, Ruiz-Zarzuela I, Cunningham D, Vendrell D, Muzquiz JL. The role of probiotics in aquaculture. Vet Microbiol 2006;114:173e86. [8] Gatesoupe FJ. The use of probiotics in aquaculture. Aquaculture 1999;180: 147e65.
A. Das et al. / Fish & Shellfish Immunology 35 (2013) 1547e1553 [9] Arguelles-Arias A, Ongena M, Halimi B, Lara Y, Brans A, Joris B, et al. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microb Cell Fact 2009;8:63. [10] Zhao P, Quan C, Wang Y, Wang J, Fan S. Bacillus amyloliquefaciens Q-426 as a potential biocontrol agent against Fusarium oxysporum f. sp. spinaciae. J Basic Microbiol 2013. http://dx.doi.org/10.1002/jobm.201200414 [in press]. [11] Cao H, He S, Wei R, Diong M, Lu L. Bacillus amyloliquefaciens G1: a potential antagonistic bacterium against eel-pathogenic Aeromonas hydrophila. Evid Based Complement Alternat Med 2011:7. http://dx.doi.org/10.1155/2011/ 824104. Article ID 824104. [12] Luis-Villaseñor IE, Macías-Rodríguez ME, Gómez-Gil B, Ascencio-Valle F, Campa-Córdova AI. Beneficial effects of four Bacillus strains on the larval cultivation of Litopenaeus vannamei. Aquaculture 2011;321:136e44. [13] Devi TB, Kamilya D, Abraham TJ. Dynamic changes in immune-effector activities of Indian major carp, catla (Catla catla) infected with Edwardsiella tarda. Aquaculture 2012;366e367:62e6. [14] Balcazar JL, Rojas-Luna T, Cunningham DP. Effect of the addition of four potential probiotic strains on the survival of Pacific white shrimp (Litopenaeus vanamei) following immersion challenge with Vibrio parahaemolyticus. J Invertebr Pathol 2007;96:147e50. [15] Irianto A, Austin B. Use of probiotics to control furunculosis in rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 2002;25:333e42. [16] Kamilya D, Ghosh D, Bandyopadhyay S, Mal BC, Maiti TK. In vitro effects of bovine lactoferrin, mushroom glucan and Abrus agglutinin on Indian major carp, catla (Catla catla) head kidney leukocytes. Aquaculture 2006;253:130e9. [17] Green IC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum S. Analysis of nitrate, nitrite and (15N) nitrate in biological fluids. Anal Biochem 1982;126:131e8. [18] Mohanty BR, Sahoo PK. Immune responses and expression profiles of some immune-related genes in Indian major carp, Labeo rohita to Edwardsiella tarda infection. Fish Shellfish Immunol 2010;28:613e21. [19] Ellis AE. Lysozyme assay. In: Stolen JS, Fletcher EC, Aderson DP, Robetrson DS, van Muiswinkel WB, editors. Techniques in fish immunology. USA: SOS Publications; 1990. p. 101e3. [20] Gornall AC, Bardawill CJ, David MM. Determination of serum proteins by means of the biuret reaction. J Biol Chem 1949;177:751e67. [21] Sahu MK, Swarnakumar NS, Sivakumar K, Thangaradjou T, Kannan L. Probiotics in aquaculture: importance and future perspectives. Indian J Microbiol 2008;48:299e308. [22] Chen XH, Koumoutsi A, Scholz R, Schneider K, Vater J, Süssmuth R, et al. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. J Biotechnol 2009;140:27e37. [23] Austin B, Stuckey LF, Robertson PAW, Effendi I, Griffith DRW. A probiotic strain of Vibrio alginolyticus effective in reducing diseases caused by Aeromonas salmonicida, Vibrio anguillarum and Vibrio ordalii. J Fish Dis 1995;18: 93e6. [24] Aly SM, Ahmed YA, Ghareeb AA, Mohamed MF. Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics, on the immune response and resistance of Tilapia nilotica (Oreochromis niloticus) to challenge infections. Fish Shellfish Immunol 2008;25:128e36. [25] Giri SS, Sukumaran V, Oviya M. Potential probiotic Lactobacillus plantarum VSG3 improves the growth, immunity, and disease resistance of tropical freshwater fish, Labeo rohita. Fish Shellfish Immunol 2013;34:660e6. [26] Secombes CJ. Isolation of salmonid macrophages and analysis of their killing activity. In: Stolen JS, Fletcher TC, Anderson DP, Robertson BS, van Muiswinkel WB, editors. Techniques in fish immunology, vol. I. Fair Haven: SOS Publications; 1990. p. 137e54. [27] Diaz-Rosales P, Arijo S, Chabrillon M, Alarcon FJ, Tapia-Paniagua ST, MartinezManzanares E, et al. Effects of two closely related probiotics on respiratory burst activity of Senegalese sole (Solea senegalensis, Kaup) phagocytes, and
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
1553
protection against Photobacterium damselae subsp. piscicida. Aquaculture 2009;293:16e21. Sharifuzzaman SM, Austin B. Influence of probiotic feeding duration on disease resistance and immune parameters in rainbow trout. Fish Shellfish Immunol 2009;27:440e5. Salinas I, Cuesta A, Esteban MA, Meseguer J. Dietary administration of Lactobacillus delbrueckii and Bacillus subtilis, single or combined, on gilthead seabream cellular innate immune responses. Fish Shellfish Immunol 2005;19: 67e77. Sun Y-Z, Yang H-L, Ma R-L, Lin W-Y. Probiotic applications of two dominant gut Bacillus strains with antagonistic activity improved the growth performance and immune responses of grouper Epinephelus coioides. Fish Shellfish Immunol 2010;29:803e9. Yin Z, Lam TJ, Sin YM. Cytokine-mediated antimicrobial immune response of catfish, Clarias gariepinus, as a defence against Aeromonas hydrophila. Fish Shellfish Immunol 1997;7:93e104. Villamil L, Tafalla C, Figueras A, Novoa B. Evaluation of immunomodulatory effects of lactic acid bacteria in turbot (Scophthalmus maximus). Clin Diagn Lab Immunol 2002;9:1318e23. Zhao Y, Zhang W, Xu W, Ma K, Zhang Y, Liufu H. Effects of potential probiotic Bacillus subtilis T13 on growth, immunity and disease resistance against Vibrio splendidus infection in juvenile sea cucumber Apostichopus japonicus. Fish Shellfish Immunol 2012;32:750e5. Dalmo RA, Ingebrigtsen K, Bogwald J. Non-specific defence mechanisms in fish, with particular reference to the reticuloendothelial system (RES). J Fish Dis 1997;20:241e73. Newaj-Fyzul A, Adesiyun AA, Mutani A, Ramsubhag A, Brunt J, Austin B. Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Oncorhynchus mykiss, Walbaum). J Appl Microbiol 2007;103:1699e706. Salinas I, Abelli L, Bertoni F, Picchietti S, Roque A, Furones D, et al. Monospecies and multispecies probiotic formulations produce different systemic and local immunostimulatory effects in the gilthead seabream (Sparus aurata L.). Fish Shellfish Immunol 2008;25:114e23. Son VM, Changa CC, Wu MC, Guu YK, Chiu CH, Cheng W. Dietary administration of the probiotic, Lactobacillus plantarum, enhanced the growth, innate immune responses, and disease resistance of the grouper Epinephelus coioides. Fish Shellfish Immunol 2009;26:691e8. Panigrahi A, Kiron V, Kobayashi T, Puangkaew J, Satoh S, Sugita H. Immune responses in rainbow trout Oncorhynchus mykiss induced by a potential probiotic bacteria Lactobacillus rhamnosus JCM 1136. Vet Immunol Immunopathol 2004;102:379e88. Song Z, Wu T, Cai L, Zhang L, Zheng X. Effects of dietary supplementation with Clostridium butyricum on the growth performance and humoral immune response in Miichthys miiuy. J Zhejiang Univ Sci B 2006;7(7):596e602. Taoka Y, Maeda H, Jo JY, Kim SM, Park S, Yoshikawa T, et al. Use of live and dead probiotic cells in tilapia Oreochromis niloticus. Fisher Sci 2006;72:755e 66. Nayak SK, Swain P, Mukherjee SC. Effect of dietary supplementation of probiotic and vitamin C on the immune response of Indian major carp, Labeo rohita (Ham). Fish Shellfish Immunol 2007;23:892e6. Abraham TJ, Babu CHS, Mondal S, Banerjee T. Effects of dietary supplementation of commercial human probiotic and antibiotic on the growth rate and content of intestinal microflora in ornamental fishes. Bangladesh J Fish Res 2007;11:57e63. Kim DH, Austin B. Innate immune responses in rainbow trout (Oncorhynchus mykiss, Walbaum) induced by probiotics. Fish Shellfish Immunol 2006;21: 513e24. Brunt J, Austin B. Use of a probiotic to control lactococcosis and streptococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 2005;28:693e701.
Contents lists available at ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Full length article
Effects of potential probiotic Bacillus amyloliquifaciens FPTB16 on systemic and cutaneous mucosal immune responses and disease resistance of catla (Catla catla) Anushree Das a, Khriezhato Nakhro a, Supratim Chowdhury b, Dibyendu Kamilya a, * a
Department of Fish Health and Environment, College of Fisheries, Central Agricultural University, Lembucherra, Post Box No. 60, Agartala 799 001, Tripura, India Department of Fish Processing Technology, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, 5 e Budherhat Road, Chakgaria, P.O. e Panchasayar, Kolkata 700 094, West Bengal, India b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 June 2013 Received in revised form 12 August 2013 Accepted 27 August 2013 Available online 4 September 2013
Effects of dietary administration of Bacillus amyloliquefaciens FPTB16 on systemic and mucosal immunity and disease resistance of catla (Catla catla) against Edwardsiella tarda infection were evaluated in the present study. The laboratory maintained B. amyloliquefaciens was used to study antagonistic activity against fish pathogenic bacteria by agar well diffusion assay. Healthy catla were challenged by this bacterium for determination of its safety. For preparation of probiotic supplemented diet, the bacteria were added to the basal diet (control) at three different inclusion levels i.e., 1 109, 1 108 and 1 107 CFU/g diet. Fish (weight 25e30 g) were fed with these diets and various immune parameters and disease resistance study were conducted at 4 weeks and 8 weeks post-feeding. The bacterial antagonism study showed inhibition zone against E. tarda, Aeromonas hydrophila, Vibrio parahaemolyticus and V. harveyi. B. amyloliquefaciens was harmless to catla as neither mortalities nor morbidities were observed after the challenge. Study of different systemic and mucosal immunological parameters viz. superoxide anion production and nitric oxide production, myeloperoxidase content, lysozyme activity and total protein content showed significant enhancement (p < 0.05) in fish fed with 108 and 109 CFU/g B. amyloliquefaciens at both time points with the highest values observed in case of 109 CFU/g. For fish fed with 107 CFU/g B. amyloliquefaciens, all the parameters showed significant enhancement (p < 0.05) at both time points except the lysozyme activity of serum at 8 weeks. Diet containing 108 and 109 CFU/g B. amyloliquefaciens significantly enhanced (p < 0.05) the resistance of catla against bacterial challenge at both time points. These results collectively suggest that B. amyloliquefaciens is a potential probiotic species and can be used in aquaculture to improve health status and disease resistance with an optimal dietary supplementation of 109 CFU/g. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Bacillus amyloliquefaciens FPTB16 Catla (Catla catla) Probiotic Immune response Disease resistance
1. Introduction With the ever increasing demand for fish and aquatic food products, there has been a shift in aquaculture practices, moving from extensive systems towards the semi-intensive and intensive systems. With the increasing intensification and commercialization of aquaculture production, disease has become a major stumbling block in the fish farming industry [1]. The widespread use of broad-
* Corresponding author. Tel.: þ91 381 2865 264, þ91 381 2865 513; fax: þ91 381 2865 291, þ91 381 2917 048. E-mail addresses: [email protected], dibyendu_kamilya@ yahoo.co.in (D. Kamilya). 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.08.022
