Diet supplementation of Pediococcus pentosaceus in cobia (Rachycentron canadum) enhances growth rate, respiratory burst and resistance against photobacteriosis

Cobia (Rachycentron canadum) is a tropical and subtropical marine fish, and has only one species under Genus Rachycentridae. Due to its good-quality meat and extraordinarily high growth rate, cobia became an important cultured fish in Taiwan during 1990s [1]. Following the successful development of cobia aquaculture technology in Taiwan, cobia now becomes one of the highest priority species for large-scale commercial aquaculture in the Americas and the Caribbean [2,3]. However, the outbreaks of photobacteriosis in sea-cage reared cobia have frequently occurred in Taiwan since 1999, which resulted in vast economic loss [4].

The causative agent of photobacteriosis is Photobacterium damselae subsp. piscicida (Pdp), a rod-shaped gram-negative bacterium, which can induce whitish tubercles in the internal organs of chronically infected fish [5,6]. Photobacteriosis is a worldwide disease, and has provoked mass mortality in many other cultured marine fish, especially at the early grow-out stage, including cobia, sea bass, flounder, breams and yellowtail [7e10]. The major control of photobacteriosis has been usage of antibiotics, but more and more drug-resistant strains of Pdp were isolated from medicated or moribund cobia cultured in Taiwan [7]. Several Pdp vaccines and vaccination programs were developed [11e15]; however, the effi- cacy of the vaccines available on market is not consistent in the field. Consequently, probiotic is considered an alternative for the control of photobacteriosis.

Probiotics have been applied in aquaculture, and there are multiple mechanisms of health improvement by probiotics [16e18]. For example, some probiotics can interact or antagonize with other enteric bacteria through colonization resistance, or direct inhibit and reduce the incidence of opportunistic pathogens. Probiotics may also enhance the health of host via physiological or immune modulation. Some probiotics can assist the process of digestion via producing extracellular enzymes or products necessary for hosts.

Lactic acid bacteria (LAB) have been used as probiotics in many studies, and 1e10% of enteric bacteria were reported to exhibit the probiotic potential [19]. The production of organic acid and bacteriocin of LAB are able to inhibit or directly kill many microbes. The resistance capability of LAB against gastric acid and bile enhances its livability through the digestive tract. In this study, a strain of lactic acid bacteria LAB 4012 was isolated from the intestine of adult cobia, and its classification was identified. In order to evaluate the probiotic potential of LAB 4012 in cobia, the inhibition ability of LAB 4012 metabolites on Pdp growth in vitro, and the feeding impact of LAB 4012 on cobia growth rate, respiratory burst, the resistance against Pdp immersion challenge, and the synergetic protection efficacy with Pdp vaccine were examined.

Pdp strain P40, a gift from Dr. Chen-Chun Ku (National Penghu University, Taiwan), is cultured in brain heart infusion (BHI) broth (Becton, Dickinson and Company) with 20% salinity at 28 ºC. The identification and characterization of Pdp strain P40 has been described in previous paper [7].

The LAB 4012 strain was provided by SynBioTech company (Taipei, Taiwan). The LAB 4012 was isolated from the intestine of a 3-day fasted adult cobia by Dr. Hung-Hsi Hu, and cultured in MRS broth (Fluka) at 37 ºC, and was proved to be able to resist the treatments of gastric acid (pH 2.0) and 0.3% bile salt. The species name of LAB 4012 was determined by sequencing LAB 16S ribosomal DNA (rDNA) and comparing with the reference strains derived from the GenBank. PCR was carried out using forward primer 5'-AGAGTTTGATCATGGCTCAG-3' and reverse primer 5'- AAGGAGGTGATCCAGCC-3' [20] on PCR System 2700 (Applied Biosystems), and PCR products were sequenced by ABI 3730 (Applied Biosystems). Phylogenetic analysis of LAB 4012 16S rDNA with reference strains was performed using software MEGA 5.0.

To examine the impact of LAB 4012 culture supernatant on the growth of Pdp, LAB 4012 was cultured in MRS broth at 37 C for 18 h. Then, the culture broth was collected by centrifugation at 1600 g for 15 min at 4 C. The culture supernatant was passed through by 0.45 mm pore-size filter unit (Minisart). After filtrating, 1 mL of tested supernatant was added into 4 mL of Brain Heart Infusion (BHI) (Difco) and then inoculated with 0.1 mL of Pdp with the optical density value (OD₅₉₅) of 1.

