Dietary supplementation of Pediococcus pentosaceus enhances innate immunity, physiological health and resistance to Vibrio anguillarum in orange-spotted grouper (Epinephelus coioides)

1. Introduction

Grouper is an economically important fish species for aquaculture in Southeast Asian countries. Due to the intensive culture, groupers have been suffering from not only viral but also bacterial diseases, such as vibriosis, resulting in serious economic loss. Vibriosis is also known as fatal hemorrhagic septicemia, and the causative agents of vibriosis belong to the genus Vibrio, such as Vibrio alginolyticus, Vibrio harveyi, and Vibrio anguillarum. The Vibrio spp. infect fish through the skin or oral intake. An outbreak of vibriosis usually occurs when fish are immunocompromised, under stress, and overcrowded at water temperature over 15 C [1,2]. The typical clinical signs are lethargy and ulceration, and high numbers of Vibrio spp. can be found in the blood and the hematopoietic tissues. Moreover, the intestine of the infected fish was filled with yellow liquid and associated with gastroenteritis [3]. However, in acute epizootics, the infected fish always die without showing any clinical signs [1,4,5]. V. anguillarum is used for challenge test in this study, and is a gram-negative, curved rod, non-spore forming, and facultative anaerobic bacterium with flagella. In aquaculture, V. anguillarum can infect more than 50 marine and brackish economic fish species, and has caused high morbidity and mortality rates [5,6].

For many years, the control strategies on vibriosis mostly employ chemotherapeutants, such as antibiotics and disinfectants. However, widespread use of medicines has resulted in antibiotic contamination of the environment and fish [7], and led to increasing the frequencies of antibiotic resistant bacteria in aquaculture systems [8e17]. Therefore, alternative ways for the control of vibriosis must be developed, such as the use of antimicrobial peptides, vaccines, and probiotics. Probiotic-feeding is a good strategy to control the disease because it improves host health and immunity, rendering the host more pathogen-resistant.

In our previous studies, Pediococcus pentosaceus strain 4012 (LAB4012), a lactic acid bacterium, was isolated from the intestine of cobia (Rachycentron canadum). In cobia, feeding with LAB4012 can enhance the growth rate and respiratory burst activity and significantly decrease the cumulative mortality after challenge with Photobacterium damselae subsp. piscicida (Pdp) [18]. However, the potential of dietary LAB4012 to protect groupers from vibriosis is still unknown. Therefore, we examined the inhibitory activity of LAB4012 supernatant against V. anguillarum in vitro and the protection efficacy of LAB4012-feeding on groupers against V. anguillarum challenge. Furthermore, we analyzed the impact of LAB4012- feeding on the growth rate, gene expression levels of pro-/antiinflammatory cytokines, peripheral blood cell counts, and the phagocytic activity and respiratory burst activity of head-kidney phagocytes in groupers. This study provides the information about the influence of LAB4012-feeding on grouper innate immunity and the effect of LAB4012-feeding on protecting groupers from V. anguillarum infection.

2. Materials and methods

2.1. Orange-spotted groupers and bacteria

Orange-spotted groupers obtained from Merit Ocean Biotech INC (Taiwan) were reared in aerated seawater with 25% salinity at 26 ºC. Fish health was evaluated by observation of fish morphology, behavior, mortality, and appetite. In addition, five fish were randomly sampled and confirmed healthy by pathology examination and microbiology tests which were conducted by plating the homogenate of head-kidney, spleen, and liver on tryptic soy agar (TSA, Difco) and thiosulfate citrate bile salts sucrose (TCBS) agar (Difco), the Vibrio selective medium agar. Besides, the fish were confirmed to be betanodavirus- and fish iridovirus-free by realtime PCR (Section 2.6).

Pediococcus pentosaceus strain 4012 (LAB4012) was a gift from Dr. Hung-Hsi Hu (National Penghu University, Taiwan), and was cultured in MRS broth (Merck) at 37 ºC [18]. V. anguillarum was a gift from Dr. Chen-Chun Ku (National Penghu University, Taiwan) and was cultured in TCBS agar (Difco) or tryptic soy broth (TSB) (Becton, Dickinson and Company) with 15% salinity at 30 ºC. The identity of V. anguillarum used in the present study was confirmed by specific primers reported in Hong et al. (2007) [19]. For preparation of challenge test, V. anguillarum was cultured on tryptic soy agar (TSA) (Difco) for overnight, and harvested by flushing with PBS. After 3 cycles of PBS washing-and-pelleting by centrifuging at 6230 g for 3 min, the pellet of V. anguillarum was resuspended in PBS for the challenge test.