spectrum chemotherapeutics to combat such health related problems has led to the development and spread of drug resistant pathogens, environmental hazards and food security problems [2]. Use of probiotics has been gaining importance for diseases prevention in aquaculture as an eco-friendly alternative to antibiotics and other drugs [3,4]. Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host [5]. Probiotics exert beneficial effects on the host by providing nutrients and enzymatic contribution to digestion, improving water quality, enhancing growth, inhibiting pathogenic microorganisms and enhancing immune responses [6,7]. Apart from lactic acid bacteria, the most commonly used probiotics in fish farming industry belong to gram positive spore
1548
A. Das et al. / Fish & Shellfish Immunology 35 (2013) 1547e1553
forming Bacillus spp [3]. Bacillus preparations are resistant to the environment, have a long lasting shelf life and the beneficial roles of these bacterial species in the aquaculture field are well established [3,8]. Within the Bacillus genus, Bacillus amyloliquefaciens is a closely related species to Bacillus subtilis with potent biocontrol ability against a number of plant and post-harvest pathogens [9,10]. However, no information is available about B. amyloliquefaciens as a biocontrol agent for aquatic pathogens except the recent report where the potential antagonistic activity of B. amyloliquefaciens against eel-pathogenic Aeromonas hydrophila has been demonstrated [11]. In another study, the bacterium has also been found to improve the survival of Litopenaeus vannamei larvae with antagonistic activity against different Vibio spp [12]. However, to the best of our knowledge, no reports are available to describe its potential probiotic nature in finfish. Thus, the aim of the present study was to evaluate the efficiency of B. amyloliquefaciens to act as a potential probiotic, especially in terms of improving immunity and disease resistance in one of the commercially important Indian major carp species, catla (Catla catla).
2. Materials and methods 2.1. Experimental fish and rearing Healthy catla (weight 25e30 g) were collected from a local fish farmer. After collection they were brought immediately and acclimatized in 1000 L fibre-reinforced plastic (FRP) tanks. The fish were acclimatized at ambient temperature (26 1 C) with aeration and were fed twice daily with a basal pelleted diet @3% of body weight. The physicochemical parameters of water were maintained throughout the period of the experiment. Water exchange (upto 50%) was done to remove the waste feed and fecal matter, as and when required. 2.2. Bacteria B. amyloliquefaciens FPTB16 (GenBank Accession: KF319058), isolated from an indigenous fermented fish product ‘Shidal’, was used in all the experiments. This bacterium was obtained from the Department of Fish Processing Technology, West Bengal University of Animal and Fishery Sciences. The Edwardsiella tarda strain (ET-PG-29), known to be pathogenic to catla [13] is available in our laboratory. Other indicator strains such as A. hydrophila, Vibrio harveyi and V. parahaemolyticus were also obtained from Department of Fish Processing Technology, West Bengal University of Animal and Fishery Sciences. The bacteria were maintained in the laboratory by subculturing in nutrient agar (NA).
2.4. Safety of the probiotic strain The safety of B. amyloliquefaciens was evaluated by using a challenge study. Ninety healthy catla were distributed in six tanks equally (15 fish per tank) and were acclimatized for one week. The bacteria were grown in nutrient broth for 48 h at 30 C and the bacterial pellets were obtained by centrifugation at 3000 rpm for 30 min. Bacterial cells were washed twice with sterile saline solution (0.85% NaCl) and then resuspended in the same solution to obtain a bacterial suspension with the concentration of 109 CFU/ml. The fish from three tanks were intraperitoneally (i.p.) injected with 0.2 ml of B. amyloliquefaciens fresh culture suspension containing 109 bacteria ml1. Rest three tanks served as a control and i.p. injected with 0.2 ml sterile saline. All groups were fed on basal diet and kept under observation for 15 days and the mortality and morbidity were recorded. 2.5. Feed preparation and determination of the survival of the probiotics bacteria in feed The basal pelleted diet was prepared from locally available ingredients. Proximate analysis of the basal diet revealed 21.08% crude protein, 6.14% crude lipid, and 13.48% ash, 6.11% moisture, 14.28% crude fibre and 38.91% nitrogen free extract (digestible carbohydrate). The basal diet was used as control diet. Three probiotic supplemented diets, designated as D1, D2 and D3, were prepared with three levels of inclusion of B. amyloliquefaciens, i.e. 1 107, 1 108 and 1 109 CFU/g diet, respectively. The survival of the supplemented bacteria in the diet was assessed following storage at 4 C and at room temperature (26 C) on weekly basis for four weeks [15]. One gram of the diet was homogenized in 9.0 ml sterile saline solution, and serial dilutions down to 104 were prepared and 0.1 ml was spread onto triplicate plates of nutrient agar. The colonies were counted after incubation for 24 h at 30 C. Based on the survivability data feeds were prepared on weekly basis to ensure high probiotic levels in the diet. 2.6. Feeding schedule Three hundred catla fingerlings without the sign of any disease (gross and microscopic examination of different tissues from representative samples) and with no previous history of infection were divided into four equal groups. The groups were subdivided into 3 equal subgroups of 25 fish in each tank to determine the probiotic effect. The basal diet was fed to all fish during the acclimatization period. The fish were provided with aeration and water was renewed daily. Three groups of fish were fed with three probiotic supplemented diets (107, 108 and 109 CFU/g diet). The 4th group was given basal diet without probiotics (control). The fish were fed twice daily (at 9:00 h and 17:00 h) @3% of the body weight for 8 weeks. All the diets were prepared once a week and stored at 4 C.
