Open access peer-reviewed chapter

Use of Probiotic Bacteria against Bacterial and Viral Infections in Shellfish and Fish Aquaculture

By Héctor Cordero, María Ángeles Esteban and Alberto Cuesta

Submitted: September 20th 2013Reviewed: October 3rd 2013Published: February 19th 2014

DOI: 10.5772/57198

Downloaded: 4469

1. Introduction

The term “probiotic” was firstly used to denominate microorganisms that have effects on other microorganisms [1]. Etymologically, the term “probiotic” was originated from the Latin word “pro” which means “for” and the Greek word “bios” which means “life”. The best known definition for probiotics was developed by the Food and Agriculture Organization (FAO), that defined them as live microorganisms which when administered in adequate amounts confer a health benefit on the host [2]. According to this description, the potential benefits are varied, and if probiotics were administered to shellfish or fish under intensive culture they could improve their production. It is known that virus and bacterial diseases/infections are one of the most important problems in aquaculture production at present. Probiotics can provide some solutions to this problem through different mechanisms or properties such as the production of inhibitory compounds such as bacteriocins, competition for adhesion sites with opportunistic or pathogen microorganisms, competition for nutrients with other bacteria or an improvement of the immune status (e.g. increase of production of immunoglobulins, acid phosphatase, antimicrobial peptides, improvement of cellular activities, etc.) [3-10]. Several reviews have already documented the benefits of probiotics in shellfish and fish but they mainly focused on their effects in the immune response. Thus, hypothetical and desired results of administering probiotics to shellfish or fish in culture will be improving their antiviral and antibacterial defences, which is the focus of the present review. Firstly, a brief description of probiotics is included, and then a review of the main used probiotics against pathogenic virus and bacteria for shellfish and finally, the same for fish. The novelty of this review is based on the shared ability of probiotics to control both viral and bacterial diseases in shellfish and fish often share, which could be the basis for sustainable aquaculture.

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2. Probiotic bacteria

There is a great diversity of tested probiotic bacteria, but only few of them have become in commercial probiotics (Table 1). Thus, further studies are mandatory to expand the use of laboratory described microorganisms with probiotic effects to the commercial level and then be used in the aquaculture industry. The procedure to test and market a probiotic is resumed in Figure 1.

Commercial nameAnimal/HumanReference/Comments
AlCareTMMammalianContains Bacillus licheniformis
Alibio®Fish[30]
Bactisubtil®HumanContains Bacillus cereus
Bactocell® PA 10Fish[42]
BaoZyme-AquaFishContains Bacillus subtilis
BGY-35Fish[51]
Biogrow®MammalianContains Bacillus subtilisand B. licheniformis
Bio-Kult®HumanContains B. subtilis
BioPlus® 2BFish[73]
Biosporin®HumanContains B. subtilisand B. licheniformis
Biostart®FishContains a mix of Bacillusspp. and Paenobacillussp.
Biovicerin®HumanContains B. cereus
Bispan®HumanContains Bacillus polyfermenticus
Cernivet®Fish[85]
DomuvarHumanContains Bacillusspp.
Ecomarine®Shellfish
Esporafeed Plus®SwineContains B. cereus
LactobacilFish[45]
LactopureMammalianContains Lactobacillus sporogenes
Liqualife®FishContains Bacillusspp.
Neoferm BS 10MammalianContains Bacillus clausii
NeolactofloreneHumanContains Lactobacillusspp. and Bacillusspp.
Promarine®Shellfish
SanoCare®FishContains Bacillusspp.
SanoGuard®FishContains Bacillusspp.
SanoLife®FishContains Bacillusspp.
SporolacFish[45]
Sustenex®HumanContains Bacillus coagulans
Toyocerin®Fish[85]

Table 1.

List of commercial probiotics, including those for shellfish and fish.

Probiotics are usually consisting on bacteria but some other microorganisms such as yeast, microalgae or even some fungi. They are mainly used as living cells but some studies have also shown their benefits when supplied as heat-inactivated cells (also known as heat-killed cells), formalin-killed (FKC), freeze-dried, dead cells or cell-free supernatant (CFS). Among the vast number of probiotic species used most information relies on the use of Bacillussp. and Lactobacillussp. Different administration modes have been checked, as bath, intraperitoneal or intramuscular injection and in diet being the bath and diet those preferred for the use in the aquaculture. Moreover, more recently, for oral dietary administration the probiotics can be encapsulated in different ways. Besides that, Artemiaand rotifers (two main diets larvae in marine larviculture) are usually enriched with probiotics in order to produce benefits in the fish/shellfish larvae.

Figure 1.

Process for making commercial probiotics.

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3. Probiotics against virus in shellfish

Viral infections are one of the most important problems in aquaculture production. In the case of shellfish, probiotics might provide a good preventive solution to this problem since they promote the innate immune response, which is the only one attributed to be responsible for the resistance in these animals.

Mainly seven viral diseases are known in shellfish which are: white spot syndrome virus (WSSV), lymphocystis disease virus (LCDV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), taura syndrome virus (TSV), yellow head disease virus (YHV), infectious myonecrosis virus (IMNV) and Macrobrachium rosenbergiinodavirus (MrNV). Unfortunately, all the studies have focused on the potential preventive effects of few probiotics on the pacific white shrimp (Litopenaus vannamei) resistance against WSSV. In a single study it was demonstrated that bath treatment of L. vannameispecimens with the probiotic Vibrio alginolyticusat a dose of 105 cfu ml-1 showed a higher rate survival against WSSV compared to those non exposed to the probiotic [11]. Interestingly, most of the information comes from studies using dietary administration of the probiotics which results the most desired for aquaculture of shellfish. It has been reported that survival of L. vannameispecimens fed supplemented diets containing 105 cfu g-1 of a mixture formed by lactic acid bacteria (BAL3, BAL7, BC1 and CIB1) failed to protect against WSSV infections [12]. By contrast, dietary administration of 1010 cfu g-1 of BacillusOJ in L. vannameispecimens produced significantly higher survival after challenge by WSSV [13]. It has also been reported that dietary administration of Pediococcus pentosaceusand Staphylococcus hemolyticusto L. vannameispecimens showed a decrease in the prevalence of WSSV, but not IHHNV [14]. Further studies including more shellfish species and virus are necessary in order to find potential solutions for the viral diseases found under their intensive culture.