2.2. The inhibition activity of LAB4012 culture supernatant on the growth of V. anguillarum

A colony of LAB4012 was inoculated in 3 ml MRS and cultured for overnight. The overnight-cultured LAB4012 was transferred to 100-fold volume of MRS broth, and 15 ml of culture suspension was collected every hour from 1 to 18 h by centrifuging at 1600 g for 15 min at 4 ºC and filtrating through the filter with pore size of 0.45 mm (Sartorius stedim biotech). The pH value and the concentration of lactic acid in each LAB4012 supernatant was respectively determined by using pH meter (JENCO) and Lactate Assay Kit (Biovision).

To examine the impact of LAB4012 culture supernatant, lactic acid, and the pH value on the growth of V. anguillarum, four kinds of tested medium and one control medium were prepared, including (1) LAB4012 culture supernatants, separately collected at 1e18 h post-culture, (2) MRS broth, pH adjusted to 4.2 with HCl, (3) 18hcultured supernatant of LAB4012, pH adjusted to 6.3 with NaOH, (4) MRS broth containing 73 mM lactic acid, and (5) the control medium, i.e., MRS broth with pH 6.3. To examine the influence of each tested medium on the growth of V. anguillarum, the 0.1 ml V. anguillarum with the optical density value at 595 nm (OD₅₉₅) of 1 was inoculated into 4 ml TSB supplemented with 1 ml tested medium. After incubation at 30 ºC for 1e8 h, the biomass (measured at OD₅₉₅) of V. anguillarum in each tested and control medium were determined by MRX II ELISA reader (DYNEX technologies).

2.3. Preparation of LAB4012-contained diet

The diet supplemented with LAB4012 was prepared as described below. LAB4012 was cultured in MRS broth until the OD₆₀₀ value of liquid culture reached 2 (approx. 10⁹ CFU ml^-1 ). LAB4012 was harvested by centrifugation at 1600 g for 15 min at 4 ºC, resuspended in PBS, and then sprayed on the commercial diet at a dose of 109 CFU g^-1 . The prepared diet was preserved at 4 ºC for 3 days at most. The 3-day-preserved diet was dissolved in PBS, and the numbers of alive LAB4012 were calculated to be 6 108 CFU g^-1 at least.

2.4. V. anguillarum challenge test

To evaluate the protection efficacy of LAB4012-feeding against V. anguillarum infection, groupers with average body weight of 2 g were divided into 4 groups. Two groups (N ¼ 20) were fed with 4% body weight of LAB4012-contained diet, and the others fed without LAB4012 were served as negative control. After 3-week feeding, the groupers were IP-challenged with V. anguillarum at doses of 5 x 10⁵ and 6 x 10⁵ CFU per fish. Groupers were no more fed with LAB4012 after V. anguillarum challenge, and the cumulative mortality of groupers was recorded for 7 days.

2.5. Growth rate and immune gene expression

Groupers with average body weight of 1.5 g were daily fed with LAB4012-contained diet at a rate of 4% body weight for 4 weeks. The average body weight of 50 groupers was determined at 1e4 weeks post-feeding (wpf). The percent weight gain was calculated as: [(final body weight e initial body weight)/(initial body weight)] x 100%. Five groupers of each group were randomly sampled at 1 to 4 wpf, and the immune gene expression of headkidney and spleen was analyzed by real-time PCR (Section 2.6).

Groupers with average body weight of 8.3 g were daily fed with LAB4012-contained diet at a rate of 2% body weight for 1 week, and then intraperitoneally (IP) challenged with a non-lethal dose of V. anguillarum (6.3 x 10⁴ CFU per fish). At 1-3 days post-infection (dpi), three fish of the fed and non-fed groupers were randomly sampled, and the immune gene expression of head-kidney and spleen was analyzed by real-time PCR (Section 2.6).

2.6. Reverse transcription and real-time PCR

The acid guanidinium thiocyanate-phenol-chloroform extraction method [20] was used to extract the total RNA from organs. Reverse transcription was carried out by incubating 6 ml total RNA at 42 ºC for 1 h in 30 ml of 1 x reaction buffer containing 0.3 mM oligo dT20, 0.4 mM dNTP, 11.7 mM DTT, 40 U ribonuclease inhibitor rRNasin (Promega), and 60 U MMLV reverse transcriptase (Promega). Real-time PCR was conducted to determine the expression level of immune gene. The sequences of primer sets for immune genes are listed in Table 1. An aliquot (0.25 ml) of the cDNA was added into a real-time PCR mixture with a final volume of 10 ml containing 0.5 mM forward and reverse primers in 1 iQ SYBR Green Super-Mix (Bio-Rad). The amplification was carried out in CFX384 Real-Time PCR Detection System (Bio-Rad) with an initial denaturing step of 95 ºC for 3 min, followed by 40 cycles of 95 ºC for 20 s, 60 ºC for 20 s, 72 ºC for 20 s, and fluorescence detection at 80 º for 20 s. All samples were analyzed in triplicate. Each immune gene expression levels were normalized with internal control (actin).

