Potential,probiotic,and,health,fostering,effect,of,host,gut-derived,Enterococcus,faecalis,on,freshwater,prawn,,Macrobrachium,rosenbergii

Shrmin Sultn Khushi, Mohmm Sifuin Sumon, Mirj Kizer Ahmme,b,c,M Nzmul Hsn Zilni, Ftem Ahmme,e, Stephen G. Giteru, M Golm Srower,＊

a Fisheries and Marine Resource Technology Discipline, Khulna University, Khulna, 9208, Bangladesh

b Department of Food Science, University of Otago, Dunedin, 9016, New Zealand

c Department of Fishing and Post-harvest Technology, Faculty of Fisheries, Chittagong Veterinary and Animal Sciences University, Khulshi, 4225, Bangladesh

d Department of Pharmacy, Jashore University of Science and Technology, Jashore, 7408, Bangladesh

e Department of Chemistry, University of Otago, Dunedin, 9016, New Zealand

f Food & Bio-based Products Group, AgResearch Limited, Palmerston North, 4442, New Zealand

Keywords:Antagonistic effect Biochemical PCR Digestive enzyme activity Immunity Feed

ABSTRACT The present study investigated the potential probiotic effect of Enterococcus faecalis against a pathogenic microorganism, Vibrio harveyi occurring in the giant freshwater prawn (Macrobrachium rosenbergii). V. harveyi was isolated from the intestine of M. rosenbergii through biochemical tests and PCR-based molecular assays. An in-vitro challenge was conducted by mixing isolated probiotic (5.12 log CFU/g) and V. harveyi (3.09 log CFU/g).Feeds incorporated with E. faecalis (9.02 log CFU/g) were applied to the treatment group during in vivo test in the aquarium and earthen pond (70 m2, 1 m depth). Pathogenic bacteria were found to be reduced after 8 h (from 5.02 to 3.62 log CFU/g) and 60 days (from 5.44 to 2.15 log CFU/g) of probiotic administration in the in vitro and in vivo test, respectively. The E. faecalis mixed feed also showed higher weight gain (WG%, 481.22% ±17.71%;SGR%, 2.93% ± 0.18%) and digestive enzymes activities (amylase, 1.26 ± 0.08 unit/mg; protease, 2.78 ±0.02 unit/mg) compared to control group (WG%, 371.31% ± 13.35%; SGR%, 2.30% ± 0.32%; amylase, 0.7 ± 0.03 unit/mg; protease, 1.82 ± 0.02 unit/mg). Furthermore, the probiotic also improved the immune response by augmenting NGH (from 73.33% ± 0.23% to 74.63% ± 0.11%) and SGH (from 22.24% ± 0.06% to 23.13% ±0.08%) in the treatment group. Therefore, E. faecalis could be recommended to use against bacterial infections of the M. rosenbergii.

Vibriosis has continued to pose a significant threat to the success, and economic sustainability of prawn farming (Hossain et al., 2017).Vibriosp., the causative bacteria for vibriosis, is identified as a predominant microbe in prawn hatcheries (Ahmmed, Ahmmed, Khushi, et al., 2020;Khan & Mahmud, 2020). The microbe can affect the prawn reproduction cycle, including the killing of the larvae and adult prawns (Sumon et al.,2018). In most cases, chemotherapeutics, for instance, antibiotics, have been used to controlV. harveyiand other potential prawn pathogens(Ahmmed, Ahmmed, Shah, & Banu, 2018). However, these agents not only enhance drug resistance among the pathogenic bacteria but also potentially cause accumulation of harmful chemical residues in the consumers (Pham et al., 2018; Thornber et al., 2019). Hence, probiotics have recently attracted interest as potential alternatives to chemotherapeutics (Sumon et al., 2018). The microbes investigated as potential sources of probiotic treatments against the proliferation ofVibriosp. inM. rosenbergiiincludeBacillus pumilusH2 (Gao et al., 2017),Bacillus cereus(Vidal et al., 2018),Clostridium butyricum(Sumon et al., 2018) andLactobacillussp. (Ahmmed et al., 2018). The genusEnterococcuscan also exhibit anti-Vibrio effects (Hanchi, Mottawea, Sebei, & Hammami,2018).