The original pH in MRS without LAB 4012 was pH 6.2; however, the pH of the LAB 4012 culture supernatant decreased to 4.1 after 18 h of growth, and the concentration of lactic acid in LAB 4012 supernatant was quantified to be 73 mM by Lactate assay kit (Biovision). To examine the influence of pH and lactic acid on the Pdp growth, additional two tested groups were included. One was an 18 h-culture supernatant of LAB 4012 with adjusted pH value of 6.2 by NaOH, and another tested group contained MRS medium supplemented with lactic acid at a final concentration of 73 mM. Similarly, 1 mL of each test supernatant was added into 4 mL of Brain Heart Infusion (BHI) (Difco) and inoculated with 0.1 mL of Pdp (OD₅₉₅ = 1). The bacterial mass of Pdp after different treatments at each time point was measured at OD₅₉₅ by MRX II ELISA reader.

The LAB 4012 was cultured in MRS broth at 37 ºC until the OD₅₉₅ of culture reached 2.0, and then concentrated by centrifuging at 1500 x g for 15 min. After washing with phosphate buffered saline (PBS) three times, LAB 4012 pellet was re-suspended in PBS (OD₅₉₅ = 2) and sprayed onto the commercial dry feed at a ratio of 1:3 (w/w). The prepared LAB-containing feed was preserved at 4 ºC and used up within five days. The titer of live LAB in the prepared feed was examined by re-dissolving the prepared feed in PBS for CFU counting.

Two groups of cobia were used for LAB 4012 feeding test separately with body weight of 4.6 g and 35 g. All cobia used in this study were raised from Pingtong, Taiwan. Fish were held in FRP (Fiberglass Reinforced Plastics) tanks, supplied with aerated 30& salinity sea water at 25 C, and fed with commercial dry feed (Omega 3rd Marine fish feed, Golden Prawn Enterprise Corp). The fish condition was monitored, and only the healthy fish were used for in vivo test. The dose of LAB in the prepared feed was 109 CFU g1 , and the fish were fed twice daily.

All challenge tests in this study were conducted by immersion. The fish were fasted for one day before challenge test. The fish were placed in the sea water at 20 ºC, and the immersion time of Pdpcontaining sea water was 2 min. At the end of challenge test, all dead fish were sampled and examined for white tubercles in the internal organs. Furthermore, Pdp was re-isolated from the kidney and spleen, and identified using Bionor Mono-Pp kit. Accumulated mortality was recorded for ten days, and the relative percentage of survival (RPS) was calculated according to the formula: RPS = [1 - (Mortality of test group/Mortality of control group)] x 100.

The fish with average body weight of 4.6 g were randomly divided into LAB-fed group (LAB+) and control group (LAB-). To detect the LAB 4012 in the intestine of cobia, microbial analyses were performed at three time points: before the feeding trial, at the end of the 2-week feeding, and one week post the end of feeding trial. Five fish were sacrificed at each time point. The intestine was dissected into pieces and put into 5 mL of MRS (the selective medium for lactic bacteria) to release the bacteria into medium. After appropriate dilution, the supernatant was plated onto the MRS agar plate, and incubated at 37 ºC. The isolated bacteria colonies grown on MRS agar were identified by sequencing the 16S rDNA as described in Section 2.1.

The number of fish in LAB+ and LAB- group was 51, and the fish in each group were further divided into 3 subgroups (17 fish per subgroup) so that all tests were performed in triplicate. In LABþ group, the fish were fed with 5% body weight of LAB 4012-mixed feed (10⁹ CFU g^-1 ) for two weeks, while the fish in control group were fed with feed without LAB 4012. The growth rate of the fish, the RB of PBL, and the mortality after Pdp immersion challenge test were monitored at the end of the 2-week feeding trial.

The RB of PBLs was determined by the nitroblue tetrazolium (NBT) assay described by Choudhury et al. (2005) [21]. To prepare PBLs, blood samples were collected from the cobia. Six fish taken from respective group were sacrificed after bleeding. The blood samples were incubated with equal volume of 0.02 M EDTA and red blood cell (RBC) lysis buffer (Roche) at 37 ºC for 1 h. The PBLs suspension was then added into a 96-well ELISA plate (50 mL/well) and incubated at 37 ºC for 1 h for cell adhesion. Afterward, the supernatant was removed and the cells were washed by PBS three times and incubated with 0.2% NBT for 1 h. Then, the cells were fixed by 100% methanol for 3 min, followed with 30% methanol washes three times. After air-drying, 60 μL of 2 N potassium hydroxide and 70 μL of dimethyl sulphoxide were added into each well for color development, and OD₄₉₀ was measured by ELISA reader (MRX II, Dynex Technologies).