To detect betanodavirus, total RNA of the brain was extracted and transcribed into cDNA by primer NNV RNA2 R. To detect grouper iridovirus (GIV) and red sea bream iridovirus (RSIV), the total DNA of the viscera was extracted by a Genomic DNA Mini Kit (Geneaid). Detection of betanodavirus and fish iridovirus was performed by real-time PCR with the primer sets described in Table 1. The real-time PCR program is the same as above. The sensitivity of viral detection in our real-time PCR assay is 5 viral copies.

Dietary supplementation of Pediococcus pentosaceus enhances innate immunity, physiological health and resistance to Vibrio anguillarum in orange-spotted grouper (Epinephelus coioides) - Image 1

2.7. Blood cell count

Two groups of groupers (N ¼ 3) with average body weight of 288 g were daily fed with or without LAB4012-supplemented diet at a rate of 1% body weight for 1 week, respectively, and then the blood was sampled from the caudal vein using sterile syringe prerinsed with anticoagulant (20 mM EDTA in PBS, pH 8). The blood samples were then mixed with equal volume of anticoagulant. After blood sampling, the groupers were immediately IPchallenged with a non-lethal dose of V. anguillarum (10⁶ CFU per fish). At 1 dpi, the blood was sampled again from the caudal vein of the infected groupers. The numbers of erythrocytes in blood samples were counted with a hemacytometer. After erythrocytes were lysed by red blood cell lysis buffer (154 mM NH₄Cl, 14 mM NaHCO3, 0.1 mM EDTA, pH 7.3), the numbers of leukocytes were counted by hemacytometer.

2.8. Isolation of head-kidney phagocytes

Three V. anguillarum-infected groupers with or without LAB4012-feeding (Section 2.7) were sacrificed with MS-222 (Merck) overdose at 1 dpi, and the head-kidney was removed and placed in anticoagulant (20 mM EDTA in PBS, pH 8). Cell suspensions were collected by forcing the head-kidney through a 100-mm mesh with a syringe plunger. Cell suspension (6 ml) was layered over 3 ml of Lymphoprep (Axis-Shield PoC AS), and the separation procedure of phagocytes was followed by the product information sheet. The purified phagocytes were re-suspended with L-15 medium containing 20% FBS and seeded into 8 wells of 96-well black plate to analyze phagocytic activity and 96-well clear plate for respiratory burst activity analysis (10⁶ cells per well). To adhere to the 96-well plate, the phagocytes were incubated at 28 ºC for overnight, and the non-adherent cells were then removed by washing with L-15 medium 3 times. The phagocytic activity and respiratory burst activity of the phagocytes were analyzed as described in Sections 2.9 and 2.10.

To normalize the phagocytic activity and respiratory burst activity of the phagocytes, the amount of phagocytes adhering to the 96-well black and clear plate were determined by crystal violet staining. The adherent phagocytes in 4 wells of 96-well plate were washed by 50% methanol in PBS once and then stained with 0.5% crystal violet in methanol for 30 min. After washing with tap-water and air-drying, the crystal violet in the cells was dissolved with 100 ml of 20% acetic acid, and the dissolved crystal violet was measured by MRX II ELISA reader (DYNEX technologies) at OD₅₉₅.

2.9. Phagocytic activity

The phagocytic activity of the phagocytes in 4 wells of 96-well black plate was analyzed with pHrodo BioParticles Conjugates for Phagocytosis (invitrogen). The procedure of analysis was followed by the product information sheet. The phagocytic activity was calculated as the fluorescent intensity of internalized Escherichia coli divided by OD₅₉₅ of crystal violet in the adherent phagocytes (Section 2.8). The average phagocytic activity of the head-kidney phagocytes in the groupers without LAB4012-feeding was regarded as 1.

2.10. Respiratory burst activity

The phagocytes in the 96-well clear plate (Section 2.8) were stained with 100 ml of 0.2% nitroblue tetrazolium (NBT) in L15 medium for 1 h. Subsequently, cells were washed once with 70% methanol, fixed with 100% methanol for 3 min, and then washed 3 times with 70% methanol. After air-drying, formazan was dissolved by adding 120 ml of 2 M KOH and 140 ml of DMSO. The OD₅₉₅ of the formazan was measured by MRX II ELISA reader (DYNEX technologies). The respiratory burst activity was calculated as OD₅₉₅ of formazan divided by OD₅₉₅ of crystal violet in the adherent phagocytes (Section 2.8). The average respiratory burst activity of the head-kidney phagocytes in the groupers without LAB4012- feeding was regarded as 1.