2.3. Antagonism study Agar well diffusion test was conducted to study antagonistic activity of B. amyloliquefaciens following a previously described method [14] with some modification. The antagonistic activity was examined against indicator pathogenic bacteria like E. tarda, A. hydrophila, V. harveyi and V. parahaemolyticus. Briefly, a culture of the indicator bacteria was independently spread on nutrient agar plates, and then wells of 4e5 mm diameter were made on solidified agar. The base of the wells was sealed with one drop of molten agar. Wells were then filled with 100 ml of overnight bacterial culture. The plates were incubated at 30 C and zones of inhibition around the wells were observed and recorded after 24e48 h.
2.7. Measurement of systemic and cutaneous mucosal immune responses Different systemic and cutaneous mucosal immune responses were measured separately for probiotic treated and control fish after 4 weeks and 8 weeks of feeding. 2.7.1. Sampling, serum collection, mucus collection and isolation of head kidney (HK) leucocytes At each time point, 3 fish were randomly sampled from each tank, thus a total of 9 fish were sampled for each treatment. Before the separation of the HK, anaesthetized fish (using clove oil
A. Das et al. / Fish & Shellfish Immunology 35 (2013) 1547e1553
@100 ml L1) were bled directly from the heart/caudal vein and allowed to clot at room temperature. Serum was collected from the clotted blood and stored at 20 C in sterilized vial until further use. Mucus was sampled by gentle scraping from the fish body. An equal volume of phosphate buffered saline (PBS, pH 7.2) containing 0$02% azide was added to collected mucus, vortexed for 1 min and stored at 4 C overnight. Mucus was then centrifuged at 4000 rpm for 10 min and supernatant collected. The processed mucus samples were stored at 20 C until further use. Isolation of HK leukocytes was done following the method described previously [16]. Following blood collection, HK was removed aseptically from the treatment and control fish and the cell suspension was obtained in complete RPMI-1640 medium. The cell suspension was washed by the centrifugation for 10 min at 1000 rpm and the pellet was resuspended in RPMI. It was then carefully layered on the top of the lymphocyte isolation medium, HiSep (Hi-media) and centrifuged at 1500 rpm for 30 min. Cells at the medium-Hisep interface were transferred into the clean tubes and washed twice by the centrifugation at 1000 rpm for 10 min. Purified leucocytes were counted using a haemocytometer and the cell viability was determined by the trypan blue exclusion test.
1549
control tanks and distributed into another 12 tanks for challenge study. Challenge was performed by injecting each fish intramuscularly with 0.2 ml of E. tarda (1 107 CFU ml1). The same challenge test was repeated 8 weeks later using same number of fish. The challenged fish were kept under observation for 15 days and the dead fish were removed from the tank. The cause of death and pathological changes were verified by re-isolation of bacteria from dead/infected samples. The mortalities were recorded and the survival percentage was determined. 2.9. Statistical analysis Statistical analysis of data was performed using SPSS-15.0 for windows software (SPSS Inc., Chicago, IL, USA). Results are presented as mean standard deviation (SD). Data were analyzed by the one-way analysis of variance (ANOVA) and Tukey’s range test. Probability levels of 0.05 were used to find out the significance in all cases. 3. Results 3.1. Antagonism study and safety of B. amyloliquefaciens
2.7.2. Assessment of systemic and mucosal immunological parameters Production of intracellular superoxide anion by the HK leukocytes was evaluated by nitroblue tetrazolium reduction assay [16]. Production of nitric oxide (NO) was assessed by measuring absorbance of nitrite from the leukocyte culture supernatant using Griess reaction [17]. The total myeloperoxidase content of serum and mucus was determined as described by Mohanty and Sahoo [18]. The lyzozyme activity of serum and mucus was measured following a turbidimetric method, modified to a microtitre plate assay [19]. Lysozyme activity was expressed as units ml1 where one unit was defined as the reduction in absorbance of 0.001 min1. The protein content of serum and mucus was estimated following the biuret method [20].
The in-vitro antimicrobial assay by agar well diffusion technique showed that the B. amyloliquefaciens induced an inhibition zone against E. tarda, A. hydrophila, V. harveyi and V. parahaemolyticus of 8.9 mm, 9.1 mm, 9.3 mm and 6.6 mm, respectively. The intraperitoneal injection of B. amyloliquefaciens was harmless to the catla as neither mortalities nor morbidities were observed during the 15 days of post-challenge observation. 3.2. Survivability of the probiotics bacteria in feed The storage of feed supplemented by B. amyloliquefaciens showed an average 44.11% reduction in viable cells when stored at room temperature (28 C) for 4 weeks. However, the average reduction was 18.61% when feed was stored at 4 C for 4 weeks.