To examine whether feeding LAB 4012 could enhance humoral immune response, fish with average body weight of 35 g were randomly divided into three groups: V+LAB+, V+LAB-, and V-LAB- groups. Each group contained 12 fish. All experiments were performed in triplicate. In V+LAB+ and V+LAB- groups, the fish were immunized with inactivated Pdp, and only the fish in V+LAB+ group were fed with LAB 4012-mixed commercial feed from Day 2 on after immunization until the day before challenge test. The total feeding period lasted for five weeks. In V-LAB- group, the fish were mock-immunized with PBS and fed with commercial feed without LAB 4012. The blood samples were collected from each group at 2, 3, and 4 weeks post vaccination, and the levels of Pdp-specific Ig were determined by ELISA. All fish were challenged with Pdp at the end of the 5-week feeding period.

For vaccine preparation, Pdp (strain P40) broth was inactivated by adding formalin to a final concentration of 0.5%, and reacted for 12 h at 28 ºC. After inactivation, the bacteria were spread on the agar plates to confirm the safety of the vaccine. The inactivated Pdp was precipitated by centrifugation and washed with PBS three times. The inactivated Pdp was adjusted to the proper concentration, and mixed with adjuvant ISA 763 AVG (Seppic) at 1:1 ratio (v/ v). Cobia (n = 24) with average body weight (BW) of 35 g were intraperitoneally (IP) injected with 0.1 mL of inactivated Pdp vaccine at a dose of 0.333 mg wet weight of Pdp per BW of fish, and the negative control fish (n = 12) were injected with PBS. The fish received booster shots 21 days post-immunization.

For quantification of anti-Pdp immunoglobulin, blood samples were collected at 0, 14, 21 and 28 dpi, and clotted at 4 ºC for overnight. The titer of cobia Pdp-specific antibodies was determined via indirect ELISA. A portion of 100 μL (10⁸ CFU mL^-1 ) of inactivated Pdp solution was coated in a 96-well ELISA microplate (NUNC) at 4 ºC for overnight. The ELISA plate was washed with PBST (PBS containing 0.1% Tween-20) three times, and blocked with 3% BSA (150 mL/well) at 37 ºC for 2 h. Cobia antiserum (100 mL per well) was added and incubated for 1 h. Following washing for three times, rat antiserum against cobia Ig (a gift from Dr. Chun-Shun Wang, National University of Kaohsiung) was added (100 μL per well) and incubated for 1 h. After washes, alkaline phosphatase-conjugated goat anti-rat IgG (Jackson Immuno Research) was added, at a dilution factor of 1000, and incubated for 1 h. Following three washes, the enzymatic activity was determined using 0.1% PNPP (Peirce) as substrate in a 30-min reaction in the dark; OD₄₀₅ was measured on an ELISA reader (MRX II, Dynex Technologies).

All trials were analyzed using one-way ANOVA to determine the significant differences between treatment and control groups. Three significance levels of p = 0.05, 0.01 and 0.001 were used.

The phylogenetic analysis of the LAB 4012 16S rDNA against the reference strains in GenBank, including minimum evolution and maximum parsimony (bootstrap value = 100) revealed that strain 4012 was Pediococcus pentosaceus (Fig. 1). The accession number of LAB 4012 16S rDNA is JN674456 in Genbank.

The concentration of lactic acid in the culture supernatant of LAB 4012 after 18 h-growth was found to be 73 mM. Two 18 hculture supernatants of LAB 4012, at pH 4.1 and adjusted pH 6.2, and the medium containing 73 mM lactic acid were separately added into Pdp culture broth at a 1:4 ratio (v/v). The control group was LAB-free MRS medium at pH 6.2. The 6 h-growth curves of Pdp recorded are shown in Fig. 2. The culture supernatant of LAB 4012 at pH 4.1 could inhibit nearly 97% growth of Pdp, but the inhibition activity diminished completely when the pH was adjusted to 6.2. The pH of the medium containing 73 mM lactic acid was 4.6, and its inhibition activity on Pdp growth was approximately half of that of LAB 4012 culture supernatant at pH 4.1.

 

No LAB 4012 could be isolated from the intestine of the cobia with body weight of 4.6 g before the feeding trial, but was found from the intestine of the fed-fish post 2-week feeding. However, LAB 4012 was no more detectable in the intestine of the fed-fish at one week post the end of feeding trial.