3. Results

3.1. The growth inhibitory activity of LAB4012 culture supernatant against V. anguillarum

The culture supernatant of LAB4012 incubated for 5 h (LAB-5h, log phase) and 18 h (LAB-18h, stationary phase) were respectively collected to examine the inhibitory activity of LAB4012 culture supernatant against the growth of V. anguillarum. The pH value of MRS, the culture medium for LAB4012, was initially 6.3. After LAB4012 was cultured, the pH values of LAB-5h and LAB-18h became 5.0 and 4.2, respectively. To realize whether LAB-5h and LAB-18h could inhibit V. anguillarum growth, V. anguillarum was cultured in the medium supplemented with MRS (pH 6.3), LAB-5h (pH 5.0) and LAB-18h (pH 4.2), respectively. The biomass of V. anguillarum was detected at the indicated time points (Fig. 1). The results showed that LAB-18h exhibited higher growth inhibitory activity against V. anguillarum than LAB-5h, indicating that a higher growth inhibitory activity against V. anguillarum might be associated with a lower pH value of LAB4012 culture supernatant (Fig. 1).

Lactic acid bacteria can secret organic acids into the extracellular metabolites, making the culture medium more acidic. Lactic acid is one of the important organic acids in the metabolites of LAB4012. The concentration of lactic acid in LAB-18h was determined to be 73 mM. To elucidate the impact of lactic acid on the growth of V. anguillarum, MRS containing 73 mM lactic acid was added into the culture medium of V. anguillarum. Afterward, the growth curve of V. anguillarum was analyzed. The results revealed that MRS containing 73 mM lactic acid completely inhibited the growth of V. anguillarum as LAB-18h exhibited (Fig. 2), indicating that lactic acid in LAB-18h plays an important role in the growth inhibitory activity against V. anguillarum.

To understand whether the hydrogen ion derived from the organic acids in the LAB-18h was a critical factor to the growth inhibitory activity against V. anguillarum, the LAB-18h (pH 4.2) was adjusted to pH 6.3 (LAB-18h-NaOH) to analyze the growth inhibitory activity against V. anguillarum. The result showed that the growth curves of V. anguillarum cultured in the medium containing LAB-18h-NaOH (pH 6.3) and MRS (pH 6.3) were similar (Fig. 3), revealing that the reduction in acidity made the LAB-18h losing its growth inhibitory activity against V. anguillarum. Moreover, when MRS pre-adjusted to pH 4.2 (MRS-HCl) was supplemented into the culture medium of V. anguillarum, it inhibited the growth of V. anguillarum as LAB-18h (pH 4.2) exhibited (Fig. 3). It was suggested that the hydrogen ion derived from the organic acids in the LAB18h was a vital factor to the growth inhibitory activity against V. anguillarum.

The lactic acid concentration in the LAB4012 culture supernatant increased along with the culturing time from 1 to 18 h, accompanying the decrease of pH value (Fig. 4). The LAB4012 culture supernatant with longer incubating time resulted in higher growth inhibitory activity against V. anguillarum (Fig. 4). Furthermore, the 9 h-cultured supernatant of LAB4012, which contained 55 mM lactic acid, completely inhibited V. anguillarum growth (Fig. 4).

3.2. The protection of LAB4012-fed groupers against V. anguillarum infection

After 3-week feeding with LAB4012, the groupers were IPchallenged with 2 doses of V. anguillarum (5 x 10⁵ and 6 x 10⁵ CFU per fish). The results showed that the cumulative mortalities of LAB-fed groupers (10% for 5 x 10⁵ CFU and 15% for 6 x 10⁵ CFU) were significantly lower than those of groupers without LAB4012-feeding (40% for 5 x 10⁵ CFU and 65% for 6 x 10⁵ CFU) (Fig. 5). The RPS values in the challenge doses of 5 x 10⁵ and 6 x 10⁵ CFU were respectively 75 and 77. Therefore, LAB4012-feeding protected the groupers from V. anguillarum infection.

3.3. The effect of LAB4012-supplementation on the growth rate of orange-spotted grouper

Groupers ate the LAB4012-supplemented diet well, similar to the normal feed. The groupers were fed with fixed amount (4% body weight) of diet every day. All the daily diets were eaten up by the groupers, and no more diet was supplied. The growth rate of groupers fed with LAB4012 for 4 weeks were shown in Fig. 6. The result revealed that the percent weight gain of groupers fed with LAB4012 (101.2%) was significantly higher than that of control fish (80.9%) at 4 weeks post-feeding (wpf).

