Wednesday, April 24, 2019

Dietary Administered Bacillus sp. PP9 Enhances Growth, Nutrition and Immunity in Cirrhinus mrigala (Hamilton)

Partha Bandyopadhyay  Biplab Sarkar  Arabinda Mahanty  Raja M. Rathore  Bidhan Chandra Patra

Received: 29 December 2014 / Revised: 23 April 2015 / Accepted: 20 May 2015 / Published online: 21 June 2015
The National Academy of Sciences, India 2015

Abstract Bacillus sp. PP9, a noble bacterial isolate of mrigal (Cirrhinus mrigala) gut was investigated for its efficacy as a dietary probiotic against mrigal (C. mrigala) through a dose dependent impact assessment. Mrigal fin-gerlings (avg.wt. 2.5 ± 0.20 g) were fed with three dif-ferent doses (2 9 104, 2 9 105 and 2 9 106 CFU) of Bacillus sp. PP9 admixed with 100 g feed for a period of 60 days. It was found that the feed with Bacillus concen-tration of 2 9 104 CFU exhibited significantly higher growth, maximum RNA DNA ratio, lower food conversion ratio in comparison to other two feed types. Enhanced intestinal protease and a-amylase activity followed by maximum hepatic glutamic oxaloacetic transaminase and glutamate pyruvate transaminase levels were also moni-tored in this specific feed type. Important hematological parameters like hemoglobin percentage, total erythrocyte count, total leukocyte count, corpuscular hemoglobin also indicated a healthy trend in this dose. Furthermore, the

1       Aquaculture Research Unit, Department of Zoology, Vidyasagar University, Midnapore 721102, West Bengal, India

2       School of Biotechnology, KIIT University, Bhubaneswar 751024, India

3       Nutrimar As, 7266 Kverva, Norway

4       Central Inland Fisheries Research Institute, Barrackpore, Kolkata 700120, India

5       National Institute of abiotic stress management, Baramati, Pune, Maharashtra, India

6       Present Address: Finray Biotech INC, Vedant Complex, NH NO. 8, Survey NO 552, Bapod, Near Hanumanji Temple, Waghodia Chowkdi, Barada – 390019. India.

same dietary treatment exhibited highest levels of total serum protein, albumin globulin ratio and serum bacteri-cidal activity. The growth, nutrition and immunological parameters showed a declining trend with increase in the bacterial concentration as well as in the control group (without probiotic). In summary, dietary supplement of 2 9 104/100 g appears as a potential probiotic dose of Bacillus sp. PP9 for growth, nutritional efficacy as well as immunological modulation of C. mrigala and denotes a recommended concentration for its future field applications.

Keywords Bacillus sp PP 9 Feed formulations Cirrhinus mrigala Probiotics Growth


In recent times, aquaculture appears as one of the fastest growing animal food producing sector and is projected as efficient agricultural subsidiary for its contribution in high revenue generation, rural livelihood and employment cre-ation. But like other farming practices, aquaculture system is also stressed with infections and diseases causing major obstacles to its productivity and sustainability [1]. It is very important to design quality health management systems in fish to improvise disease resilience as well to recover from pathogenic infestation. In recent years, applications of vaccine, antibiotics and probiotics are accepted therapeutic tools to boost fish health. Among such methods, the pro-phylactic use of probiotics has gained more attention [2] due to its easy availability and handling, wide applicability as well as ecological viability. One obvious reason for selecting probiotics is its unique applicability at larval as well as early fry and fingerlings stages where vaccines

cannot be delivered. At these stages, mortality is high due to proliferation of opportunistic pathogens even at low initial infection pressure [3]. Application of antibiotics has been a matter of concern in contemporary times, due to emergence of resistant varieties, environmental issues and export related regulations. In such cases, alternative strategies like application of probiotics and other nutri-tional interventions are very useful and being applied [4].