To date, few species of the genusEnterococcushave been investigated for their probiotics effects. These include E. faecalis, E. faecium, E. lactis,E. hirae, E. durans(Baccouri et al., 2019), which were shown to be effective against pathogenic bacteria.E. faecalieswas reported to possesses healthy benefits against chronic sinusitis and bronchitis, lessened gastrointestinal tract infections in human and also immunological stimulation in the farmed animal (Hanchi et al., 2018; Zhang et al.,2018). Zymetin, a feed additive comprising a range of beneficial bacteria, includingBacillus mesentericus,Streptococcus faecalis,Clostridium butyricum, proteases, lipases and beer yeast has also been investigated(Azad et al., 2019; Ghosh et al., 2016).

The giant freshwater prawn,M. rosenbergiiis one of the most favoured prawn species for farming, but its economic feasibility depends upon critical health management protocols. The prawn is a potent source of protein, minerals (magnesium, phosphorous, calcium and manganese), vitamins (A and D), and long-chain unsaturated fatty acids,including eicosapentaenoic acid (EPA, C20:5, and DHA, C20:5) (Ferdose& Hossain, 2011; Muralisankar, Bhavan, Radhakrishnan, Santhanam, &Jayakumar, 2017). It also contains an elevated level of phospholipid(36–42% of the total lipid) mostly concentrated in the ovary, which has a vital role in brain and mental health (Ahmmed, Ahmmed, Tian, et al.,2020). Therefore, this species has high demand both in the domestic and international markets. Owing to its physical characteristics, environmental adaptation and high commercial value, the farming of giant freshwater prawn is escalating in the subtropical climates (Khemundu &Banerjee, 2019). This popularity is attributable to the favourable agro-climatic conditions and ease of accessibility to its nutritional requirements in most coastal regions (Ahmed, Thompson, & Glaser, 2018).Moreover, owing to its economic significance, private and government hatcheries continue to establish their capacity to supply the prawn post-larvae, and other demands related to fish consumption (Ahmmed et al., 2017; Bala, Mallik, Saclain, & Islam, 2017).

Thus far, the knowledge on the antagonistic effect of theE. faecalieson theV. harveyiinM. rosenbergiiis still limited.Therefore, the present study aimed at isolatingE. faecaliesfrom the gut ofM. rosenbergiiand evaluate its antibacterial effect onV. harveyiboth inin-vitroandin-vivo.Also, the health-promoting impact ofE. faecalies,including the growth,immunity, and digestive enzyme activity ofM. rosenbergiiwere also investigated.

2.1. Ethical approval

The experiment was carried out according to the guidelines of the‘Biosafety and Ethics Committee,’ Fisheries and Marine Resources Technology (FMRT) Discipline, Khulna University, Bangladesh.

2.2. Gut separation and preparation of bacterial stock solution

The intestinal tract was separated fromMacrobrachium rosenbergii(26.34 ±1.3 g, 13 ±1.5 cm, N =30), collected from a local shrimp pond(22.7417◦N, 89.5167◦E), following an improved-traditional culture system without no probiotics or antibiotics throughout the culture period. One gram of intestinal tract tissue was homogenized with 1 mL alkaline peptone saline water using a tissue homogenizer (Bioneer,South Korea) and centrifuged for 10 min at 3000 ×gusing a bench-top centrifuge machine (Thermofisher Scientific, United States). The resultant supernatant was kept at −80◦C and used as a stock solution for further analysis.

2.3. Isolation of Enterococcus faecalis

The probiotic(E. faecalis)was isolated from the intestinal extracts following the method described by Sha, Wang, Liu, Jiang, and Wang(2016). Briefly, an aliquot of 0.1 mL diluted (1:10) with saline peptone was inoculated in Kenner Fecal Streptococcal media (Merck, Germany)with bacteriological agar. After the incubation period of 48 h at 37◦C,the red-coloured bacterial colonies counted flowed by their inoculation inE. faecalisbroth media and incubation at 37◦C for 2 days.E. faecaliswas identified by the level of turbidity and development of a yellow-brown colouration. After that, the isolated colonies ofE. faecaliswere aseptically preserved under frozen conditions (−20◦C) until required for further analysis using biochemical tests and Polymerase Chain Reaction (PCR).