3.4. Gene expression of pro-/anti-inflammatory cytokines in LAB4012-fed and non-fed groupers

The gene expression levels of pro-inflammatory cytokines (TNF1, TNF-2 and IL-1b) and an anti-inflammatory cytokine (TGF-b) in the head-kidney and spleen of the LAB4012-fed and non-fed groupers were determined at 1 to 4 wpf. In the head-kidney, regardless being fed with or without LAB4012, the gene expression levels of TNF-1, TNF-2 and IL-1b showed no differences between groups (Fig. 7A). The gene expression level of TGF-b1 in control groupers at 2 wpf exhibited a 2-fold increase and then decreased at 3e4 wpf; however, TGF-b1 gene expression of LAB4012-fed groupers increased and maintained 2-fold level at 3e 4 wpf (Fig. 7A). In the spleen, the gene expression levels of TNF-1 and TNF-2 at most time points had no differences between LAB4012-fed and non-fed groupers except that TNF-2 gene expression level at 4 wpf in LAB4012-fed groupers was lower than that of control groupers (Fig. 7B). The expression levels of IL-1b gene in LAB4012-fed groupers increased 3-fold higher than that of control groupers at 1 wpf and then decreased to the level of control groupers at 1 wpf (Fig. 7B). The TGF-b1 gene expression levels in groupers without LAB4012-feeding fluctuated from 1 to 4 wpf, but those of LAB4012-fed groupers at 1e4 wpf maintained 1/3 or 1/2 level, compared with that of control groupers at 1 wpf (Fig. 7B).

Click here to enlarge the image

We further analyzed the influence of 1-week feeding with LAB4012 on the expression of immune genes in the groupers postV. anguillarum infection. After 1-week LAB4012-feeding, the groupers were challenged with V. anguillarum at a non-lethal dose. In the head-kidney, the gene expression levels of TNF-1, TNF-2 and IL1b in LAB4012-fed groupers were significantly lower than those of non-fed groupers at 2 dpi; however, the gene expression levels of TGF-b1 in LAB4012-fed groupers at 1 and 3 dpi were significantly higher than that of non-fed groupers (Fig. 8A). In the spleen, the TNF-2 gene expression level in the LAB4012-fed groupers was significantly lower than that in the control groupers at 1 dpi, but the IL-1b gene expression level was significantly higher than that in the control groupers. In addition, the TGF-b gene expression level in the LAB4012-fed groupers was significantly lower at 1 dpi and became considerably higher at 2e3 dpi, compared to that of control groupers (Fig. 8B).

Click here to enlarge the image

3.5. Blood cell counts of LAB4012-fed and control groupers

After 1-week LAB4012-feeding, the erythrocyte count (5.36 10⁸ cells ml^-1 ) of LAB4012-fed groupers was significantly higher than that (1.06 10⁸ cells ml^-1 ) of control groupers (Fig. 9A), but the leukocyte counts between the two groups showed no difference (Fig. 9B). At 1-day post-V. anguillarum infection with a non-lethal dose, either erythrocyte or leukocyte counts increased in both LAB4012-fed and control groupers; however, the erythrocyte and leukocyte counts in LAB4012-fed groupers were all significantly higher than those in control groupers (Fig. 9).

3.6. Phagocytic activity and respiratory burst activity of headkidney phagocytes in V. anguillarum-infected groupers with or without LAB4012-feeding

The groupers were fed with or without LAB4012 for 1 week and then challenged with V. anguillarum at a non-lethal dose. The phagocytic activity of head-kidney phagocytes in LAB4012-fed groupers was 4-fold higher than that in the groupers without LAB4012-feeding (Fig. 10A). However, no difference was observed in the respiratory burst activities of head-kidney phagocytes in LAB4012-fed and control groupers (Fig. 10B).

4. Discussion

In our previous study, the culture supernatant of LAB4012 was found to inhibit the growth of Photobacterium damselae subsp. piscicida [18], and the present study revealed that the supernatant of 18h-cultured LAB4012 also suppressed the growth of V. anguillarum. The components in the LAB4012 culture supernatant that contribute to the inhibition of V. anguillarum growth was investigated in this study. We demonstrate that the lactic acid in LAB4012 culture supernatant plays an important role in inhibiting the growth of V. anguillarum in vitro. Lactic acid is a potent outer membrane-disintegrating agent which can cause LPS release and damage the outer membrane of gram-negative bacteria, including Vibrio spp. [21]. LAB4012 could be detected in the grouper intestine after LAB4012-feeding (data not shown). Therefore, we suggested that the lactic acid produced by LAB4012 might make the numbers of pathogenic bacteria moderate in the intestine, which exhibited competitive exclusion of pathogenic bacteria [22]. Additionally, the LAB-18h containing 73 mM lactic acid generated a pH value of 4.1, while the pH of MRS supplemented with 73 mM lactic acid decreased only to 4.6 (Fig. 2), indicating that other kinds of organic acids were produced in the metabolites of LAB4012.