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4. Probiotics against bacteria in shellfish

In the case of bacterial diseases much more studies have focused on the benefits of the use of probiotics for shellfish species. Moreover, and in contrast to the viral pathogens described above, more shellfish species have focused the studies about the use of probiotics. Herein we will summarize the main findings about the potential use of probiotics against bacterial diseases grouped by shellfish species.

A first attempt to describe the probiotic potential of a microorganism comes from in vitrostudies. Thus, it has been demonstrated that Pseudoalternomonassp. strains DIT09, DIT44 and DIT46 isolated from Peromytilus purpuratusshowed bacteriostatic anti-Vibrio parahaemolyticusactivity [15] but their in vivoeffects have not been tested yet. In a similar way, Roseobactersp. strain BS107 isolated form the scallop (Pecten maximus) showed antibacterial activity against several pathogenic Vibriosp. [16] as well as the probiotic Alteromonas haloplanktisobtained from Argopecten purpuratuslarvae specimens [17]. Further preliminary studies of this kind are worthy to be taken in the future and prior to those conducted in vivo.

Several studies have been conducted in bivalves. In the case of Pacific oyster larvae (Crassostrea gigas) exposed to 105 cfu ml-1 of the pathogenic Vibrio tubiashiireached a total mortality in just 2 days, whilst in combination with 104 cfu ml-1 of the probiotic Aeromonas mediaA199 strain the larvae prolonged their viability up to 144 hours indicating its benefits when used by bath [18]. By contrast, C. virginicaspecimens fed supplemented diets containing 104 cfu ml-1 of Vibriosp. OY15 for three weeks showed no effect in survival ratio after challenge with Vibrio sp. M183 [19]. It has been reported [20] that abalone (Haliotis discus hannai) specimens fed supplemented diet with 109 cfu g-1 of Shewanella colwellianaWA64 and Shewanella oyellanaWA65 for four weeks showed a better survival rate (with mortalities of 27%-50% in WA64, and 30%-43% in WA65 compared with 77%-80% in the control group) when infected with Vibrio harveyi. In other research with other abalone specie, Haliotis midaespecimens fed supplemented diet with a mix of three unknown probiotic strains (SY9, SS1 and AY1) at doses of 107 cfu ml-1 for two weeks showed a better survival ratio (62%) than control group specimens after intra-mantle injection of Vibrio anguillarum[21]. Further studies are still needed to broad the use of probiotics in bivalves against bacterial diseases.

Among the shellfish, most of the studies have at this respect focused on shrimps. Thus, western king prawn (Penaeus latisulcatus) specimens fed 20×105 cfu kg-1 diet of Pseudomonas aeruginosaand Pseudomonas synxanthafor eighty-four days and afterwards challenged with V. harveyi. P. aeruginosa-supplemented diet improved the survival rate of the western king prawns more effectively than P. synxantha-supplemented diet, and furthermore, administration of both probiotics in combination resulted in better results than when administering separately [22-23].

Most of the studies administering probiotics have been developed in white shrimp (Litopenaeus vannamei) at different development stages. For example, Bacillus subtilisE20 administered in the diet at 106, 107 and 108 cfu kg-1 increased the survival rates at 13.3%, 16.7% and 20% respectively, after the injection of pathogenic V. alginolyticus[24]. In juvenile specimens, commercial white shrimp fed supplemented diet with 105 cfu g-1 diet of Bacillus subtilisUTM126 achieved a mortality of 18% against pathogenic infection of vibrios (including V. harveyi, V. alginolyticusand V. parahaemolyticus) while the control group mortality exceeded of 50% [25]. In other research, juvenile specimens fed supplemented diets containing V. alginolyticusUTM 102, B. subtilisUTM 126, Roseobacter gallaeciensisSLV03 or Pseudomonas aestumarinaSLV22, separately, at doses of 105 cfu g-1 diet for four weeks showed low mortality (between 17%-22%) after immersion with Vibrio parahaemolyticusPS-017 compared with the control group (33%) [26]. In adult specimens of L. vannameifed supplemented diet with 3×105 cfu of the probiotic Vibrio gazogenesper shrimp showed a decrease of mortality after infection with Vibriospp. (including V. harveyi, V. anguillarumand V. alginolyticus) [27]. In addition, the inhibitory effect was also demonstrated in a in vitroassay [27]. Other recent work [28] has been carried out with white shrimp fed a supplemented diet containing 105 cfu g-1 (BM5) and 108 cfu g-1 (BM8) (two Bacillus subtilisstrains) for 2 months, and afterwards each shrimp was injected with 107 cfu of Vibrio harveyi. Results indicate that cumulative mortality of the control group was 63.3%, whereas in the groups fed probiotics were of 20% and 33.3%, for the group fed BM8 or BM5 strains, respectively. Cumulative mortality also decreased in white shrimp fed a supplemented diet with 1010 cfu kg-1 of Lactobacillus plantarumafter injection with V. alginolyticus[29]. Moreover, the administration of a mixture of Bacillus(B. endophyticusYC3-b, B. endophyticusC2-2 and B. tequilensisYC5-2) to the water at doses of 0.1×106 cfu ml-1 to juvenile specimens resulted in a high survival ratio (33%) compared with the control group (9.5%) after challenge with V. parahaemolyticus. However, a commercial probiotic (Alibio) at the same dose that the Bacillusmix had no effect in survival ratio compared with the control group in Litopenaeus vannameispecimens [30]. L. vannameispecimens fed diet supplemented with two potential probiotics (strains C2 and B6) achieved a better survival ratio (44% and 50%) than control group (21%) after infection with Vibrio harveyiin stages from Myosis 3 to postlarvae 1 [31]. Strikingly, other microorganisms such as yeast have been also assayed as potential probiotics. Unfortunately, L. vannameispecimens fed Saccharomyces cerevisiae, Phaffia rhodozymaand Saccharomyces exiguusshowed no significant different in survival ratio after infection with V. harveyicompared with control group specimens [32].