In last decade, an upsurge in probiotics research has been observed and numerous trials have been conducted with its supplements to improve health status and produc-tivity of farmed animals. Appropriate probiotics applica-tions have shown an improvement in gut microbial balance leading to enhanced food absorption [5] and reduced pathogenic problems in gastrointestinal tract [6]. Though multiple probiotics species have been screened and evalu-ated including Lactobacillus sp., Bacillus sp. and various mixed cultures [4, 79] specially in economically impor-tant terrestrial animals, yet reports on commercial appli-cation in aquaculture varieties are less. Among them, Bacillus possesses important properties like adhesion, immune-stimulation and bacteriocin secretion etc. as a potential probiotic candidate [8]. However, endeavors to search for new Bacillus strains with improved performance are still continued. Bacillus sp. PP9 was isolated and screened from the gut of Cirrihinus mrigala and thereafter, identified through molecular biology tools as a new pro-biotics species [8]. The present study was aimed to eval-uate the efficacy and performance of Bacillus sp.PP9 strain on growth, nutrition and immunity of C. mrigala finger-lings, as a noble probiotic candidate.

Material and Mmethods

Fish Sample Collection

Cirrihinus mrigala fingerlings were collected from Back-ery Aqua Farm, a carp culture centre located near Midna-pore city, West Bengal (22L250 N and 87L200 E), India. Fishes (Weight 2.5 ± 0.020 g; Length 7.1 ± 0.2 cm) were released into continuous flow through aquaria (200 l capacity) and were acclimatized for 15 days in laboratory conditions. Before acclimatization, fish were dipped in 0.9 % potassium permanganate to dispel any external infections.

Preparation of Experimental Feeds

Fish feeds were prepared using locally available ingredi-ents including mustard oilcake (40 %), fish meal (40 %), rice polish (13 %), tropica flour (5 %), vitamin and mineral mixture (1 %) and cod liver oil (1 %). Feed were

formulated by ‘‘square-method’’ applying determined pro-tein values of the ingredients. Dough was prepared with careful blending of ingredients and the feeds were pelleted separately with ‘hand pelletizer’. The pellets were dried in an oven at 38 LC to less than 10 % moisture and stored in airtight containers at ambient temperature.

Preparation of Experimental Diet

Probiotic bacterium Bacillus sp. PP 9, an isolate of C. mrigala gut, was incubated in nutrient broth at 31 LC for

48   h. The bacterial cultures were suspended in sterile saline water after washing with 1 % sodium chloride [4]. Experimental feed were prepared by absorbing suspension of the mentioned probiotic strain. The suspended bacterial

cultures were sprayed over the earlier prepared feeds in the concentration of 2 9 104 (M1), 2 9 105 (M2) and 2 9 106

(M3) Bacillus sp. CFUs per 100 g feed excluding the control feed (MC). The feeds were kept at 4 LC inside vacuumed plastic containers and the bacterial concentra-tion were monitored at a weekly interval.

Design of Experimental Trial

Three aquaria (100 L capacity) were allotted for each treatments as well as control group with 30 L water and constant aeration facility. Fifteen fishes were kept in each aquarium. The physico-chemical properties of water were monitored weekly as per recommended protocol of APHA (2012) [10]. Fishes were fed two times a day (morning and evening) at 4 % body weight and the leftover feeds were siphoned out after 1 h of dispension into the aquarium. Fifty percent of the used water was exchanged regularly with fresh water. Experiments were conducted at room temperature for 60 days regime. The net weight was measured at every 15 days and feed quantity was read-justed. Evaluation of the dietary performances were con-ducted through estimation of nutritional indices like live weight gain (LWG), average daily growth (ADG), feed conversion ratio (FCR), specific growth rate (SGR) and protein efficiency ratio (PER) as per standard protocols [4]. At the end of experiment, three fishes from the each treatment and control group were randomly selected and sacrificed with the application of high dose anesthesia (MS222; Sigma Chemicals, India) and different tissue parts (liver, intestine etc.) were collected for further assessment.

Proximate Analysis

Proximate analyses of feed ingredients and fecal matter were performed according to the method of AOAC [11]. Crude protein and fat contents were estimated by micro-Kjeldahl and soxhlet methods respectively. Crude fiber content of the

defated samples were determined following the protocol of Patra et al. [12]. Ash content was determined by incinerating the samples in muffle furnace (at 500 ± 50 LC for 10 h). For fecal matter estimation, pooled fecal matter was collected into petri dishes from aquarium bed by a pipette. The col-lected material was dried in oven at 55 LC and stored in air-tight containers until further analysis.