After two weeks of feeding, the average BW gain in LAB 4012-fed (LAB+) group was significantly higher than that of non-fed (LAB-) group, p < 0.05 (Fig. 3), and the RB of LAB+ group was found significantly higher than that of LAB- group (Fig. 4). The protection effect of LAB 4012 against Pdp infection was evaluated by the immersion challenge at the end of 2-week feeding. White tubercles were recorded in the kidney and spleen in all moribund fish, and Pdp was reisolated from the internal organs containing white tubercles. The accumulated mortality of LAB 4012-fed group was 20% versus approx. 80% in control group (Fig. 5), indicating that feeding cobia with LAB 4012 significantly reduced the mortality after Pdp challenge.

 

In Fig. 6, the Pdp-specific Ig levels in the immunized cobia increased in two weeks following vaccination, and were signifi- cantly different from that of the non-vaccinated fish. However, the difference in Pdp-specific Ig levels between LAB-fed and non-fed immunized fish was found statistically insignificant.

To test if feeding cobia with LAB 4012 could improve the survival rate of vaccinated fish, fish in the V+LAB+, V+LAB- and V-LAB- groups were immersion-challenged with Pdp (1.5 10⁵ CFU mL^-1 ). The accumulated mortality of V+LAB- group was found to be 37% lower than that of V-LAB- group, indicating that vaccinating inactivated Pdp would significantly elevate the survival rate against Pdp infection. Furthermore, the accumulated mortality of V+LAB+ group was recorded to be 22% lower than that of V+LAB- group (Fig. 7).

 

 

It has been reported that some components in the LAB metabolites are able to kill or inhibit the growth of some pathogens, such as bacteriocin, proteases, lysozymes and hydrogen peroxide [17,18]. In most cases, bacteriocin secreted by lactic acid bacteria can directly inhibit gram-positive bacteria, but can affect gramnegative species only when their outer membrane is injured [22]. LAB 4012 is identified to be Pedicoccus pentosaceus (Accession number JN674456), belonging to Lactobacilles of Lactobaciilaceae, and the bacteriocin in this family is pediocin. It has been reported that lactic acid can damage the outer membrane of gram-negative bacteria, and then bacteriocin can be react on the injured bacteria [23]. However, pediocin gene was not detectable in LAB 4012 strain (data not shown), implying that it is not involved in the inhibition activity on Pdp growth in the LAB 4012 culture supernatant.

The 18 h-culture supernatant of LAB 4012 exhibited the lowest pH (4.1) and the highest inhibition activity on Pdp growth; however, the adjusted LAB 4012 culture supernatant pH at 6.2 did not inhibit Pdp growth. We therefore suggested that acidic pH resulted from the metabolic acids in LAB culture supernatant is essential for the depression of Pdp growth in vitro. Short-chain fatty acids, such as lactic acid and acetic acid, are the major metabolites of lactic acid bacteria responsible for the antimicrobial activity against Escherichia coli O157:H7 in the intestine [24]. Lactic acid (LA) produced by probiotic Lactobacillus strains was revealed to be bacteriocidal effect on E coli when LA concentration in the co-cultured medium exceeds 65 mM [24,25]. In the present study, we did not co-culture LAB 4012 with Pdp, instead, we added the culture supernatant of LAB 4012 into Pdp culture medium at a ratio of 1:4 from the beginning of Pdp growth.

The pH of MRS medium containing 73 mM lactic acid was 4.6, a little higher than that of LAB 4012 culture supernatant (4.1), and its inhibition efficacy on Pdp growth was about 50% lower than that of LAB 4012 culture supernatant, suggesting that other metabolic acids in the culture supernatant of LAB 4012 are either responsible for the acidification of culture supernatant and the depression on Pdp growth. The tolerance to gastric acid and bile and the adherence of the intestinal epithelium cells are important characteristics of probiotics for passing through the digestive tract and colonizing in the intestine [26]. At the beginning of LAB 4012 feeding, none of the LAB 4012 could be isolated from the intestine of the cobia. After a 2- week feeding, LAB 4012 became available from the fed-fish and grew on MRS agar. It therefore reconfirmed that LAB 4012 exhibited tolerance against gastric acid and bile salt, and suggestively remained resident in the digestive tract of the fish post feeding and displayed its positive impact on the fed-cobia. It has been reported that Pdp accumulation could be found in the liver, spleen, kidney and the intestinal space of the fish in the acute form of Pdp infection [27]. Since LAB 4012 could reside in the intestine of fed-cobia, its metabolic acids (e.g. lactic acid) may have chance to inhibit Pdp directly in the intestine. However, most microbes are transient in aquatic animals [28]. The level of the intestinal LAB 4012 also diminished within one week after the LAB-feeding ceased; therefore, continuously feeding cobia with LAB 4012 to maintain the protection against Pdp infection is advisable.