Black tiger shrimp (Penaeus monodon) has also received much attention. Thus, P. monodonspecimens exposed to 106 cfu ml-1 of B. subtilisBT23 for 5 days (long-term treatment) or for 1 hour (short-term treatment), and thereafter challenged with V. harveyi, showed a decrease in their cumulative mortality in both groups (32% and 60%, respectively) [33]. In other research, P. monodonjuvenile specimens fed Bacillussp. S11 at 1010 cfu g-1 diet for one month and infected with V. harveyi,combined with ozone addition, showed a significant increase in the survival ratio (75%) compared with the control group and not fed with probiotics [34]. Also in juvenile specimens fed supplemented diet containing Lactobacillus acidophilus04 at dose of 105 cfu g-1 for one month showed a higher survival ratio (80%) than the control group (13.3%) after challenged with Vibrio alginolyticus[35]. In postlarvae specimens, dietary administration of Paenibacillussp. EF012164 and Bacillus cereusDQ915582 at doses of 104 and 105 cfu ml-1 caused lower mortality after infection with Vibrio harveyiand Vibriospp. (without statistical analysis) [36]. In other work, Penaeus monodonpostlarvae specimens fed supplemented diet with 109 cfu g-1 diet of two strains of Synechocystissp. (C51 and C54) separately for twenty days showed significantly better survival after infection with Vibrio harveyiMCCB 111 than those fed without probiotics [37]. Also in postlarvae specimens, dietary administration of Bacillussp. P11 at 109 cfu g-1 caused a high survival ratio (66%) compared with the control group (0%) after 9 days of infection with Vibrio harveyiand Vibriospp. [38]. Dietary administration of Artemia-encapsulated Bacillussp. S11 showed an increased survival of Penaeus monodonwhen infected with Vibrio harveyiD331 [39]. Finally, dietary administration to P. monodonwith 103 cfu ml-1 of Pseudomonassp. PM11 and Vibrio fluvialisPM17 for 45 days did not alter the mortality after challenge with Vibrio anguillarum[40]. As it has been widely shown in shellfish and fish the use of low or suboptimal dosages of probiotics have no biological role, and in this case protective effect against pathogens.

Other shrimp species have received little attention. In the Indian white shrimp (Penaeus indicus) juvenile specimens fed diets supplemented with Lactobacillus acidophilus, Streptococcus cremoris, Lactobacillus bulgaricus56 or L. bulgaricus57 at doses of 5×106 cfu g-1 for 4 weeks and infected with Vibrio alginolyticusshowed a higher survival rate (56% - 72%) compared with that observed in specimens of the control group (20%) [41]. Similarly, in blue shrimp (Litopenaeus stylirostris) specimens fed supplemented diet of 107 cfu g-1 of Pediococcus acidilacticifor 4 weeks and infected with Vibrio nigripulchritudoSFn1 showed a mortality level of 25% in the probiotic-treated group while in non-treated group the mortality was of 41.7% [42]. It was also reported that Penaeus chinensispostlarvae specimens exposed to ArthrobacterXE-7 at dose of 106 cfu ml-1 and pathogenic Vibriossp. (Vibrio parahaemolyticus, Vibrio anguillarumand Vibrio nereis) showed a significant higher survival ratio than specimens exposed to pathogenic Vibriosspp. alone [43].

Marron (Cherax tenuimanus) specimens fed five probiotics (Bacillussp. AQ2, Bacillus mycoidesA10, Shewanellasp. A12, Bacillus subtilisPM3 and Bacillussp. PM4) separately showed no significant differences in survival rate. However, the total haemocyte count was significantly higher in all probiotic-treated groups compared with the control group after injection with 2×108 cfu ml-1 of Vibrio mimicus[44].

Overall, studies have shown that probiotics are good alternative to protect shellfish against pathogenic bacteria, namely against Vibriosp. pathogens, the most important in the culture of shellfish. However, further studies are necessary to broad the probiotic candidates and the shellfish species prior they are applied to aquaculture from a practical point of view. Moreover, the mechanisms behind this protection are generally ignored and deserve deeper evaluation.

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5. Probiotics against virus in fish

Viral diseases are major problems in fish farming since there is a lack of suitable antiviral agents and a very limited number of effective vaccines. Moreover, there are few studies about the effects of probiotics against viral infections in fish. Olive flounder (Paralychthys olivaceus) and grouper (Epinephelus coioides) are the two main species which have been studied. Olive flounder specimens fed 2.4×108 cfu g-1 of Lactobacil and/or Sporolac (commercial acid lactic bacteria) were infected with lymphocystis disease virus (LCDV) [45]. Lowest mortality rate was seen in groups fed Lactobacil (30%) or Lactobacil and Sporolac (25%) supplemented diets followed by groups receiving Sporolac alone (45%) compared to those groups fed without probiotics that showed a mortality of 80%. Evaluating the disease resistance of grouper through probiotics against virus infection, a recent study has demonstrated that specimens fed a supplemented diet with 108 cfu g-1 of B. subtilisE20 for 28 days showed a survival rate of 50% higher than the control group for seven days post-infection with iridovirus [46]. In another study, grouper specimens fed a diet containing L. plantarumat 108 cfu kg-1 and challenged with an iridovirus showed an increase in the survival of 36.7% compared to the survival rate in control group [47]. Similar results were obtained when grouper specimens were fed S. cerevisiaesupplemented diet (5.3×107 cfu kg-1 for four weeks) and afterwards infected with a grouper iridovirus (GIV). Specimens of treated group showed a higher survival ratio (43.3%) than specimens in the control group (16.7%) [48]. Viral pathogens diversity and impact in the actual aquaculture deserves further characterization of the potential benefits of probiotics for economically important cultured fish world-wide.