2.8. Challenge test
3.3. Immune responses
Four weeks after the start of the feeding experiment, 10 fish were collected from each of the probiotic supplemented and
3.3.1. Superoxide anion (O2) production The superoxide anion production increased significantly (p < 0.05) in all the diet groups when compared with that of the control (Fig. 1). These increments were evident for all the groups after 4 weeks and 8 weeks of probiotic feeding. The maximum values were obtained for the highest level of probiotic inclusion to the diet (D3, 1 109 CFU/g diet), whereas the lowest values were obtained for lowest probiotic dose (D1, 1 107 CFU/g diet). The values were higher for all the dietary groups after 8 weeks of feeding than the 4 weeks of feeding.
Fig. 1. Superoxide anion production by catla head kidney leucocytes. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
3.3.2. NO production The NO production showed similar trend with that of the superoxide anion production. The increments were statistically significant (p < 0.05) for all the probiotic supplemented groups after 4 weeks and 8 weeks of feeding (Fig. 2). The maximum values were obtained in the highest level of probiotic inclusion to the diet (D3, 109 CFU/g diet), whereas the lowest values were obtained for lowest probiotic dose (D1, 107 CFU/g diet). Similar to superoxide anion production the values were higher for all the dietary groups after 8 weeks of feeding than the 4 weeks of feeding. 3.3.3. Myeloperoxidase content of serum and mucus The total myeloperoxidase content of both serum and mucus increased significantly (p < 0.05) in all the dietary groups. The
1550
A. Das et al. / Fish & Shellfish Immunology 35 (2013) 1547e1553
Fig. 2. Nitric oxide production by catla head kidney leucocytes. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
myeloperoxidase content of serum was higher in the group D3 (109 CFU/g diet) after 4 weeks of feeding than the 8 weeks of feeding (Fig. 3). In contrary, the myeloperoxidase content of mucus for all the dietary treatment groups was higher after 8 weeks of feeding than the 4 weeks of feeding (Fig. 4). At both point of time, an increase in serum and mucosal myeloperoxidase content was observed with the increasing supplementation level of B. amyloliquefaciens. 3.3.4. Lysozyme activity of serum and mucus The lysozyme activity of catla serum increased significantly (p < 0.05) after 4 weeks of feeding with different probiotic supplemented diets when compared to that of the control (Fig. 5). Statistically significant increase (p < 0.05) of serum lysozyme activity was also observed after 8 weeks of feeding with 108 and 109 CFU/g B. amyloliquefaciens. However, the increment was not statistically significant (p > 0.05) for the dietary group D1 (107 CFU/ g diet). The lysozyme activity of catla mucus increased significantly (p < 0.05) after 4 and 8 weeks of probiotic feeding for all the treatment groups (Fig. 6). The values of lysozyme activity of mucus were higher for all the dietary groups after 8 weeks of feeding than the 4 weeks of feeding.
Fig. 3. Myeloperoxidase activity of catla serum. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
fed control diet exhibited lowest survival rate (i.e. 20%) after 8 weeks of feeding.
4. Discussion Use of probiotics in aquaculture is now increasingly being considered as an eco-friendly approach to mitigate health related problems. Disease preventing abilities of probiotics are achieved through enhancement of immunity and exclusion of pathogens. Among probiotics, Bacillus strains are gaining more and more importance and are widely used in aquaculture. The present study describes the effects of a little explored potential probiont B. amyloliquefaciens in ameliorating the health of catla in an 8weeks feeding trial. A common way to select probiotics is to perform in vitro antagonism tests, in which pathogens are exposed to the candidate probiotics or their extracellular products [21]. In the present work B. amyloliquefaciens showed antagonistic activity against the pathogenic indicator bacteria like E. tarda, A. hydrophila, V. harveyi and V. parahemolyticus The inhibitory activity of
3.3.5. Total protein content of serum and mucus After both 4 and 8 weeks of feeding, the total protein content of serum and mucus from 3 treatment groups were significantly higher (p < 0.05) than that of the control (Figs. 7 and 8). Fish fed with 109 CFU/g B. amyloliquefaciens showed the highest level of protein content of serum and mucus at both the time points. The protein contents of serum from all the dietary groups were always higher than that of mucus at all the time points.