Biochemical Analysis

After 60 days of trial period, DNA and RNA contents in the liver were estimated following the method of Munro and Fleck [13]. The intestinal protease and a-amylase activities were determined by the standard method prescribed by Bernfeld [14]. Briefly, the a- amylase activity was deter-mined in triplicate by using 1 % soluble starch, as substrate, with 3,5-dinitrosalicyclic acid (DNS) at 550 nm. Specific a-amylase activity is reported as U = lg of maltose min-1 - mg-1 of protein present in the enzyme extract tested at 37 LC. Liver glutamic oxaloacetic transaminase (GOT) and gluta-mate pyruvate transaminase (GPT) activities were deter-mined following the method of Bergmeyer and Bernt [15].

Hematological Assays

At the end of 60 days trial, blood samples were collected through cardiac puncture before sacrificing the fishes for other assays and collected into heparinised vials. Hema-tological parameters were estimated according to the method of Decie and Lewis [16]. The total serum protein (TSP) and albumin contents were estimated following the method of Lavanya et al. [17]. The globulin content was calculated as the difference between the total proteins and the albumins.

Immunological Assays

At the end of feeding trials, fishes from the experimental aquaria of each group were bled to collect serum samples and analyzed for agglutination titre using microtitre plates [18]. Collected sera were stored at -0.20 LC until further analysis. Serum bactericidal activity was estimated by the prescribed method of Kajita et al. [19].

Above all, major water quality parameters were main-tained in ambient range during the entire experimental period (Temperature-28 ± 3.25 LC; pH-7.48 ± 0.21; Total alkalinity (ppm)-140.25 ± 5.6; Dissolved Oxygen (ppm) -4.6 ± 0.42; Total ammonia (ppm) -0.13 ± 0.02.

Statistical Analysis

Descriptive statistics was employed to analyze the experi-mental data by using the SPSS 17. For evaluating the

significant differences (p \ 0.05) in the treatments of dietary performances, nutritional indices, enzymatic activities, RNA DNA ratio and immunological parameters, Duncan multiple Range Test (DMRT) were performed.

Results and Discussion

In aquaculture, probiotics have captured special attention for its efficacious function for supplementary synthesis of digestive enzymes [20], and improvement in growth per-formance of the farmed species [21, 22]. Bacillus have been widely used as putative probiotics for its beneficial nutritional impact, antagonism against microbial pathogens and easy supplementation [23, 24], as reported for larval development of Gilthead sea bream (Sparus aurata, L.) by Arıg et al. [25]. The purpose of current experiment was to evaluate the efficacy of Bacillus sp. PP9 on growth, nutrition and immunological status in C. mrigala at an effective dose.

Proximate Analysis of Feed and Fecal Matter

The proximate composition of prepared feed, its ingredi-ents and concentration of the probiotics are presented in Fig. 1. All the prepared feed were isocaloric and equivalent in terms of P/E ratio and nitrogen free extract (NFE) content. As shown in Fig. 1a, the proximate composition of ingredients applied in preparing experimental feeds were quite different. The crude protein percentage of rice polish, mustard oil cake, and fish meal was 13.10, 39.20 and 49.16, whereas the crude lipid percentage was 5.22, 11.15, and 6.58 respectively. The percentage of average crude protein on dry matter basis was 36.90 whereas the crude lipid percentage was around 9.82 (Fig. 1b). Bacillus concentra-tion of three different probiotic feed formulations are pre-sented in Fig. 1c.

Proximate analysis of the fecal matter of different treatments and control group showed significant difference in the protein, fat and ash content (Fig. 2). Fecal matter proximate analysis revealed significantly (p B 0.05) higher nitrogen excretion in fish fed with MC (13.15 ± 0.040 %) and least in fish fed with M1 (12.41 ± 0.050 %). The crude lipid content varied between 3.64 ± 0.011 % (M1) and 3.98 ± 0.012 % (MC).

Fish Growth and Nutrition Analysis

In relation to all the feeding regimes, growth of C. mrigala was observed highest (p B 0.05; DMRT) in the M1 group (5.68 ± 0.018 g), while lowest (5.37 ± 0.022 g) in MC. Growth in terms of weight gain percent was significantly higher (127.20 ± 0.532 %) in M1 fed fish and least in MC

(114.80 ± 0.460 %) (Table 1). In addition, highest SGR, PER and lowest FCR were observed in M1 fed C. mrigala, whereas reverse trends were observed in MC fed fishes. M2 and M3 fed groups showed an intermediary trend in per-formance between M1 and MC in a dose dependant manner where M2 performed better than M3 (Table 1).