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6. Probiotics against bacteria in fish

By far, the effects of probiotics on fish have received most of the investigations. Among the fish studied, the rainbow trout (Oncorhynchus mykiss) has been the most evaluated. Many different probiotic bacteria have been tested and two of the best studied are Bacillus subtilisand Lactobacillus acidophilus,two lactic acid bacteria which showed in vitroinhibition against Aeromonas hydrophila[49]. Furthermore, B. subtilisavoids the development of Pseudomonas fluorescenswhile L. acidophilushad also antimicrobial activity against Streptococcus iniae. The information relative to the use of probiotics as a beneficial treatment of fish against bacterial pathogens is described below and summarized (Table 2).

Fish testedProbioticPathogenSurvivalCites
Anguilla anguillaEnterococcus faeciumSF68Edwardsiella tarda 981210L1Significant increase for SF68 and no difference for B. toyoi[85]
Bacillus toyoi
Anguilla japonicaLactobacillus pentosusPL11Edwarsiella tardaSignificant increase[87]
Carassius auratusAeromonas hydrophilaA3-51 formalin-inactivatedAeromonas salmonicidaSignificant increase[90]
Carassius auratusBacillussp., Lactobacillussp., Streptococcus faecium, and Saccharomyces cerevisiaePseudomonas fluorescens58CNo differences[89]
Xiphophorus helleri
Clarias gariepinusLactobacillus acidophilusStaphylococcus xylosusSignificant increase[91]
Aeromonas hydrophilagr2
Streptococcus agalactiae
Dicentrarchus labraxVagococcus fluvialisVibrio anguillarumSignificant increase[107]
Epinephelus coioidesLactobacillus plantarumStreptococcussp.Significant increase[47]
Saccharomyces cerevisiaeStreptococcussp.Significant increase[48]
Bacillus subtilisE20Streptococcussp.Significant increase[46]
Gadus morhuaCarnobacterium divergensVibrio anguillarumSignificant increase[57]
Aeromonas salmonicida
Labeo rohita
Bacillus subtilisAeromonas hydrophilaNo difference[96]
Pseudomonas aeruginosaVSG-2Aeromonas hydrophila MTC1739Significant increase[98]
Lactobacillus plantarumVSG-3Aeromonas hydrophilaSignificant increase[97]
Miichthys miiuyClostridium butyricumCB2 as alive and dead cellsVibrio anguillarumSignificant increase[94]
Aeromonas hydrophila
Mycteroperca rosaceaDebariomyces hanseniiCBS-8000339Aeromonas hydrophilaAH-315No difference[50]
Oncorhynchus mykissClostridium botyricumVibrio anguillarumSignificant increase[95]
Streptococcus iniaeDan-1 formalin inactivatedStreptococcus iniaevirulentSignificant increase[80]
Pseudomonas fluorescensAH2Vibrio anguillarumSignificant increase[72]
Lactobacillus rhamnosusATCC 53103Aeromonas salmonicidassp. salmonicidaSignificant increase[67]
Aeromonas hydrophilaA3-51
Vibrio fluvialisA3-47S
Carnocteriumsp. BA211
Unidentified coccus A1-6
Aeromonas salmonicidaSignificant increase[60]
Aeromonas hydrophilaA3-51
Vibrio fluvialisA3-47S
Carnocteriumsp. BA211
Unidentified coccus A1-6
formalin-inactivated
Aeromonas salmonicidaSignificant increase[62]
Bacillus subtilis Bacillus licheniformisYersinia ruckeriSignificant increase[73]
Carnobacterium maltaromaticumB26
Carnobacterium divergensB33
Yersinia ruckeri
Aeromonas salmonicida
Significant increase[75]
Lactococcus lactisssp. lactisCFLP100
Leuconostoc mesenteroidesCLFP196
Lactobacillus sakeiCLFP201
Aeromonas salmonicidassp. salmonicidaCLFP501Significan increase[63]
Bacillussp. JB-1
Aeromonas sobriaGC2
Streptoccocus iniaeSignificant increase[64]
Lactococcus garvieae
Vibrio anguillarum
Vibrio ordalii
Aeromonas salmonicida
Yersinia ruckeri
Bacillus subtilisAB1 as live, sonicated and formalized cells and cell-free supernatantAeromonassp.Significant increase[82]
Brochothrix thermophastaBA211
Aeromonas sobriaGC2
Aeromonas bestiarumORN2Significant increase[65]
Brochothrix thermophastaBA211
Aeromonas sobriaGC2
Ichthyophthrius multifiliisSignificant increase for GC2 and no difference for BA211[65]
Leuconostoc mesenteroidesCLFP196Lactococcus garvieaeSignificant increase[68]
Lactobacillus plantarum
CLFP238
Enterobacter cloacaeYersinia ruckeriSignificant increase[74]
Bacillus mojavensis
KocuriaSM1Vibrio anguillarumSignificant increase[69-71]
Lactobacillus plantarumCLFP238Lactococcus garvieaeCLFP LG1Significant increase[66]
Lactococcus lactisCFLP100
Leuconostoc mesenteroidesCLFP196
Pseudomonassp. M174 and M162Flavobacterium psychrophilumSignificant increase[79]
Enterococcus faecalisinactivatedAeromonas salmonicidaSignificant increase[81]
Oplegnathus fasciatusLactobacillus sakeiBK19Edwarsiella tardaNo difference[88]
Oreochromis niloticusLactobacillus acidophilus, Bacillus subtilis, Clostridium butyricumand Saccharomyces cerevisiaeEdwardsiella tardaSignificant increase[86]
Bacillus subtilisAeromonas hydrophila,Significant increase[49]
Lactobacillus acidophilus
Pseudomonas fluorescens
Streptococcus iniae
OreochromisSaccharomyces cerevisiaeAeromonas hydrophilaSignificant increase[51]
Pseudomonas fluorescens
Flavobacterium columnare
Paralichthys olivaceusZooshikellasp. JE-34Stretoccocus iniaeSignificant increase[93]
Bacillus subtilisStreptococcus iniaeSignificant increase (except for B. licheniformis)[92]
Bacillus pumilus
Bacillus licheniformis
Salmo salarVibrio alginolyticusAeromonas salmonicida256/81Significant increase[52]
Vibrio anguillarumVIB256
Vibrio ordalii17K
Vibrio alginolyticusYersinia ruckeriEx5No difference[52]
Pseudomonas fluorescensAH2Aeromonas salmonicidaNo difference[55]
Salmo truttaLactococcus lactisssp. lactisCLFP100Aeromonas salmonicidaSignificant increase[83]
Leuconostoc mesenteroidesCLFP196
Salvelinus fontinalisS1, S5, S9 and S10Flavobacterium columnareSignificant increase[84]
Scophthalmus maximusRoseobactersp. strain 27-4Vibrio anguillarumSignificant increase[108]
Phaeobactersp.Vibrio anguillarumUnmeasured[102]
Ruegeriasp.
Lactobacillus plantarumVibriosp.Significant increase[99]
Carnobacteriumsp.
Roseobactersp.
Solea senegalensisShewanella putrefaciensPdp11Photobacterium damselaessp. piscicidaSignificant increase[104-105]
Shewanella balticaPdp13
Sparus aurataShewanella putrefaciensPdp11Vibrio anguillarumDC11R2Significant increase[103]
Bacillus subtilisPhotobacterium damselaessp. piscicidaNo effect[109]