LAB4012 was identified as Pediococcus pentosaceus [18]. Pediococcus spp. has potential to secret the bacteriocin, i.e., pediocin [23]. However, neither pediocin gene was detected in LAB4012 with the method described by Todorov and Dicks (2009) [24], nor pediocin was purified from LAB4012 by using the method published by Yang et al. (1992) [25] (Data not shown). Therefore, it implies that pediocin is not involved in the inhibitory growth activity of LAB4012 culture supernatant against V. anguillarum.

In this study, feeding with LAB4012 was able to benefit the growth rate of orange-spotted groupers. Many studies have concluded that fish growth can be improved by feeding lactic acid bacteria, such as orange-spotted grouper fed with Lactobacillus plantarum [26], flounder fed with Lactobacillus spp. [27], Atlantic cod fed with Carnobacterium divergens [28], gilthead sea bream fed with Lactobacillus spp. [29], and nile tilapia fed with Lactobacillus acidophilus or Lactobacillus rhamnosus GG [30,31]. Advancing fish growth rate and food utilization via probiotic-feeding may be related to the effects of probiotic actions, including competitive exclusion of pathogenic bacteria and improvement of nutrition by producing the hydrolytic enzymes from probiotics to decompose the indigestible components and by supplying the fatty acid and vitamins [22,29,32e34].

LAB4012-feeding increases not only the growth rate but also the erythrocyte count (Fig. 9A). Cetin et al. (2005) [35] has reported that the counts of erythrocyte in turkeys also increased after feeding with probiotics, including Lactobacillus acidophilus, Lactobacillus casei, Enterotococcus faecium and Bifidobacterium thermophiles, respectively. Similarly, administration of diet with Lactobacillus acidophilus in health adult dogs significantly increases the concentration of erythrocyte [36]. Some studies reported that feeding with probiotics elevated numbers of erythrocytes in fish similar to higher vertebrates, including Oscar Astronotus ocellatus fingerlings fed with protexin [37], rainbow trout fed with probiotics, A1-6, A3-47S, A3-51, BA211, Kocuria SM1 [38e40], and goldfish fed with Aeromonas hydrophila A3-51 [41]. The increase of erythrocyte counts is suggested to reflect the improvement of the physiological function.

V. anguillarum infection stimulated the proliferation of both erythrocytes and leukocytes in the peripheral blood of groupers (Fig. 9). However, when compared with the non-fed groupers, LAB4012-feeding enhanced the groupers to proliferate more erythrocytes and leukocytes in peripheral blood and significantly improve the phagocytosis of head-kidney phagocytes as soon as one day post-V. anguillarum infection (Figs. 9 and 10A). Hence, LAB4012-feeding is suggested to facilitate the LAB4012-fed groupers to eliminate pathogens more quickly.

LAB4012-feeding increased the resistance of groupers against V. anguillarum infection (Fig. 5). The contributions of LAB4012- feeding to the protection of groupers against V. anguillarum is possibly related to three reasons. One is the enhancement of growth rate which strengthens the immunity of LAB4012-fed groupers. The second is that the high leukocyte counts in peripheral blood and phagocytic activity in head-kidney phagocytes of the infected groupers are very helpful to eliminate pathogens more efficiently. The third reason may be related to the better regulation of cytokine expressions for antagonizing the pathogens. LABfeeding has been reported to regulate cytokine gene expression and make animals more resistant to pathogens [42,43]. In this study, LAB4012-feeding to non-infected groupers did not affect the gene expression levels of TNF-1, TNF-2, and IL-1b in the headkidney, but it up-regulated the gene expression of TGF-b1, an anti-inflammatory cytokine (Fig. 7A). In addition, once the LAB4012-fed groupers were challenged with V. anguillarum, the gene expressions of cytokines were efficiently regulated in the head-kidney, including the decreased gene expression levels of pro-inflammatory cytokines (TNF-1, TNF-2, and IL-1b) and the increased gene expression levels of anti-inflammatory cytokine (TGF-b1) (Fig. 8A). Similar phenomenon has been observed that feeding with Lactobacillus plantarum CFLP 3 could protect the rainbow trout from Lactococcus garvieae infection, and decrease TNF-a and IL-1b gene expression in the head-kidney [42]. How the regulation of pro-/anti-inflammatory cytokines antagonizes V. anguillarum in the groupers needs further investigation.

In summary, this study revealed that lactic acid in LAB4012 metabolites played an important role in antagonizing V. anguillarum in vitro. Feeding with LAB4012 could enhance the growth rate of groupers and the erythrocyte counts, elevate proliferation of leukocytes and phagocytic activity of head-kidney phagocytes post-V. anguillarum infection, and protect the groupers from vibriosis. LAB4012 was demonstrated to be a viable probiotic for groupers, and dietary supplementation of LAB4012 would be a good alternative prophylaxis method to protect groupers against V. anguillarum infection.