3.4. Challenge test Diet containing 108 and 109 CFU/g B. amyloliquefaciens significantly enhanced (p < 0.05) the resistance of catla against E. tarda challenge at both time points (Fig. 9). However, diet containing 107 CFU/g B. amyloliquefaciens increased the survival percentage of catla than that of the control, but it was not statistically significant (p > 0.05). The highest post-challenge survival rate (p < 0.05) was observed in fish group fed diet containing 109 CFU/g B. amyloliquefaciens (i.e. 76.67%) after 8 weeks of feeding. The fish
Fig. 4. Myeloperoxidase activity of catla mucus. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
A. Das et al. / Fish & Shellfish Immunology 35 (2013) 1547e1553
Fig. 5. Lysozyme activity of catla serum. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
1551
Fig. 7. Total protein content of catla serum. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
B. amyloliquefaciens has also been demonstrated against A. hydrophila [11] and against V. harveyi and V. parahemolyticus [12]. The result indicated that the probiont has potential antagonistic activity against a diverse group of pathogenic bacteria. The inhibitory activity of B. amyloliquefaciens may be attributed to its ability to produce a vast array of secondary metabolites with antimicrobial activity [22]. Another criterion to select probiotics is that the probiont should not be pathogenic to the hosts and this should be confirmed prior to acceptance [21]. The probiont used in the present study was not non-pathogenic in catla as mortalities and/or morbidities were not observed during the 15 days post-challenge. The present finding agreed with Austin et al. [23] and Aly et al. [24] who showed the safety of probiotics via intramuscular and intraperitoneal injection in Atlantic salmon and tilapia, respectively. The storage of the probiotic-supplemented diet, under cold temperature demonstrated the durability of B. amyloliquefaciens in the feed. There were a greater number of viable cells in the probiotic supplemented diet stored in the refrigerator at 4 C than that stored at room temperature (26 C). Such decline in viability of the bacteria is common is probiotic supplemented diet. Therefore, fresh diets were prepared at weekly interval to ensure high probiotic
levels in the diets. Similar way of preparing diet has also been reported from other works [24,25]. Probiotics interact with different immune cells to enhance immune responses. Respiratory burst activity of HK leukocytes is one of the important innate immune parameters which is widely used as an indicator of immunocompetence, especially when provoked by stimulators [26]. Increased superoxide anion production in the present study indicated inducement of one of the innate immunity components following probiotic administration. The respiratory burst activities by fish leukocytes following probiotics treatment are often contradictory. Some studies indicate that probiotics do not have significant impact on this innate defense mechanism of fish [27,28], while several other studies showed significant increase in respiratory burst activity in fish by various probiotics [25,29,30]. Apart from superoxide anion, NO is another effector molecule associated with activated fish granulocytes, and is involved in the immune response [31]. In the present study, the trend of nitric oxide production was similar to that of superoxide production following probiotic supplemented diet administration. However, studies on NO production following probiotic application are
Fig. 6. Lysozyme activity of catla mucus. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
Fig. 8. Total protein content of catla mucus. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
1552
A. Das et al. / Fish & Shellfish Immunology 35 (2013) 1547e1553
Fig. 9. Effects of dietary administration of B. amyloliquefaciens on the post-challenge survival of catla after infection with Edwardsiella tarda. Control: fish fed with basal diet. D1, D2 and D3: fish fed with basal diet supplemented with B. amyloliquefaciens @1 107, 1 108 and 1 109/g diet, respectively. Data are presents as mean SD. Asterisks indicate statistically significant difference (*p < 0.05) when compared with control.
limited. In one study, increased NO production following lactobacillus lactis admiministration was observed in turbot (Scophthalmus maximus) [32]. In another study, sea cucumbers fed with B. subtilis at 109 CFU/g diet also showed significantly higher NO production than the control diet [33]. The probiotic administration enhanced the synthesis of reactive oxygen and nitrogen species in activated leukocytes which were persistent throughout the feeding period. This may be attributed to the weekly addition of young culture of B. amyloliquefaciens to the diets thereby maintaining a high level of probionts in the diet. The myeloperoxidase is an important enzyme that utilizes oxidative radicals to produce hypochlorous acid to kill pathogens. During oxidative respiratory burst, it is mostly released by the azurophilic granules of neutrophils [34]. In the present study myeloperoxidase content of serum as well as mucus was significantly higher after 4 and 8 weeks of feeding with B. amyloliquefaciens supplemented diet. Similar result of elevated myeloperoxidase level in serum was observed for B. subtilis in rainbow trout (Oncorhynchus mykiss) [35], B. subtilis in gilthead seabream (Sparus aurata L.) [36] and Lactobacillus plantarum in grouper (Epinephelus coioides) [37]. However, to our knowledge, no reports are available regarding the myeloperoxidase content of cutaneous mucus of fish fed with probiotic diet. Lysozyme is an important humoral component of the systemic as well as mucosal immune system and act as defensive factor against invasive microorganisms in vertebrates [19]. In the present study, enhanced serum and mucosal lysozyme activity was recorded in all the treatment groups throughout the study period. Different probiotic species have been reported to trigger the lysozyme level in teleosts. Elevated serum lysozyme activity were observed by probiotics in rainbow trout [38], in brown trout, Salmo trutta [14] and in grouper [37]. Apart from serum, probiotics have also been reported to enhance the lysozyme level in skin mucosa of fish [39,40]. After 4 and 8 weeks of feeding, the total protein content of serum and mucus from treatment groups were significantly higher than that of the control. Similar increment of the serum protein content was observed in rohu (Labeo rohita) fed with B. subtilis supplemented diet [41]. The rise in total protein level may be a joint contribution form group of serum and mucosal proteins like
agglutinins, lectins, lysozyme, immunoglobulins etc. which are important defense molecules. Increase in total protein level in both serum and mucus corroborates the fact that these defense molecules were synthesized in ample quantity as a result of the stimulation of the systemic as well as mucosal immunity following dietary administration of B. amyloliquefaciens. In aquaculture, probiotics help in achieving natural resistance and high survivability of larvae and post larvae of fishes [42]. The effectiveness of probiotics in terms of protection against infectious pathogens is often attributed to the elevate immunity. In the present study dietary administration of B. amyloliquefaciens significantly increased the survival rates of catla challenged with Edwardsiella tarda. In a previous study protection was achieved in eel (Anguilla anguilla) against Aeromonas hydrophila infection after the fish were fed with diet supplemented with B. amyloliquefaciens @3 109 CFU/g [11]. Protection against edwardsiellosis [40,41], enteric red mouth disease [43], furunculosis [15], lactococossi and streptococcosis [44] and aeromoniasis [35] have successfully been accomplished through probiotics feeding. The results indicate the involvement of an activated and prolonged immune status of catla to resist E. tarda infection. As a final note, the present study delineates the efficacy of B. amyloliquefaciens as a potential probiotic for use in aquaculture. This probiont with an optimal dietary supplementation of 109 CFU/ g was effective in maintaining an elevated immunity level persistently, thereby conferring resistance against edwardsiellosis. We suggest long term feeding with frequent addition of fresh preparation of probiotics in the diet to maintain the persistent activation of immune cells throughout the feeding period. However, more studies are required to elucidate other mode of action of this probiont, especially its effect on growth, colonization in gut etc. to further characterize its efficacy.