Table 2.

Overview of the effects of probiotics against bacteria in fish.

Few works have evaluated the disease resistance of grouper (Epinephelus coioides) through probiotics against the pathogenic Streptococcussp. Thus, dietary treatment of grouper specimens fed Lactobacillus plantarumat 106 to 108 cfu kg-1 [47] or 108 cfu g-1 of Bacillus subtilisE20 [46] showed a better survival rate than the control. Moreover, the yeast Saccharomyces cerevisiaehas shown probiotic effects in the grouper. Feeding with 5.3×107 cfu kg-1 yeasts four weeks showed a higher survival ratio (56.6%) than the control group (20%) after infection with Streptococcussp. [48].

Leopard grouper (Mycteroperca rosacea) specimens fed supplemented diet with 106 cfu g-1 of Debaryomyces hanseniiCBS 8339 for five weeks showed an increase in immunoglobulin M (IgM), catalase (CAT) and superoxide dismutase (SOD) after infection with Aeromonas hydrophilaAH-315 and there was no mortality in any group [50].

Nile tilapia (Oreochromis niloticus) fed supplemented diet containing 0.5×107 cfu g-1 of a mixture of B. subtilisand L. acidophilus, or 107 cfu g-1 of each bacteria alone, for two months showed a higher relative level of protection against Aeromonas hydrophila, Pseudomonas fluorescensand Streptococcus iniaecompared to the control group [49]. The results were even better when fish were fed a commercial probiotic supplemented diet containing S. cerevisae. Similar results were also obtained in another two experiments using as a challenge an injection of 2×107 cfu ml-1 of P. fluorescensand fish immersion with 2×109 cfu ml-1 of Flavobacterium columnare[51].

Probiotic bacteria identified as Vibrio alginolyticuswas inoculated intramuscular or intraperitoneally in atlantic salmon (Salmo salar) at doses of 4×106 cfu ml-1 followed by a bath for ten minutes in a suspension of the same probiotic with 108 cfu/ml and seven days later fish were challenged with Aeromonas salmonicida256/81, Vibrio anguillarumVIB256, Vibrio ordalii17K or Yersinia ruckeriEx5 [52]. So, this work indicated that application of the probiotic to salmon specimens induced a decrease in mortalities after challenge with Aeromonas salmonicida256/81, and to a lesser extent with Vibrio anguillarumVIB256 and Vibrio ordalii17K and does not reduce mortality with Yersinia ruckeriEx5. In this sense, competition in vitrostudies will help to elucidate these in vivoresults. In other work [53] atlantic salmon specimens were fed a supplemented diet with 5×108 cells ml-1 of the microalgae Tetraselmis suecicafor 14 days were challenged with fish pathogens. Results showed that use of T. suecicaas a probiotic supplement was successful in preventing mortalities caused by Aeromonas hydrophila, Aeromonas salmonicida(strains LL and NG), Serratia liquefaciens, Vibrio anguillarum, Vibrio salmonicidaand Yersinia ruckeritype I. Salmo salarfry specimens which were fed Lactobacillus plantarumat dose of 2.5×109 cfu g-1 and infected with Aeromonas salmonicidaAL2020 showed a cumulative mortality lower than infected control group [54]. Pseudomonas fluorescensAH2 at doses of 103-105 cfu ml-1 in water did not confer protection against Aeromonas salmonicidain Salmo salarspecimens [55]. It has been also reported in vitrothat the pathogen Vibrio anguillarumLFI1243 showed a complete inhibition of growth in presence of Carnobacterium divergensstrains [56]. This is in accordance with another study showing that Carnobacteriumsp. isolated from salmon inhibited the growth of both Vibrio anguillarumand Aeromonas salmonicidain intestinal fish mucus [57]. Interestingly, Carnobacterium divergensisolated from Salmo salarspecimens were also tested as fed probiotics in atlantic cod (Gadus morhua) specimens which showed lower mortalities.