Acknowledgments

The authors thank SynBioTech for the donation of LAB4012 (Pediococcus pentosaceus) and appreciate Mr. C. J. Cheng for English revision. This study was partially supported by a grant from Academic Sinica (Grant number 100S0020012) under the Development Program of Industrilization for Agriculture Biotechnology.

References

[1] Austin B, Austin DA. Bacterial fish pathogens: diseases of farmed and wild fish. 4th ed. New York-Chichester: Springer-Praxis Publishing; 2007.

[2] Thune RL, Stanley LA, Cooper RK. Pathogenesis of gram-negative bacterial infections in warmwater fish. Annu Rev Fish Dis 1993;3:37e68.

[3] Lee KK, Liu PC, Chuang WH. Pathogenesis of gastroenteritis caused by Vibrio carchariae in cultured marine fish. Mar Biotechnol 2002;4:267e77.

[4] Actis LA, Tolmasky ME, Crosa JH. Fish and disorders: viral, bacterial and fungal infections. Wallingford, UK: CABI International; 1999.

[5] Toranzo AE, Magarinos B, Romalde JL. A review of the main bacterial fish diseases in mariculture systems. Aquaculture 2005;246:37e61.

[6] Buller NB. Bacteria from fish and other aquatic animals: a practical identifi- cation manual. Wallingford, UK: CABI Publishing; 2004.

[7] Alderman DJ, Hastings TS. Antibiotic use in aquaculture: development of resistance e potential for consumer health risks. Int J Food Sci Technol 1998;33:139e55.

[8] Colquhoun DJ, Aarflot L, Melvold CF. gyrA and parC mutations and associated quinolone resistance in Vibrio anguillarum serotype O2b strains isolated from farmed Atlantic cod (Gadus morhua) in Norway. Antimicrob Agents Chemother 2007;51:2597e9.

[9] Rahim Z, Sanyal SC, Aziz KM, Huq MI, Chowdhury AA. Isolation of enterotoxigenic, hemolytic, and antibiotic-resistant Aeromonas hydrophila strains from infected fish in Bangladesh. Appl Environ Microbiol 1984;48:865e7.

[10] Hjeltnes B, Anderson K, Egidius E. Multiple antibiotic resistance in Vibrio salmonicida. Bull Eur Assoc Fish Pathol 1987;7:85.

[11] Gomezgil BRS, Grobois FAA, Jarero JR, Vega MDH. Chemical disinfection of Artemia nauplii. J World Aquacult Soc 1994;25:574e83.

[12] Bruun MS, Schmidt AS, Madsen L, Dalsgaard I. Antimicrobial resistance patterns in Danish isolates of Flavobacterium psychrophilum. Aquaculture 2000;187:201e12.

[13] Miranda CD, Zemelman R. Bacterial resistance to oxytetracycline in Chilean salmon farming. Aquaculture 2002;212:31e47.

[14] Sahul H, Balasubramanian G. Antibiotic resistance in bacteria isolated from Artemia nauplii and efficacy of formaldehyde to control bacterial load. Aquaculture 2000;183:195e205.

[15] Spanggaard B, Jørgensen F, Gram L, Huss HH. Antibiotic resistance in bacteria isolated from three freshwater fish farms and an unpolluted stream in Denmark. Aquaculture 1993;115:195e207.

[16] Sandaa RA, Torsvik VL, Goksøyr J. Transferable drug resistance in bacteria from fish-farm sediments. Can J Microbiol 1992;38:1061e5.

[17] Husevag B, Lunestad BT, Johannessen PJ, Enger O, Samuelsen OB. Simultaneous occurrence of Vibrio salmonicida and antibiotic-resistant bacteria in sediments at abandoned aquaculture sites. J Fish Dis 1991;14:631e40.

[18] Xing CF, Hu HH, Huang JB, Fang HC, Kai YH, Wu YC, et al. Diet supplementation of Pediococcus pentosaceus in cobia (Rachycentron canadum) enhances growth rate, respiratory burst and resistance against photobacteriosis. Fish Shellfish Immunol 2013;35:1122e8.

[19] Hong GE, Kim DG, Bae JY, Ahn SH, Bai SC, Kong IS. Species-specific PCR detection of the fish pathogen, Vibrio anguillarum, using the amiB gene, which encodes N-acetylmuramoyl-L-alanine amidase. FEMS Microbiol Lett 2007;269:201e6.

[20] Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162: 156e9.