In the present study, higher growth was noted in C. mrigala fed with M1 as evaluated by weight gain per-centage (WGP), SGR, when compared with other probiotic treatments as well as control. This result focuses on important inference that the probiotic concentration of

2   9 104 per 100 gm feed, might be supportive for optimum dietary utilization due to a positive correlation between the WGP and protease activity. Similar findings were reported earlier by using particular concentration of Bacillus sp. S11

Table 1 Initial and final body weight, live weight gain, weight gain percentage, FCR, SGR and PER of Cirrhinus mrigala after 60 days trial period (values with the same letter are not significantly different among treatments at 0.05 level)

Fish weight (g)

Live wt. gain (g)
Weight gain (%)


2.5 ± 0.020
5.37 ± 0.022
2.87 ± 0.009
114.80 ± 0.460a
1.11 ± 0.005a
1.27 ± 0.006a
1.93 ± 0.008a
2.5 ± 0.015
5.68 ± 0.018
3.18 ± 0.012
127.20 ± 0.532b
1.02 ± 0.004b
2.06 ± 0.010b
2.13 ± 0.008b
2.5 ± 0.012
5.60 ± 0.014
3.10 ± 0.007
124.00 ± 0.520c
1.02 ± 0.005b
1.34 ± 0.006c
1.82 ± 0.007c
2.5 ± 0.018
5.54 ± 0.015
3.04 ± 0.010
121.60 ± 0.348d
1.03 ± 0.004d
1.32 ± 0.006c
1.33 ± 0.006d

 in Penaeus monodon [26]. FCR and PER are reported to be decreased with increasing protein contents [27] and the effects may differ with the species. In the current study, although all the feeds were isonitrogenous but the con-centration of probiotics in M1 feed might have contributed for maximum nutrient utilization. Lesser nitrogenous con-tent in egestion were observed in M1 fed groups which can be attributed to suitable probiotic concentration. This is also obvious from the results that greater nitrogenous egestion was recorded in MC fed fishes that exhibited a comparatively low feed utilization.

Biochemical Analysis

As represented in Fig. 3, maximum RNA DNA ratio (1.38 ± 0.005) was registered in the fish fed with M1 followed by M2, M3 and the lowest (1.16 ± 0.006) in the MC group. RNA DNA ratio is an important indicator of growth [27]. The ratio was highest in the M1 with higher dietary utilization and growth performances. Zehra and Khan [28] reported an enhanced RNA DNA ratio through dietary lysine enrichment in catla (Catla catla) fingerlings. The results of present study are highly corroborative with such findings where supplement of 2 9 104 Bacillus sp.

PP   9 cells 100 g-1 of feed (M1) projected better RNA DNA ratio and growth.

Intestinal protease and a amylase activities were sig-nificantly highest (p B 0.05) in M1 fed group, whereas lowest in MC fed fishes (Fig. 4a, b). Hepatic GPT and GOT levels were observed significantly higher in M1 fed fishes as compared to the group fed with MC (Fig. 4c). Earlier reports revealed the fact that the activity of protease enhances with high dietary protein [29], but in this experiment all feeds were iso-nitrogenous and M1 showed greater protease activity that might be related to greater dietary protein utilization. Intestinal mucosa is one of the origin of carbohydrate digestive enzymes in fish and the amylase activity is correlated with the carbohydrate content

Fig. 3 RNA DNA ratio in fishes fed with different feeds. Different letters (a, b, c, d) above the bars corresponds to statistical difference (p \ 0.05)

of the diet [30]. Hence, M1 fed fishes exhibited greater a amylase activity which indicates greater carbohydrate uti-lization. The present study also showed greater a-amylase activity in all test feeds as compared to initial activity. These results indicate the efficacy of other probiotic con-centrations to utilize the carbohydrate content of the diet. Hepatic GOT and GPT activity in fish also increased in probiotics supplemented diet over the initial level. The highest level of GOT and GPT in M1 group signifies better utilization of dietary protein. Within the protein, most of the amino acids undertake transamination reactions and transaminases are primarily located in both mitochondria and cytosol of a cell are induced by high protein diet [31]. Thus a positive correlation between hepatic GOT, GPT level and dietary protein could be extrapolated. In other way, M2 and M3 fed groups showed a dose dependant declination in DNA:RNA, intestinal protease and a amy-lase activity and hepatic GOT and GPT levels but showed better activity than in control.