The most studied fish specie regarding the potential benefits of probiotics is the rainbow trout (Oncorhynchus mykiss). In vitrostudies have demonstrated the competitive adhesion and production of antagonistic compounds by some lactic acid bacteria (Lactococcus lactisssp. lactisCLFP100, Lactococcus lactisssp. cremorisCLFP102 and Lactobacillus curvatusCLFP150) against fish pathogens, including Aeromonas salmonicidassp. salmonicidaCLFP 501, Carnobacterium piscicolaCLFP 601, Lactococcus garvieaeCLFP LG1, Vagococcus salmoninarumCLFP 602, Yersinia ruckeriATCC 29473 and Vibrio anguillarumLa192 [58]. In another in vitroassay authors checked the inhibitory effect of Carnobacteriumsp. and Pseudomonassp. isolated from gut of rainbow trout against Vibrio anguillarum, although there was no correlation with the in vivostudy since the same probiotic failed to protect them against Vibrio anguillaruminfection [59]. In rainbow trout specimens fed 107 cfu g-1 of four putative probiotics (Aeromonas hydrophila, Vibrio fluvialis, Carnobacteriumsp. and an unidentified coccus) showed a better survival after intraperitoneal injection of Aeromonas salmonicida[60]. However, the same dietary doses of Carnobacterium inhibensand Vibrio alginolyticusconferred a lower protection against Aeromonas salmonicida. These results were correlated with other two studies [52, 61]. In rainbow trout fingerlings, the same four putative probiotics seen previously [60] but administered as formaline-inactivated bacteria showed a lower mortality (4%, 4%, 8% and 0%, respectively) after challenge with Aeromonas salmonicida[62] suggesting that the use of dead probiotics has also many benefits for fish. Dietary administration of lactic acid bacteria (Lactococcus lactisssp. lactisCLFP 100, Leuconostoc mesenteroidesCLFP 196, and Lactobacillus sakeiCLFP 202) at doses of 106 cfu g-1 for 2 weeks showed a survival rate of 97.8%-100% (versus 65.6% in the control group) when trout specimens were challenged with Aeromonas salmonicidassp. salmonicidaCLFP 501 [63]. It has been reported that dietary supplementation with Bacillussp. JB-1 and Aeromonas sobriaGC2 at doses of 2×108 and 107 cfu g-1, respectively for two weeks led to a higher survival rates in trout after challenge with Streptococcus iniaeand Lactococcus garvieaeat doses of 2×107 cfu ml-1, and Vibrio anguillarum, Vibrio ordalii, Aeromonas salmonicidaand Yersinia ruckeriat doses of 3×108 cfu ml-1 [64]. Thus, survival rates in specimens fed control diets were 0%-20% whereas in specimens fed probiotic-diets survival rate was 100% in all treatments (with JB-1 and GC2) with all pathogens bacteria except for Vibrio anguillarum(87% and 94% respectively) and Yersinia ruckeri(94% in GC2 diet). In other study it has been found that dietary administration of Aeromonas sobriaGC2 at dose of 108 cfu g-1 and Brochothrix thermosphastaBA211 at dose of 1010 cfu g-1 for two weeks showed a higher survival rate (76% and 88%) than in control group (22%) after intramuscular injection with Aeromonas bestiarumORN2 [65]. In the same experiment, it was demonstrated that GC2 probiotic exerts resistance also against ichthyophthiriasis (caused by the parasite Ichthyophthirius multifiliis) however BA211 strain had no effect against this pathogen. An in vitroassay tested the inhibitory ability of Lactobacillus plantarumstrains, Lactococcus lactisstrains and Leuconostoc mesenteroidesstrains against Lactococcus garvieaeCLFP LG1 [66]. Other research [67] reported that rainbow trout specimens fed Lactobacillus rhamnosusATCC 53103 at doses of 109 and 1012 cfu g-1 for fifty-one days obtained a reduced mortality (18.9% and 46.3%, respectively) compared with the control group (52.6%) when were infected with Aeromonas salmonicidassp. salmonicida.An in vivoassay against lactococcosis, dietary administration with lactic acid bacteria (Leuconostoc mesenteroidesCLFP 196, and Lactobacillus plantarumCLFP 238) at doses of 106 cfu g-1 for four weeks showed a decrease in cumulative mortality (46% and 54%) compared with the control group (78%) in trout specimens after injection with Lactococcus garvieae[68]. Following with the development of protection in rainbow trout, specimens were fed a supplemented diet with 108 cfu g-1 of KokuriaSM1 for four weeks and after replacement for control diet they were infected with Vibrio anguillarumevery week [69]. Interestingly, this relative protection was maximum (87%) just after the end of the probiotic-supplemented diet that was disappearing with the time and was of 71%, 68%, 62% and 36% after two, three, four and five weeks after cessation of probiotic, respectively, representing a sign of gradual loss of effect [70, 71]. In other research, O. mykissspecimens exposed to Pseudomonas fluorescensAH2 at 105 cfu ml-1 for 5 days or added in situwhen challenged with Vibrio anguillarumshowed a higher survival ratio (56% and 65%, respectively) than specimens exposed to Vibrio anguillarumwithout probiotic (50%) [72]. Dietary administration of BioPlus2B, wich contains two probiotic bacteria (Bacillus subtilisand Bacillus licheniformis) for four weeks resulted in a better survival ratio (41.7%) compared with Ergosan-diet (8.9%) and control diet (9%) in trout specimens after intraperitoneal injection of Yersinia ruckeri[73]. Following with the protection against yersiniosis, dietary administration of 108 cfu g-1 of Enterobacter cloacaeand Bacillus mojavensisseparately for two months achieved a high survival ratio (99.2%) compared with the control group (35%) when infected with Yersinia ruckeri[74]. In addition, in other research, dietary administration of 107 cfu g-1 of Carnobacterium maltaromaticumB26 and Carnobacterium divergensB33 separately for two weeks conferred protection against Yersinia ruckeriwith a high survival ratio of 73% and 80% respectively, compared with the control group (13%); and the same probiotics (B26 and B33) also provided protection against Aeromonas salmonicidawith a survival ratio of 80% in both cases compared with the control group (20%) [75]. Flavobacterium psychrophilumis the causative agent of coldwater disease (CWD), also known as rainbow trout fry syndrome (RTFS). Although many types of salmonids are susceptible to RTFS, rainbow trout can be especially impacted due to direct mortality or deformities in surviving specimens leading to economic losses in aquaculture [76, 77]. In order to establish strategies of resistance against CWD with probiotics, in two studies [78, 79] it was demonstrated the ability of Pseudomonassp. M174 and M162 to inhibit Flavobacterium psychrophilumin vitro. In addition, others in vivoexperiments, rainbow trout specimens fed supplemented diet with Pseudomonassp. M174 (at 4×106) and M162 (at doses of 5×107-2×109 cfu g-1) showed a decrease in cumulative mortality after infection with Flavobacterium psychrophilumJIP02/86. Thus, cumulative mortality was 41% in the M174-diet group, 35% in the M162-diet group, and 57% in control groups. In an interesting study, oral vaccines with formalin-killed Streptococcus iniaeDan-1 at doses of 3×1011 cfu ml-1 were inoculated in Oncorhynchus mykissspecimens provided them protection against Streptococcus iniaevirulent at doses of 105 cfu ml-1 until six months later. The survival ratio was 90% in the treated group and 20% in the control group [80]. As seen in the vast literature the benefits of many probiotics in the culture of rainbow trout is achieved. Furthermore, some papers also demonstrate that probiotics do not need to be alive exclusively. Thus, trout specimens fed supplemented diet with inactivated Enterococcus faecalisat dose of 5g kg-1 feed showed lower cumulative mortality (40%) than the control group (83%) after challenge with Aeromonas salmonicida[81]. Other probiotic forms of Bacillus subtilisAB1 such as live cells, sonicated cells, formaline-dead cells and cell-free supernatant were applied as supplement in diets to rainbow trout specimens which achieved a survival of 100% in all forms of probiotic-treatments whereas the survival in control groups was 10-15% after intraperitoneal injection with a pathogenic Aeromonassp. [82].