[21] Alakomi HL, Skytta E, Saarela M, Mattila-Sandholm T, Latva-Kala K, Helander IM. Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane. Appl Environ Microbiol 2000;66:2001e5.

[22] 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.

[23] Papagianni M, Anastasiadou S. Pediocins: the bacteriocins of Pediococci. Sources, production, properties and applications. Microb Cell Fact 2009;8:3.

[24] Todorov SD, Dicks LM. Bacteriocin production by Pediococcus pentosaceus isolated from marula (Scerocarya birrea). Int J Food Microbiol 2009;132: 117e26.

[25] Yang R, Johnson MC, Ray B. Novel method to extract large amounts of bacteriocins from lactic acid bacteria. Appl Environ Microbiol 1992;58:3355e9.

[26] Son VM, Chang 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.

[27] Byun JW, Park SC, Benno Y, Oh TK. Probiotic effect of Lactobacillus sp. DS-12 in flounder (Paralichthys olivaceus). J Gen Appl Microbiol 1997;43:305e8.

[28] Gildberg A, Mikkelsen H, Sandaker E, Ringo E. Probiotic effect of lactic acid bacteria in the feed on growth and survival of fry of Atlantic cod (Gadus morhua). Hydrobiologia 1997;352:279e85.

[29] Suzer C, Coban D, Kamaci HO, Saka S, Firat K, Otgucuoglu O, et al. Lactobacillus spp. bacteria as probiotics in gilthead sea bream (Sparus aurata, L.) larvae: effects on growth performance and digestive enzyme activities. Aquaculture 2008;280:140e5.

[30] Aly SM, Abdel-Galil Ahmed Y, Abdel-Aziz Ghareeb A, 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.

[31] Pirarat N, Pinpimai K, Endo M, Katagiri T, Ponpornpisit A, Chansue N, et al. Modulation of intestinal morphology and immunity in nile tilapia (Oreochromis niloticus) by Lactobacillus rhamnosus GG. Res Vet Sci 2011;91:e92e7.

[32] Fuller R. Probiotics in man and animals. J Appl Bacteriol 1989;66:365e78.

[33] Planas M, Vazquez JA, Marques J, Perez-Lomba R, Gonzalez MP, Murado M. Enhancement of rotifer (Brachionus plicatilis) growth by using terrestrial lactic acid bacteria. Aquaculture 2004;240:313e29.

[34] Sakata T. Microbiology in poecilotherms. Amsterdam: Elsevier; 1990.

[35] Cetin N, Guclu BK, Cetin E. The effects of probiotic and mannanoligosaccharide on some haematological and immunological parameters in turkeys. J Vet Med A Physiol Pathol Clin Med 2005;52:263e7.

[36] Baillon ML, Marshall-Jones ZV, Butterwick RF. Effects of probiotic Lactobacillus acidophilus strain DSM13241 in healthy adult dogs. Am J Vet Res 2004;65: 338e43.

[37] Firouzbakhsh F, Noori F, Khalesi MK, Jani-Khalili K. Effects of a probiotic, protexin, on the growth performance and hematological parameters in the Oscar (Astronotus ocellatus) fingerlings. Fish Physiol Biochem 2011;37:833e42.

[38] Irianto A, Austin B. Use of probiotic to control furunclosis in rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 2002;25:333e42.

[39] Irianto A, Austin B. Use of dead probiotic cells to control furunculosis in rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 2003;26:59e62.

[40] Sharifuzzaman SM, Austin B. Kocuria SM1 controls vibriosis in rainbow trout (Oncorhynchus mykiss, Walbaum). J Appl Microbiol 2010;108:2162e70.

[41] Irianto A, Robertson PA, Austin B. Oral administration of formalin-inactivated cells of Aeromonas hydrophila A3-51 controls infection by atypical A. salmonicida in goldfish, Carassius auratus (L.). J Fish Dis 2003;26:117e20.

[42] Perez-Sanchez T, Balcazar JL, Merrifield DL, Carnevali O, Gioacchini G, de Blas I, et al. Expression of immune-related genes in rainbow trout (Oncorhynchus mykiss) induced by probiotic bacteria during Lactococcus garvieae infection. Fish Shellfish Immunol 2011;31:196e201.

[43] Liu W, Ren P, He S, Xu L, Yang Y, Gu Z, et al. Comparison of adhesive gut bacteria composition, immunity, and disease resistance in juvenile hybrid tilapia fed two different Lactobacillus strains. Fish Shellfish Immunol 2013;35: 54e62.

Dietary supplementation of Pediococcus pentosaceus enhances innate immunity, physiological health and resistance to Vibrio anguillarum in orange-spotted grouper (Epinephelus coioides)

Preventive measures in aquaculture: How to better protect our crops

Priya Chemicals