Acknowledgment The authors are thankful to AICRP on Post Harvest Technology (ICAR), Kolkata Centre, Department of Fish Processing Technology, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, India for providing the probiotic strain. The authors are also thankful to Vice Chancellor, Central Agricultural University, Imphal, India and Dean, College of Fisheries, CAU, Lembucherra, Tripura, India for providing necessary research facilities.
References [1] Bondad-Reantaso MG, Subasinghe RP, Arthur JR, Ogawa K, Chinabut S, Adlard R, et al. Disease and health management in Asian aquaculture. Vet Parasitol 2005;132:249e72. [2] Ai Q, Xu H, Mai K, Xu W, Wang J, Zhang W. Effects of dietary supplementation of Bacillus subtilis and fructooligosaccharide on the growth performance, survival, non-specific immune response and disease resistance of juvenile large yellow croaker Larimichthys crocea. Aquaculture 2011;317:155e61. [3] Wang YB, Li JR, Lin J. Probiotics in aquaculture: challenges and outlook. Aquaculture 2008;281:1e4. [4] Nayak SK. Probiotics and immunity: a fish perspective. Fish Shellfish Immunol 2010;29:2e14. [5] Food and Agriculture Organization of the United Nations (FAO). Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. In: The joint FAO/WHO expert consultation report on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria (October 2001) 2001. [6] Verschuere L, Rombaut G, Sorgeloos P, Verstraete W. Probiotic bacteria as biological control agents in aquaculture. Microbiol Mol Biol Rev 2000;64:655e 71. [7] Balcazar JL, de Blas I, Ruiz-Zarzuela I, Cunningham D, Vendrell D, Muzquiz JL. The role of probiotics in aquaculture. Vet Microbiol 2006;114:173e86. [8] Gatesoupe FJ. The use of probiotics in aquaculture. Aquaculture 1999;180: 147e65.
A. Das et al. / Fish & Shellfish Immunology 35 (2013) 1547e1553 [9] Arguelles-Arias A, Ongena M, Halimi B, Lara Y, Brans A, Joris B, et al. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microb Cell Fact 2009;8:63. [10] Zhao P, Quan C, Wang Y, Wang J, Fan S. Bacillus amyloliquefaciens Q-426 as a potential biocontrol agent against Fusarium oxysporum f. sp. spinaciae. J Basic Microbiol 2013. http://dx.doi.org/10.1002/jobm.201200414 [in press]. [11] Cao H, He S, Wei R, Diong M, Lu L. Bacillus amyloliquefaciens G1: a potential antagonistic bacterium against eel-pathogenic Aeromonas hydrophila. Evid Based Complement Alternat Med 2011:7. http://dx.doi.org/10.1155/2011/ 824104. Article ID 824104. [12] Luis-Villaseñor IE, Macías-Rodríguez ME, Gómez-Gil B, Ascencio-Valle F, Campa-Córdova AI. Beneficial effects of four Bacillus strains on the larval cultivation of Litopenaeus vannamei. Aquaculture 2011;321:136e44. [13] Devi TB, Kamilya D, Abraham TJ. Dynamic changes in immune-effector activities of Indian major carp, catla (Catla catla) infected with Edwardsiella tarda. Aquaculture 2012;366e367:62e6. [14] Balcazar JL, Rojas-Luna T, Cunningham DP. Effect of the addition of four potential probiotic strains on the survival of Pacific white shrimp (Litopenaeus vanamei) following immersion challenge with Vibrio parahaemolyticus. J Invertebr Pathol 2007;96:147e50. [15] Irianto A, Austin B. Use of probiotics to control furunculosis in rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 2002;25:333e42. [16] Kamilya D, Ghosh D, Bandyopadhyay S, Mal BC, Maiti TK. In vitro effects of bovine lactoferrin, mushroom glucan and Abrus agglutinin on Indian major carp, catla (Catla catla) head kidney leukocytes. Aquaculture 2006;253:130e9. [17] Green IC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum S. Analysis of nitrate, nitrite and (15N) nitrate in biological fluids. Anal Biochem 1982;126:131e8. [18] Mohanty BR, Sahoo PK. Immune responses and expression profiles of some immune-related genes in Indian major carp, Labeo rohita to Edwardsiella tarda infection. Fish Shellfish Immunol 2010;28:613e21. [19] Ellis AE. Lysozyme assay. In: Stolen JS, Fletcher EC, Aderson DP, Robetrson DS, van Muiswinkel WB, editors. Techniques in fish immunology. USA: SOS Publications; 1990. p. 101e3. [20] Gornall AC, Bardawill CJ, David MM. Determination of serum proteins by means of the biuret reaction. J Biol Chem 1949;177:751e67. [21] Sahu MK, Swarnakumar NS, Sivakumar K, Thangaradjou T, Kannan L. Probiotics in aquaculture: importance and future perspectives. Indian J Microbiol 2008;48:299e308. [22] Chen XH, Koumoutsi A, Scholz R, Schneider K, Vater J, Süssmuth R, et al. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. J Biotechnol 2009;140:27e37. [23] Austin B, Stuckey LF, Robertson PAW, Effendi I, Griffith DRW. A probiotic strain of Vibrio alginolyticus effective in reducing diseases caused by Aeromonas salmonicida, Vibrio anguillarum and Vibrio ordalii. J Fish Dis 1995;18: 93e6. [24] Aly SM, Ahmed YA, Ghareeb AA, Mohamed MF. Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics, on the immune response and resistance of Tilapia nilotica (Oreochromis niloticus) to challenge infections. Fish Shellfish Immunol 2008;25:128e36. [25] Giri SS, Sukumaran V, Oviya M. Potential probiotic Lactobacillus plantarum VSG3 improves the growth, immunity, and disease resistance of tropical freshwater fish, Labeo rohita. Fish Shellfish Immunol 2013;34:660e6. [26] Secombes CJ. Isolation of salmonid macrophages and analysis of their killing activity. In: Stolen JS, Fletcher TC, Anderson DP, Robertson BS, van Muiswinkel WB, editors. Techniques in fish immunology, vol. I. Fair Haven: SOS Publications; 1990. p. 137e54. [27] Diaz-Rosales P, Arijo S, Chabrillon M, Alarcon FJ, Tapia-Paniagua ST, MartinezManzanares E, et al. Effects of two closely related probiotics on respiratory burst activity of Senegalese sole (Solea senegalensis, Kaup) phagocytes, and
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
1553
protection against Photobacterium damselae subsp. piscicida. Aquaculture 2009;293:16e21. Sharifuzzaman SM, Austin B. Influence of probiotic feeding duration on disease resistance and immune parameters in rainbow trout. Fish Shellfish Immunol 2009;27:440e5. Salinas I, Cuesta A, Esteban MA, Meseguer J. Dietary administration of Lactobacillus delbrueckii and Bacillus subtilis, single or combined, on gilthead seabream cellular innate immune responses. Fish Shellfish Immunol 2005;19: 67e77. Sun Y-Z, Yang H-L, Ma R-L, Lin W-Y. Probiotic applications of two dominant gut Bacillus strains with antagonistic activity improved the growth performance and immune responses of grouper Epinephelus coioides. Fish Shellfish Immunol 2010;29:803e9. Yin Z, Lam TJ, Sin YM. Cytokine-mediated antimicrobial immune response of catfish, Clarias gariepinus, as a defence against Aeromonas hydrophila. Fish Shellfish Immunol 1997;7:93e104. Villamil L, Tafalla C, Figueras A, Novoa B. Evaluation of immunomodulatory effects of lactic acid bacteria in turbot (Scophthalmus maximus). Clin Diagn Lab Immunol 2002;9:1318e23. Zhao Y, Zhang W, Xu W, Ma K, Zhang Y, Liufu H. Effects of potential probiotic Bacillus subtilis T13 on growth, immunity and disease resistance against Vibrio splendidus infection in juvenile sea cucumber Apostichopus japonicus. Fish Shellfish Immunol 2012;32:750e5. Dalmo RA, Ingebrigtsen K, Bogwald J. Non-specific defence mechanisms in fish, with particular reference to the reticuloendothelial system (RES). J Fish Dis 1997;20:241e73. Newaj-Fyzul A, Adesiyun AA, Mutani A, Ramsubhag A, Brunt J, Austin B. Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Oncorhynchus mykiss, Walbaum). J Appl Microbiol 2007;103:1699e706. Salinas I, Abelli L, Bertoni F, Picchietti S, Roque A, Furones D, et al. Monospecies and multispecies probiotic formulations produce different systemic and local immunostimulatory effects in the gilthead seabream (Sparus aurata L.). Fish Shellfish Immunol 2008;25:114e23. Son VM, Changa CC, Wu MC, Guu YK, Chiu CH, Cheng W. Dietary administration of the probiotic, Lactobacillus plantarum, enhanced the growth, innate immune responses, and disease resistance of the grouper Epinephelus coioides. Fish Shellfish Immunol 2009;26:691e8. Panigrahi A, Kiron V, Kobayashi T, Puangkaew J, Satoh S, Sugita H. Immune responses in rainbow trout Oncorhynchus mykiss induced by a potential probiotic bacteria Lactobacillus rhamnosus JCM 1136. Vet Immunol Immunopathol 2004;102:379e88. Song Z, Wu T, Cai L, Zhang L, Zheng X. Effects of dietary supplementation with Clostridium butyricum on the growth performance and humoral immune response in Miichthys miiuy. J Zhejiang Univ Sci B 2006;7(7):596e602. Taoka Y, Maeda H, Jo JY, Kim SM, Park S, Yoshikawa T, et al. Use of live and dead probiotic cells in tilapia Oreochromis niloticus. Fisher Sci 2006;72:755e 66. Nayak SK, Swain P, Mukherjee SC. Effect of dietary supplementation of probiotic and vitamin C on the immune response of Indian major carp, Labeo rohita (Ham). Fish Shellfish Immunol 2007;23:892e6. Abraham TJ, Babu CHS, Mondal S, Banerjee T. Effects of dietary supplementation of commercial human probiotic and antibiotic on the growth rate and content of intestinal microflora in ornamental fishes. Bangladesh J Fish Res 2007;11:57e63. Kim DH, Austin B. Innate immune responses in rainbow trout (Oncorhynchus mykiss, Walbaum) induced by probiotics. Fish Shellfish Immunol 2006;21: 513e24. Brunt J, Austin B. Use of a probiotic to control lactococcosis and streptococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 2005;28:693e701.