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Use of Probiotic Bacteria against Bacterial and Viral Infections in Shellfish and Fish AquacultureHector Cordero1, Maria Angeles Esteban1 and Alberto Cuesta1[1] Fish Innate Immune System Group, Department of Cell Biology and Histology, Faculty of Biology, Regional Campus of International Excellence "Campus Mare Nostrum". Dissolve two moderately heaped teaspoonful of Chocolate powder in a glass of milk or any smoothie base for a fun tasting probiotic drink.
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It is rich in associated strains of Lactobacillus acidophilus, Lactobacillus casei, Plantarium bifidus, (Bifidobacterium bifidum) Lactobacilus Bulgaricus, Lactobacilus lactis, Lactobacilus fermenti and Saccharomyces boulardii and cerevisae are used. Nature Made Triple Probiotic also contains several key minerals and vitamins known to support the immune system.
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). The fluffy sherbet like taste makes it easy to enjoy as a sherbet, dissolve it in your mouth, or mix it in milk or your favourite smoothie base which will make you crave for more. Alternatively dissolve one teaspoonful of powder in your mouth mixed with saliva and this fun tasting probiotic sherbet will simply melt away.
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IntroductionThe term “probiotic” was firstly used to denominate microorganisms that have effects on other microorganisms [1]. Among the vast number of probiotic species used most information relies on the use of Bacillus sp.
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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]. 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. As such, these products will reflect a higher Shipping Weight compared to the unprotected product.
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.
Moreover, more recently, for oral dietary administration the probiotics can be encapsulated in different ways.

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. Probiotics against virus in shellfishViral infections are one of the most important problems in aquaculture production. Several reviews have already documented the benefits of probiotics in shellfish and fish but they mainly focused on their effects in the immune response. 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. 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.
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 rosenbergii nodavirus (MrNV).
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. Unfortunately, all the studies have focused on the potential preventive effects of few probiotics on the pacific white shrimp (Litopenaus vannamei) resistance against WSSV.
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.2. Probiotic bacteriaThere 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. 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 also been reported that dietary administration of Pediococcus pentosaceus and Staphylococcus hemolyticus to L.
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.4. Probiotics against bacteria in shellfishIn 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 vitro studies. Further preliminary studies of this kind are worthy to be taken in the future and prior to those conducted in vivo. In the case of Pacific oyster larvae (Crassostrea gigas) exposed to 105 cfu ml-1 of the pathogenic Vibrio tubiashii reached a total mortality in just 2 days, whilst in combination with 104 cfu ml-1 of the probiotic Aeromonas media A199 strain the larvae prolonged their viability up to 144 hours indicating its benefits when used by bath [18].
It has been reported [20] that abalone (Haliotis discus hannai) specimens fed supplemented diet with 109 cfu g-1 of Shewanella colwelliana WA64 and Shewanella oyellana WA65 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 midae specimens 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 aeruginosa and Pseudomonas synxantha for eighty-four days and afterwards challenged with V.
In juvenile specimens, commercial white shrimp fed supplemented diet with 105 cfu g-1 diet of Bacillus subtilis UTM126 achieved a mortality of 18% against pathogenic infection of vibrios (including V. 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 subtilis strains) for 2 months, and afterwards each shrimp was injected with 107 cfu of Vibrio harveyi.
Cumulative mortality also decreased in white shrimp fed a supplemented diet with 1010 cfu kg-1 of Lactobacillus plantarum after injection with V. However, a commercial probiotic (Alibio) at the same dose that the Bacillus mix had no effect in survival ratio compared with the control group in Litopenaeus vannamei specimens [30].

Strikingly, other microorganisms such as yeast have been also assayed as potential probiotics.
Also in juvenile specimens fed supplemented diet containing Lactobacillus acidophilus 04 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]. EF012164 and Bacillus cereus DQ915582 at doses of 104 and 105 cfu ml-1 caused lower mortality after infection with Vibrio harveyi and Vibrio spp. In other work, Penaeus monodon postlarvae specimens fed supplemented diet with 109 cfu g-1 diet of two strains of Synechocystis sp. 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 harveyi and Vibrio spp. S11 showed an increased survival of Penaeus monodon when infected with Vibrio harveyi D331 [39].
PM11 and Vibrio fluvialis PM17 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 bulgaricus 56 or L.
Similarly, in blue shrimp (Litopenaeus stylirostris) specimens fed supplemented diet of 107 cfu g-1 of Pediococcus acidilactici for 4 weeks and infected with Vibrio nigripulchritudo SFn1 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 chinensis postlarvae specimens exposed to Arthrobacter XE-7 at dose of 106 cfu ml-1 and pathogenic Vibrios sp.
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 Vibrio sp.
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.5. Probiotics against virus in fishViral 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.
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. 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.6. Probiotics against bacteria in fishBy 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 subtilis and Lactobacillus acidophilus, two lactic acid bacteria which showed in vitro inhibition against Aeromonas hydrophila [49]. The information relative to the use of probiotics as a beneficial treatment of fish against bacterial pathogens is described below and summarized (Table 2).

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