2.4. Isolation of Vibrio sp.

The pathogen,Vibriosp., was isolated from the stock solution as described by Azad et al. (2019). The stock supernatant was diluted 10 times using alkaline saline peptone water and incubated for 6 ± 1 h at 37◦C to ensure selective enrichment. Subsequently, a second enrichment step was conducted using the first enriched culture, where an aliquot was diluted 10 times followed by incubation for 18 ± 1 h at 37◦C. Thereafter, a thiosulfate citrate bile salt (TCBS) agar medium was inoculated using 100 μL of the culture from the second enrichment process followed by incubation at 37◦C for 24 ± 3 h. The resultant colonies were aseptically collected and preserved by freezing to −80◦C until used in the PCR-based molecular assay and biochemical confirmatory tests forV. harveyi.

2.5. Confirmation of bacterial isolates

2.5.1. Biochemical tests

Biochemical profiling ofE. faecaliswas performed using various techniques as described by (Cowan, 2004). The tests include gram staining, microscopic morphology, Voges–Proskauer, and Methyl Red(Nimrat & Vuthiphandchai). Carbohydrate fermentation, catalase activity, salt tolerance and temperature, growth relation tests were also performed.V. harveyiwas confirmed using motility, Indole, Voges–-Proskauer, methyl red test, salt tolerance, and the effect of temperature on growth. All tests involved ten colonies of each microbe.

2.5.2. Confirmatory tests using PCR

PCR confirmatory tests were performed by culturing the bacteria in a nutrient broth followed by incubation at 37◦C for 12 h. A DNA sample was extracted in five randomly selected bacterial colonies using DNAzol reagent (Invitrogen, Life Technologies, USA). The quality and concentration of the DNA were assessed by determining the absorbance at 260–280 nm on a UV–visible spectrophotometer (Hitachi, Japan).Subsequently, the extracted DNA was dissolved in Tris-acetate-EDTA(TAE) buffer (Merck, Germany) and kept at −20◦C until the PCR analysis.

To identifyE. faecalis, two specific primers of Ent-F: 5′-ACACTTGGAAACAGGTGC-3′and Ent-R: 5′- AGTTACTAACGTCCTTGTTC-3′designed from 16 S rRNA region were used (Ryu et al., 2013). The 16 S rRNA target region was amplified through primer-directed PCR using thermostable DNA polymerase. Standard PCR mixtures (25 μL) were prepared with 1 μL template DNA (50–100 ng/mL), 1 μL Taq Polymerase(5 U/μL; Top DNA Polymerase, Bioneer, Korea), 10 μL of 10X reaction buffer, 2 μL of dNTPs, 6 μL MgCl2(25 mM), 2.5 μL of each primer, and 13 μL of deionized water. PCR amplifications were performed in a thermal cycler (C1000TM, BIO-RAD, USA) as follows: an initial denaturation step at 94◦C for 4 min and 35 cycles of 4 min at 94◦C, 1 min at 48◦C and 1 min at 72◦C succeeded by a final elongation period of 10 min at 72◦C.

Table 1 Sequence of primers and product size for each of two primer pairs in PCR.

2.6. In vitro challenge test

Thein vitrochallenge test was performed as described by Sumon et al. (2018). The confirmed colonies ofV. harveyiandE. faecaliswere cultured separately in a nutrient broth media. After incubation at 37◦C for 24 h, they were centrifuged at 10,000 ×gfor 5 min and resuspended in peptone water at an estimated concentration of 5.12 log CFU/mL and 3.09 log CFU/mL of the pathogen and probiotic, respectively. The antibacterial effect ofE. faecaliswas investigated by inoculating 1 mL of mixed culture (1:1 pathogen and probiotic) in TCBS agar and incubated for four successive time points (0, 4, 8, and 12 h). A control culture ofV. harveyi(1 mL) withoutE. faecaliswas also inoculated separately. The growth of the colonies was determined by performing a standard plate count after the incubation period of 24 h at 37◦C.

2.7. Probiotic mixed feed preparation

The feed was formulated by mixingE. faecalis(harvested through centrifugation at 3000 ×gfor 10 min) with commercial diet at a rate of 9.02 log CFU/g of feed. The loading of the colonies was standardized using UV–vis spectrophometer at 600 nm. The feed was dried at 42◦C for 2 h and packaged using aseptic technique, which was followed by at 4◦C until use (Mujeeb et al., 2010).