Hematological and Immunological Assays

Hematological parameters such as total leucocyte count (TLC), total erythrocyte count (TEC), haemoblobin (Hb), hemotcrit (Hct), mean corpuscular hemoglobin (MCV), mean corpuscular volume (MCH), and mean corpuscular volume hemoglobin (MCHC) of C. mrigala were signifi-cantly high (p B 0.05) in M1 fed fishes in comparison with other treatments (Table 2). Impacts of different feeding trials on albumin globulin ratio was observed maximum at MC fed fishes (1.890 ± 0.008) and minium in M1 (1.545 ± 0.006) (Fig. 5). The impacts of different probi-otics on non-specific and specific immunity were recorded significantly high (p B 0.05; DMRT) for NBT positive values (63.33 ± 0.326), antibody titre (125.76 ± 5.490) and lowest bactericidal activity (7.23 ± 0.010 9 103 cells ml-1) in M1 treated C. mrigala (Fig. 6).

As a liquid connective tissue, blood functions as a patho-physiological indicator and measuring blood parameters are important for diagnosing fish health status. In the current evaluation, blood parameters retained the optimum range in all the doses [32] and M1 fed fishes indicated the best positive impact. Statistically significant increase was recorded in all the parameters of non-specific and specific immunity. Highest NBT positive cells, anti-body titre (p B 0.05; DMRT) and lowest bactericidal activity, albumin globulin ratio was obtained in M1 fed fishes and these phenomenon could have been undertaken due to better lymphocyte proliferation and consequent immunoglobulin synthesis in fish. These results are cor-roborated with the report given by Iwashita et al. [33] in which three candidate probiotic species used were Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus oryzae.

Table 2 Comparative hematological parameters of Cirrhinus mrigala after 60 days of feeding trial (Figures having different letter (super-scripted) in the same row are significantly different at 0.05 level)






TEC (9 106 mm3)
± 0.004a
± 0.002b
± 0.009c
± 0.010d
TLC (9 103 mm3)
± 0.011a
± 0.001b
± 0.006c
± 0.007d
Hb (g %)
± 0.004a
± 0.006b
± 0.002c
± 0.002c
Hct (%)
± 0.023a
± 0.016b
± 0.011c
± 0.009c
MCV (lm3 cell-1)
± 0.163a
± 0.094b
± 0.306c
± 0.247d
MCH (pg cell-1)
± 0.023a
± 0.056b
± 0.073c
± 0.062d
MCHC (g 100 ml Hct-1)
± 0.068a
± 0.022b
± 0.049c
± 0.086d
NBT positive cells (%)
± 0.381a
± 0.326b
± 0.201c
± 0.406d

From multiple correlations, it was also noticed that RNA DNA ratio was negatively correlated (p B 0.01) with albumin globulin ratio and positively correlated (p B 0.05; DMRT) with antibody titre. Results of current investigation suggests that the concentration of probiotic Bacillus sp. PP9 in feed M1 was highly efficacious on improving the overall physiological and immunological status of C. mri-gala fingerlings.

In the current experiment, other two Bacillus concen-trations (M2) and (M3) also exhibited higher trends in growth, nutrition and immunological parameters in respect to control, but their performances were not the best. The dose dependent data obtained from the experiments proved the accuracy of selecting M1 as best probiotic candidate. Similar dose dependant efficacy study were also conducted by Bandyopadhay et al. [34] in applying S. cerevisiae as a feed supplemented probiotics in rohu (Labeo rohita).


The present investigations demonstrated the role of noble bacterium, Bacillus sp. PP9 to promote enhanced growth and immunity in Indian major carp, C. mrigala as a dietary probiotics. Inclusion of Bacillus sp.PP9 at a concentration of (2 9 104/100 g feed) showed highest efficacy. 2 9 104/100 g of Bacillus dose also revealed the critical point of best performance and surpassing this point would initiate inhibition of above mentioned activities in C. mrigala. But further trials should be conducted in pond conditions before its commercialization.

Acknowledgments Authors would like to express their thanks to Indian Council of Agricultural Research (ICAR), New Delhi, India for financial assistance. The authors are also thankful to Head of the Department, Department of Zoology and Vice-Chancellor, Vidyasa-gar University for providing necessary facilities.



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