The initial interest of using probiotics in aquaculture is to increase the growth of cultured species, and the enhancement of growth is probably due to increased appetite and/or digestibility of the fed-organisms [18]. Probiotics are suggested to have positive impact on digestive process by synthesizing some extracellular enzymes, such as proteases, amylases and lipases, and providing growth factors, like vitamins, fatty acids and amino acids [17,29,30]. In the present study, cobia larvae fed daily with 5% body weight of LAB 4012-containing feed (10⁹ CFU g^-1 ) for two weeks showed significantly better growth, which might result from the contribution of the enzymes and nutrients secreted by LAB 4012.

In fish, probiotics have been reported to enhance respiratory burst of phagocytes, which play a central role in non-specific cellular defense [28,31,32]. At the present study, a 2-week feeding of LAB 4012 significantly enhanced the respiratory burst of PBL from the fed-fish, and triggered effective protection of cobia against Pdp challenge (RPS = 74.3). The effect of diet supplementation of two probiotics (Pdp11 and Pdp13) belonging to the family Shewanella, has been compared in Senegalese sole (Solea senegalensis, Kaup). Only Pdp11 could induce significantly increasing of RB after 60-day feeding, and Pdp13 could not. However, similar RPS values were found in the fish separately fed with Pdp11 (RPS = 20-25) and with Pdp13 (RPS = 20-35). Therefore, it is suggested that the increased RB is not essential to enhance survival rate of the fed fish after Pdp challenge [33]. For this reason, it remains uncertain if the elevated RB after feeding with LAB 4012 is essential for the protection of cobia away from Pdp infection. Further experiments are required to test it.

Vaccinating inactivated Pdp is another prophylaxis way to control cobia disease but the adaptive immunity of cobia is not potent enough to induce long-term protection, especially when the BW of cobia is less than 50 g. Thus, administering the booster more than once to maintain the effective level of neutralizing antibodies will be necessary. However, in reality, the farmers usually transport the cultured cobia from the land-ponds in southern Taiwan to the seacages in Penghu islands during the larval stage with BW of 10-30 g. Once the larvae are transferred into the sea-cages, it becomes technically difficult to boost the vaccinated cobia afterward. Oftentimes, the outbreaks of Pdp-induced mortality repeatedly occur 2-6 months post sea-cage culture. In some studies, probiotics are found to enhance humoral immune response, and thus can be applied as adjuvant in oral vaccination [34,35]. Some studies also reported that probiotics may improve the immune response through gut-associated lymphoid tissue to help antigen uptake by dendritic cells, hence postponing the outbreaks of epidemic [36,37]. In this study, feeding with LAB 4012 did not elevate specific antibody response in vaccinated cobia, but heightened the synergetic protection against Pdp challenge by 22%. The additional protection may be contributed by the improved respiratory burst, the inhibition of Pdp growth by LAB 4012 metabolic acids, or by the competitive adherence to fish intestinal epithelium. Further experiments are required to clarify the detailed mechanism of LAB 4012-induced antagonism against Pdp infection in vivo.

In summary, a 2-week feeding of Pediococcus pentosaceus LAB 4012 strain is adequate to significantly elevate the growth rate by 12% in cobia and improve the survival rate in Pdp challenge test (RPS = 74.3). The metabolic acids from LAB 4012 after 18 h-growth exhibited bacteriocidal activity on Pdp growth. Supplementation of LAB 4012 in the feed poses great potential to compensate antibiotics shortage for disease control, and applying LAB 4012 solely or with vaccination would be beneficial in protecting cobia against Pdp infection, especially in large scale sea-cage culture.

This work is supported by a grant from the National Scientific Council (NSC 95-3114-P-346-001-MY3). The authors thank SynBioTech for the donation of LAB 4012 (Pediococcus pentosaceus), and also appreciate Mr. C. J. Cheng for English revision.

 Xing C-F, et al., Diet supplementation of Pediococcus pentosaceus in cobia (Rachycentron canadum) enhances growth rate, respiratory burst and resistance against photobacteriosis, Fish & Shellfish Immunology (2013), http://dx.doi.org/10.1016/ j.fsi.2013.07.021

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