2.8. In vivo challenge test

The probiotic effect on the pathogenic bacteria was investigated usingin vivotest in aquariums (length, 45 cm; width, 30 cm; height, 30 cm) stocked with prawns (10 prawns/tank) for 60 days. The prawn larvae, weighing 2.19 ± 0.45 g and length, 5.14 ± 0.32 cm were collected from a private local hatchery (Khulna, Bangladesh) and acclimatized for two days. Probiotics or antibiotics were not provided to the larvae in the hatchery or the laboratory. The treatment group was fed using the probiotic-loaded feed at a rate of 7%–11% of their body weight for the first month and 5%–6% per day from the next month at a frequency of thrice per day until harvested, whereas the control group was fed using the basal diet without the probiotic. The water was maintained at an optimum quality throughout the experiment (salinity,1.0 ±0.2; dissolved oxygen, 5.0 ±0.55 μg/mL; pH, 7.2 ±0.2; ammonia,0.01 ±0.002 μg/mL; Temperature, 27 ±2.5◦C) and the light was set at a 10-h/14-h light/dark cycle. Sampling was carried out at intervals of ten days to monitor the load ofE. faecalisandV. harveyi.

2.9. Pond culture

All experimental grow-out ponds (70 m2, 1 m depth) were prepared as described by Asaduzzaman et al. (2008). Randomly selected prawn larvae were released at a stocking density of 50 larvae/pond, ensuring that the ponds were free from probiotics or antibiotics contaminants.The prawns were fed two times a day with the probiotic mixed feed at 7%–11% and 5%–6% of the prawn body weight during the first 30 days and the remainder 30 days of the culturing period, respectively. The basal diet (without the probiotic) was provided to the control group at a similar rate. The growth ofV. harveyiwas determined every ten days,and the water quality was maintained at the salinity level of 1.0 ±0.5,dissolved oxygen, 5.3 ± 0.45 μg/mL, pH, 7.3 ± 0.4, ammonia, 0.02 ±0.003 μg/mL and temperature, 26 ± 3◦C. All prawns were harvested after 60 days and stored at −80◦C until used for further analysis. The growth performance after 60 days of culture was calculated as a factor of weight and specific growth rate of the cultured juveniles according to Eqs (1) and (2).

2.10. Digestive enzyme activity

Frozen prawn samples were defrosted overnight at 4◦C followed by separation of the digestive tract, which was cut into smaller fragments and homogenized (7000 rpm, 5 min) in 9 vol of Tris-HCl buffer (0.05 M;pH 7.8). The supernatant was transferred into a 50 mL Nalgene tube and further centrifuged at 4800 ×gat 4◦C for 60 min. The clarified supernatant was used for enzymatic assay by determining the amylase and protease activity as described by Lovett and Felder (1990) and Ravan,Mehrabadi, and Bandani (2009). Briefly, aliquots of 250 μL were mixed with 260 μL Tris-HCl buffer (pH 7.8) and 40 μL soluble starch followed by incubation at 35◦C for 30 min. Afterwards, 100 μL of 3,5-dinitrosalicylic acid was added, and the mixture was heated in boiling water for 20 min, then cooled for 10 min. The absorbance of the cooled samples was measured at 540 nm, and a standard curve was prepared against a range of maltose concentration (0.1–2.0 mg/mL).

The protease activity was determined according to the method described by Anson (1938). Briefly, 1 mL of the prepared sample was mixed with 5 mL casein (0.65% w/v) (Merck, Darmstadt, Germany) and equilibrate at 37◦C for 5 min. Then, 5 mL trichloroacetic acid (100 g/L)was added, and the mixture was incubated at 37◦C for 30 min followed by filtration using a 0.45 μm polyethersulfone syringe filter before transferring into a cuvette. The absorbance of the resultant filtrate was determined at 660 nm (Roy et al., 2018) and a standard curve was developed using tyrosin (Merck, Darmstadt, Germany) at a concentration of 0.05–0.5 mg/mL.

2.11. Immunity assay

The immunity of prawn larvae against pathogenic bacteria was determined using differential hemocyte count (DHC) and total haemocyte count (THC) of the hemolymph, extracted from the pericardial sinus of theM. rosenbergii. Three distinct categories of haemocyte, including non-granular haemocyte (NGH), small granular hemocyte (SGH) and large granular hemocyte (LGH) were counted in an improved Neubauer hemocytometer on a light microscope (Olympus CX-2, Japan) (Sumon et al., 2018).