Other trout species have been slightly evaluated. Thus, brown trout (Salmo trutta) specimens fed diets containing lactic acid bacteria (Lactococcus lactisssp. lactisCLFP 100 or Leuconostoc mesenteroidesCLFP 196) at doses of 106 cfu g-1 for four weeks separately, reduced the cumulative mortality after challenge with Aeromonas salmonicidafrom 37% in the control group to 15% and 9%, respectively. [83]. In the case of brook trout (Salvelinus fontinalis), specimens exposed to four potential probiotics (S1, S5, S9 and S10) separately at doses of 105 cfu ml-1 and one pathogen (Flavobacterium columnare) showed a higher survival ratio than specimens exposed to Flavobacterium columnare(without probiotics) being S9 the most successful with a cumulative mortality of only 4% [84].

Edwardsiellosis, a bacterial septicaemia caused by the Gram-negative bacterium Edwardsiella tarda, is one of the most serious bacterial diseases in cultured eels [85]. So, in a study with European eel (Anguilla anguilla), dietary administration with Enterococcus faeciumSF68 from Cernivet® and Bacillus toyoifrom Toyocerin® for 2 weeks was followed by challenge with Edwardsiella tarda981210L1. Bacillus toyoidid not protected against Edwardsiellosis whilst Enterococcus faeciumSF68 showed higher rate of survival (73%) compared with the control (45%). In the resistance of Nile tilapia (Oreochromis niloticus) against edwardsiellosis, dietary administration of a commercial mix of probiotics that contained Lactobacillus acidophilus(1.2×108 cfu g-1), Bacillus subtilis(1.6×107 cfu g-1), Clostridium butyricum(2×107 cfu g-1) and Saccharomyces cerevisiae(1.6×107 cfu g-1) for 30 days following infection with Edwardiella tarda, provided a cumulative mortality lower than positive control group [86]. Recently, it has been also reported [87] that dietary supplementation of 108 cfu g-1 of Lactobacillus pentosusPL11 in Japanese eel (Anguilla japonica) challenged with Edwardsella tardashowed an increase in growth performance compared with the control group. In the case of rock bream (Oplegnathus fasciatus) it has been also shown that dietary supplementation with 2.2×107 cfu g-1 of Lactobacillus sakeiBK19 and challenged with Edwardsiella tardaproduced a non-significant decrease in the cumulative mortality [88].

Dietary supplementation of different species of Bacillussp., Lactobacillussp., Streptococcus faeciumand Saccharomyces cerevisaehad no effect in survival ratio of ornamental fishes (Carassius auratusand Xiphophorus helleri) specimens after challenge with Pseudomonas fluorescens58C [89]. However, other study with Carassius auratusfed a supplemented diet of formalin-inactivated Aeromonas hydrophilaA3-51 for twenty days showed a decrease in cumulative mortality compared with the control group after infection with Aeromonas salmonicida[90].

African catfish (Clarias gariepinus) juvenile specimens were fed a commercial diet supplemented with 3×107 cfu g-1 of Lactobacillus acidophilusfor 12 weeks. Then, fish were intraperitoneally injected with 2×106 cfu ml-1 of Staphylococcus xylosus, Aeromonas hydrophilagr2 and Streptococcus agalactiaeseparately [91]. At one week post infection, the fish survival rate in control group and in infected groups treated with probiotic diet was 100%, whilst in the groups infected with Staphylococcus xylosus, Aeromonas hydrophilagr2 and Streptococcus agalactiaefed the non-probiotic diet, fish survival recorded was 83.3%, 76.6% and 80.0% respectively.

Olive flounder (Paralichthys olivaceus) specimens fed supplemented diet with Bacillus subtilis, Bacillus pumilusand Bacillus licheniformis, separately and at doses 1010 cfu g-1 for eight weeks showed a higher survival ratio in the case of Bacillus subtilisand Bacillus pumilus(97.3% and 98.7%, respectively) than specimens in the control group (77.3%) after immersion with Streptoccocus iniae[92]. For Bacillus licheniformisdiet, specimens did not show statistically significant differences in survival ratio (86.7%) compared with the control group (77.3%). In another study, Paralichthys olivaceusspecimens were fed a diet containing 3.4×104 (low dose), 3.5×106 (medium dose) and 3.4×108 cfu ml-1 (high dose) of Zooshikellasp. JE-34 and challenged with Streptococcus iniaeshowed their mortality reduced from 85 to htose of the controls 25-40% [93].

Chinese drum (Miichthys miiuy) specimens were also fed commercial diet supplemented with 108 cfu g-1 of Clostridium botyricumCB2 in the form of alive cells (CB) or dead cells (D-CB) for 30 days and then challenged with Vibrio anguillarumand Aeromonas hydrophila, separately. Result showed that survival in chinese drum specimens increased in both groups of probiotic diet compared with the control for both pathogen bacteria [94]. These results are according to other study [95] which demonstrated that dietary administration of Clostridium botyricumin rainbow trout (Oncorhynchus mykiss) achieved resistance against vibriosis.