2.12. Statistical analysis

The data was analyzed using Microsoft Excel (Microsoft Office, 2007;USA) and Minitab, Version 17 (Minitab, 2014a; 2014b, USA). Assumptions for normality and homogeneity of the variance were tested using Shapiro-Wilk and Levene’s tests, respectively. An independent samplet-test was used to determine the statistical difference between the control and the treatment groups using a 95% level of confidence withp<0.05, indicating significant differences. The experiments were conducted at least in duplicate using independent samples, and data were reported as mean ±standard deviation.

The probiotic (E. faecalis) and pathogenic (V. harveyi) bacterias were successfully isolated from the intestines of freshwater prawn and profiled through a series of biochemical tests. The result of biochemical tests conducted using ten colonies ofE. faecalis(left) andV. harveyi(right) are presented in Table 2. Of the entire colony,E. faecalisconstituted 70%, whereas the remainder (30%) returned positive against methyl red test and growth inhibition at high temperature and salinity, thus not classified asE. faecalis. The biochemical test showed that 80% of the colony returned positive forV. harveyi, whereas the rest(20%) revealed analogous characteristics ofVibriospecies (Vs) (Table 2).Notably, all colonies ofV. harveyiacquired green colouration and showed a positive result in the motility and MR tests.

The probiotic and pathogenic bacteria identified in biochemical tests were further confirmed using polymerase chain reaction (PCR) molecular diagnosis. The 16 S rRNA fragments of the microbes were amplified to identifyE. faecalisandVibriosp., as presented in Fig. 1. The identity ofVibriosp. andE. faecaliswas ascertained by the presence of a single band of 663bp forVibriosp. and 318bp forE. faecalis, respectively in the gel electrophoresis.

In vitroandin vivochallenge tests were performed to ascertain the antagonistic effect ofE. faecalison the growth ofV. harveyi. The results of thein vitrostudy showed that the probiotic and pathogenic bacteria had a similar population at 0 h (3.54 log CFU/g) and 4 h (4.61 log CFU/g).However, the proliferation ofV. harveyiwas significantly (t-test, p <0.05) reduced after 8 h (control, 5.02 log CFU/g; treatment, 3.62 log CFU/g) and 12 h (control, 5.63 log CFU/g; treatment, 3.23 log CFU/g) of probiotic administration (Fig. 2). The results of thein vivochallenge conducted in a glass aquarium with supplementation of the probioticloaded feed are presented in Fig. 3. The results showed that the probiotic did not affect the growth ofV. harveyiin the first 40 days of treatment with the control feed. However, the establishment of the pathogenic bacteria continuously inhibited between 40 (control, 5.49 log CFU/g; treatment, 4.43 log CFU/g) and 50 days (control, 5.44 log CFU/g; treatment, 4.35 log CFU/g) of treatment, followed by a sharp decrease (control, 5.42 log CFU/g; treatment, 2.15 log CFU/g) in the remaining ten days.

The measured weight gain (%) and specific growth rate (%) after 60 days was used to determine the effect of the probiotic-loaded treated feed on the growth ofM. rosenbergiigrowth (Fig. 4). The weight gain of the prawns fedon E. faecalisincorporated diets were found to be significantly (t-test, p < 0.05) augmented in the treatment group(481.22 ± 17.71%) compared to the control (371.31 ± 13.35%).Furthermore, higher p < 0.05 specific growth rates were also observed in the probiotic treated prawn (2.93 ±0.18%) compared to the control(2.30 ±0.30%).

The digestive enzymes, measured using the amylase and protease activity, showed performance in the probiotic-treated prawns (amylase,1.26 ±0.03 Unit/mg of the intestine; protease, 2.78 ±0.03 Unit/mg of the intestine) compared to the control (amylase, 0.07 ±0.05 Unit/mg of the intestine; protease, 1.82 ± 0.01 Unit/mg of the intestine) (Fig. 5).Therefore, the present study demonstrated the positive effect ofE. faecalison the digestive enzyme activity ofM. rosenbergii.The impact of the probiotic treated feed on the immunity ofM. rosenbergiiTHC and the percentage count DHC including NGH, SGH and LGH showed higher(p < 0.05) NGH and SGH for the treatment group compared to the control (Table 3) but THC and LGH did not differ.

The manipulation of native micro-flora using probiotics is common in aquaculture due to the tremendous beneficial effects on aquatic animals. Probiotics help to cease bacterial infection, minimise the acquisition of resistance to antibiotics and improve the immunity of cultured fish (Chauhan & Singh, 2019; Wanka et al., 2018). The present study revealed the antagonistic effect of a probioticE. faecalison apathogenic bacteriaV. harveyibothin vivoandin vitroinM. rosenbergii. Besides,

M. rosenbergiiacquired positive physiological and biological attributes that are necessary for required for better growth performance (Sumon et al., 2018).

In the present study, both the probiotic and pathogenic bacteria were identified using several biochemical tests followed by PCR-based molecular diagnosis. The findings from the biochemical and PCR tests were in agreement with the reported data ( Ahmmed et al., 2018; Cowan,2004; Ryu et al., 2013; Tarr et al., 2007), which confirmed the presence of the microbes. The PCR analysis showed a similar amplicon size for the primer sequence ofVibriosp. (663bp) andEnterococcus Sp.(318bp),confirming their presence.

The findings of the study showed a positive antagonistic effect ofE. faecalisagainstV. herveyi. A previous study by Ringø (2020) also demonstrated the antagonistic effectE. faecalisonVibriosp. in pacific white shrimp (Litopenaeus vannamei) fed withE. faecalis-supplemented diet. Additionally, Gopalakannan and Arul (2011) and Swain, Singh, and Arul (2009) also reported on the positive antibacterial effect ofE. faecalisincorporated feed againstVibriosp. in common carp and tiger shrimp.Probiotics impede the pathogenicity of the disease-causing microbes by releasing either bacteriostatic or bactericidal components (Ringø, 2020).For instance,Lactobacillussp. andClostridiumsp. produce lactic acid and butyric acid, establishing an acidic environment that leads lead to the death of pathogenic bacteria (Sumon et al., 2018). Also,E. faeciumcan release bacteriocin (Cytolysin), which has the potential to lower the pH of the environment, which affects the porosity of the cell membrane of the pathogenic bacteria, causing the death of the cells due to leakage of intracellular cell components, such as potassium ions (Ben Braïek &Smaoui, 2019; Swain et al., 2009). Therefore, a possible mechanism of action for the probioticE. faecalisis the release of bacteriocins, which forms a barrier against the growth of the pathogenic bacteria.

In the present study, the prawns fed onE. faecalis-supplemented diet demonstrated superior growth performance and a higher specific growth rate compared to the control (Fig. 4). Previous studies showed that dietary supplementation ofE. faecaliscould influence the growth performance and lower the cumulative mortality of rainbow trout(Rodriguez-Estrada, Satoh, Haga, Fushimi, & Sweetman, 2013). In similar studies, Azad et al. (2019) showed thatE. faecaliscould also be intermixed with other probiotics, resulting in better effectiveness towards the enhancement of growth and immune response inM. rosenbergii. Additionally, the probiotic effects of other microbes on the development ofM. rosenbergiipost-larvae were observed usingBacillus subtilis(Seenivasan, Radhakrishnan, Shanthi, Muralisankar, &Bhavan, 2014),Lactobacillus cremoris (Seenivasanet al., 2014;Suralikar& Sahu, 2001),Clostridium butyricum(Sumon et al., 2018) andSaccharomyces cerevisiae(Seenivasan et al., 2014). The finding of the current study agreed with previous works, where the administration of a probioticE. faecalissupplemented diet significantly (t-test, p < 0.05)improved the growth and survival of giant tiger prawn(Peneaus monodon)(Elumalai & Raffi, 2013; Shefat, 2018; Swain et al., 2009).

M. rosenbergiifed withE. faecalismixed diet showed significantly higher (t-test, p <0.05) amylase and protease activities than the control group. The probiotic bacteria produce essential extracellular digestive enzymes (Padmavathi, Bhargavi, Priyanka, Niranjan, & Pavitra, 2018;Yang et al., 2018), which act as a substrate for growth-promoting factors, such as essential fatty acids, vitamins and minerals (Fidelis, 2019).The findings of our study were comparable to the previous studies reporting increased amylase, protease, and lipase activity after administration of probiotic products ofE. faecalisinM. rosenbergii(Li et al.,2010; Nimrat & Vuthiphandchai, 2011). The higher activity of the digestive enzyme accelerates the metabolism and nutrient absorption(Sumon et al., 2018), leading to improved growth ofM. rosenbergiifed with probiotic mixed feed.