Tropical freshwater fish (Labeo rohita) specimens were fed a supplemented diet with 0.5×107, 107 or 1.5×107 cfu g-1 of Bacillus subtilisfor two weeks. After challenge by intraperitoneal injection of Aeromonas hydrophilaO:18, specimens showed increased serum bactericidal activity and granulocyte numbers in probiotic-fed groups compared with the control group [96]. In other work [97] it has been reported that L. rohitaspecimens fed dietary supplementation with 106, 108 or 1010 cfu g-1 of Lactobacillus plantarumVSG3 for two months showed a higher survival rate (37%, 77% and 63%, respectively) than the control group (14%) after injection of Aeromonas hydrophila. In addition, dietary supplementation of 107 or 109 cfu g-1 of Pseudomonas aeruginosaVSG-2 for two months showed a higher survival rate (66% and 55%, respectively) than in the control group (11%) after injection with Aeromonas hydrophilaMTCC1739. So, the appropriate administration dose was 107 cfu g-1 of Pseudomonas aeruginosaVSG-2 and 108 cfu g-1 of Lactobacillus plantarumVSG-3 which achieved the better survival rate (66% and 77%, respectively) after challenge with Aeromonas hydrophilaMTCC1739 [97, 98], demonstrating that probiotics are only effective when administered in adequate doses.

Turbot (Scophthalmus maximus) larvae specimens fed rotifers enriched with Lactobacillus plantarumand Carnobacteriumsp. at doses of 107-2×107 cfu ml-1 showed a higher survival ratio (53%) than specimens fed rotifers without probiotics (8%) [99]. Similarly, larvae specimens exposed to Roseobactersp. strain 27-4 at dose of 107 cfu ml-1 showed a significant decrease in cumulative mortality compared with control larvae specimens. In addition, this Roseobactersp. strain 27-4 was previously tested as antagonist to Vibrio anguillarum[100]. When specimens were fed rotifers enriched with Roseobactersp. strain 27-4 and infected with Vibrio anguillarum, achieved a decrease in cumulative mortality compared with specimens only infected [101]. It was demonstrated in an in vitroassay that Phaeobactersp. and Ruegeriasp. are also potential probiotics against Vibrio anguillarumin turbot [102].

Gilthead seabream (Sparus aurata) specimens were fed a commercial diet supplemented with 108 cfu g-1 of Shewanella putrefaciens(Pdp11) for 15 days and challenged with 3.7×107 cfu ml-1 of Vibrio anguillarumDC11R2a [103]. The mortality of the fish which receiving the diet supplemented with the potential probiotic Pdp11 was 10%, lower than the mortality of the fish that received the control diet (56%).

In other works [104, 105] it has been described the effect of the dietary administration of 109 cfu g-1 of Shewanella putrefaciens(Pdp11) and Shewanella baltica(Pdp13) to sole (Solea senegalensis) against Photobacterium damselaessp. piscicida. The mortality decreased after one and two months with dietary administration of both bacteria compared with the control diet.

In european seabass (Dicentrarchus labrax) juvenile specimens, it has been demonstrated that dietary intake of Artemiawith an acid lactic bacteria (Lactobacillus delbrueckiissp. delbrueckii) improved growth of specimens [106]. Dietary administration of 109 cfu g-1 of Vagococcus fluvialisduring 20 days in adults resulted in a mortality of 17.3% while in control group (without probiotic) was 30% after exposure to Vibrio anguillarum975-1 [107].

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7. Conclusions

Probiotics are usually live microorganisms that administered at adequate doses confer health benefits to the host. In this review we have focused only in those probiotics conferring protection to shellfish and fish species important for the aquaculture against viral and bacterial diseases. Some of the main conclusions are summarized below:

  • The most studied probiotics are usually Bacillusand Lactobacillusspecies.

  • Dietary administration of probiotics is the preferred for the researchers and farmers. However, bioencapsulation through Artemiamight be considered a good solution, mainly at larval stages.

  • Most of the studies have used live bacteria but other forms such as inactivated, killed, homogenized or even supernatants have also presented good probiotic properties.

  • Bacteria are the most known probiotics but other microorganisms such as yeast or microalgae are also suitable and good candidates.

  • Although probiotics have probed protection against pathogenic bacteria further evaluation of their potential against virus and parasites is deserved.

  • The concentration of the administered probiotic is essential and needs to be optimized for every situation.

  • The time of administration is also a very important factor and periods of 2 to 4 weeks of dietary administration seem to be the optimal.

  • Only a few potential probiotics tested in vitrobecome in effective probiotics in vivoand in commercial probiotics.

Further studies are still necessary to increase our knowledge about the use of probiotics to control bacterial infections in shellfish and fish but much more efforts are needed in the case of viral diseases. This is an important issue for the aquaculture industry that is continuously growing due to the fish and shellfish demand for human consume. Apart from the discovery of new or better probiotic formulations, improvement of their benefits may be helpful. Thus, better and cheaper production methods, administration ways or combination with other preventive/therapeutic measures are welcomed.

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Acknowledgments

H. Cordero wishes to thank the Ministerio de Economía y Competitividad(MINECO) for a F.P.I.fellowship. This work has been funded by grants AGL2010-20801-C02-02 (MINECO and FEDER), AGL2011-30381-C03-01 (MINECO) and 04538/GERM/06 (Fundación Séneca,Grupo de Excelencia de la Región de Murcia).

© 2014 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Héctor Cordero, María Ángeles Esteban and Alberto Cuesta (February 19th 2014). Use of Probiotic Bacteria against Bacterial and Viral Infections in Shellfish and Fish Aquaculture, Sustainable Aquaculture Techniques, Martha Patricia Hernandez- Vergara and Carlos Ivan Perez-Rostro, IntechOpen, DOI: 10.5772/57198. Available from:

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