Improved health and immunity are inter-related, and a healthy immune system helps to protectM. rosenbergiiby creating the first line of defence. In crustaceans, circulating haemocytes have an essential role in the immune response, including phagocytosis, recognition, cytotoxicity,cell-cell communication, and cytotoxicity (Johansson, Keyser, Sritunyalucksana, & So¨derh¨all, 2000). The data obtained in the present studyindicated a better immune response in probiotic treated prawns compared with the control. Kolanchinathan, Kumari, Gnanam, John,and Balasundaram (2017) and Sumon et al. (2018) reported similar observations inM. rosenbergiiwhen supplemented with probiotic strains.The results of the current study showed no abnormalities in colour,shape, movement and other phenotypic characteristics in prawns fed with probiotic mixed feed within the experimental period. Therefore,E. faecaliscould be used as a biological control agent to mitigate the pathogenicity ofVibriosp. in prawn culture.

Table 2 Biochemical tests used to identify E. faecalis and V. harveyi.

Fig. 1. PCR amplified fragment of 16s rRNA gene of Vibrio sp. (A) and Enterococcus faecalis (B) following electrophoresis in 1.5% agarose gel. (A) Lane M: 100-bp DNA ladder (size marker), Lane 1–5: isolates of Vibrio sp. (B): Lane M: 100-bp DNA ladder (size marker); lane 1–5, isolates of E. faecalis.

Fig. 2. Effect of growth of Vibrio harveyi in in-vitro challenge test. Different letter on the bar indicates significant differences (t-test, P <0.05). Each value is mean ± standard deviation of three individual replications.

Fig. 3. Effect of E. faecalis on Vibrio harveyi load in in-vivo challenge test conducted in glass aquaria. Each value is mean ± standard deviation of three individual replications.

Fig. 4. Effect of probiotic on weight gain (%) and specific growth rate (%) of M. rosenbergii. The asterisks (＊) on the top of the bar indicates significant difference between control and treatment (t-test,P < 0.05).

Fig. 5. Effect of E. faecalis incorporated feed on amylase and protease activity of M. rosenbergii. Different superscript letter on bar graph indicates significant difference between control (without probiotic) and treatment (with probiotic)(t-test, P < 0.05).

Table 3 The total haemocyte count (THC) and the percentage of differential haemocyte count (DHC) in M. rosenbergii after 60 days probiotic supplementation in earthen pond.

The present study revealed the potential probiotic effects of hostderivedE. faecalisagainst pathogenic bacteriaV. harveyiand its associated health-promoting effect onM.rosenbergii.Bothin vitroandin vivoassays showed thatE. faecalisresulted in impaired growth of the pathogenic bacteria. Moreover,E. faecalissupplemented diet enhanced the growth performance, enzymatic (amylase and protease) activity, and immunity ofM.rosenbergii.Therefore, the use ofE. faecalisin prawn farms might open a new possibility inM.rosenbergiiculture. However,the present study may be limited by the short duration low stocking density of the prawns. Although histological features of the gut ofM.rosenbergiibefore and after probiotic treatment were not determined in the current study, they may be of interest in future investigations.

Conflicts of interest

The authors declare that there is no conflict of interest regarding the publication of this article.

CRediT authorship contribution statement

Sharmin Sultana Khushi: Carried out the research work, analy.Mohammad Saifuddin Sumon: Determined enzymatic activity assay and analy. Mirja Kaizer Ahmmed: Edited the first draft and reviewed the revised and final manuscript. Md Nazmul Hasan Zilani: Prepared first draft of the manuscript and edited revised and final version. Fatema Ahmmed: Carried out in-vitro challenge test and analy. Stephen G.Giteru: Edited and Reviewed the language of the revised manuscript.Md Golam Sarower: Overall conceptualization, Supervision, and editing of the final manuscript.

Acknowledgements

The authors are grateful to the Bangladesh Fisheries Research Institute (BFRI) for the financial support of this project.