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Chapter 1

Immunostimulants, probiotics and phage therapy: alternatives to antibiotics
Indrani Karunasagar, Iddya Karunasagar, Victoria Alday-Sanz

Use of chemicals such as antimicrobials and chemotherapeutic agents in shrimp farming might lead to the selection of shrimp pathogens that are resistant to treatments and/or to problems of residues with adverse public health consequences. Antibiotic resistant luminous bacteria causing mass mortalities in shrimp larvae and postlarvae have been reported (Karunasagar et al 1984). Luminous Vibrio harveyi can form biofilms on various surfaces in hatchery environment and biofilm bacteria have an increased resistance to treatment with antibiotics and sanitizers (Karunasagar et al., 1996). Detection of residues of banned antibiotics such as chloramphenicol and nitrofuran groups or antimicrobial dyes such as malachite green and crystal voilet have led to disruptions in international trade of aquaculture products (FAO/WHO/OIE 2006). As a result, there is an increasing concern about antimicrobial resistance in aquaculture pathogens, the public health and trade impacts of the use of antimicrobials in health management in aquaculture which have led to the search of alternatives to these products

The Sniezko epidemiological triad suggests that disease in aquaculture is the result of a breakdown in the balance between the host, pathogen and environment. Most often, one encounters situations wherein presence of a pathogen can be detected in shrimp without any disease symptoms. For example, shrimp testing positive for the presence of white spot syndrome virus (WSSV) may go through a normal culture cycle, when environmental conditions are suitable (Umesha et al 2006). Therefore development of strategies for health management in aquaculture following Sniezko`s triad should consider a holistic approach to prevent diseases such as the improvement of host immune system, reduction/exclusion of pathogens and improvement of the environment in which the shrimp are cultured. Use of antimicrobial agents such as antibiotics result in a disturbance in the microbial balance in the aquatic system, since they act not only on the target pathogen, but also on the beneficial commensal microflora. Some alternatives to antimicrobials useful for shrimp health management are the use of immunostimulants which may act by improving the immune response to combat pathogens, the use of probiotics to improve the gut flora and culture conditions in different ways and through phage therapy where phages (viruses affecting bacteria) will target specifically the bacterial pathogens of shrimp.

1. Immunostimulants, studies with fish provide a lead

An immunostimulant has been defined as a chemical, drug, stressor, or action that elevates the non-specific defence mechanisms or the specific immune response (Anderson, 1992). Cultured aquatic animals depend to a large extent on non-specific defence mechanisms (Anderson, 1992) and therefore, immunostimulants could play an important role in enhancing the resistance of these animals to disease. Early work on immunostimulants was carried out in fish for which several compounds such as FK-565 (a synthetic lactoyl tetrapeptide obtained from Streptomyces olivaceogriseus), quaternary ammonium compounds, ISK (a fish by-product), yeast glucans and Levamisole (levo-isomer of tetramisole) have been reported as immunostimulants (Kitao et al., 1987; Yano et al., 1989; Robertsen et al., 1990; Nikl et al., 1991; Anderson, 1992). Many of the claimed immunostimulants are molecules derived from microbial cell wall or outer membrane with characteristic patterns such as repeating units e.g. glucans, lipoplysaccharides, peptodoglycans and have been termed "pathogen associated molecular patterns" (PAMP). Immunostimulants induce enhanced activities in the non-specific defence mechanisms such as increased oxidation activity of neutrophils and macrophages, augmented phagocytosis or potentiating cytotoxic cells (Kitao and Yoshida, 1986; Yano et al., 1989; Kajita et al., 1990; Chen and Ainsworth, 1992; Anderson, 1992; Jorgensen et al., 1993; Sakai et al., 1993; Yoshida et al., 1993; Dalmo and Bogwald, 2008). Enhanced complement activity and lysozyme activity in fish has also been reported (Engstad et al., 1992; Jorgensen et al., 1993). It has been suggested that immunostimulants may be used in anticipation of disease outbreaks to prevent losses, in cases where such events are cyclical and can be predicted (Anderson, 1992).

1.1. Immunostimulants in shrimp

The encouraging results obtained in fish systems with immunostimulants have led to similar studies in shrimp. However, while it is established that the fish have well characterised specific defence mechanism, there is no definitive evidence of such specific defences in shrimp and crustaceans. Therefore, shrimp appear to depend more heavily on non-specific defences compared with fish. However, shrimp have a wide array of non-specific factors, both cellular as well as humoral which are involved in defence against pathogens (S?derhäll and Cerenius, 1992), which are stimulated by microbial molecules.

1.2. Haemolymph factors and their induction by immunostimulants

The haemolymph factors associated with defence in crustaceans include lectins, agglutinins, precipitins, bactericidins, lysins and bacteriostatic substances (Smith and Chisholm, 1992; S?derhäll and Cerenius, 1992). Induction of such haemolymph factors in crustaceans including shrimp has been reported in literature. Injection of killed bacteria in spiny lobster, Panulirus argus induces production of bactericidins (Evans et al., 1969). Agglutinins and bactericidal substances are induced in the hemolymph following injection of Vibrio bacterin in Squilla mantis (Danielli et al., 1989). Bactericidins and other humoral factors are induced in black tiger shrimp, Penaeus monodon within day 1 of injection of heat killed Vibrio alginolyticus and bactericidins peaked at day 2 and persisted until day 5 (Adams 1991). Administration of Vibrio bacterin orally through diet in P. monodon induced bactericidin (Devaraja et al., 1998). Enhancement of bactericidal activity in the haemolymph of P.monodon can be brought about through treatment with immunostimulants such as ? glucans, zymosan or Vibrio bacterin (Sung et al., 1996). Much higher titres of vibriocidal activity of hemolymph could be observed in P. monodon fed with a diet containing ? glucans and vibrio bacterin compared to diet with either component alone suggesting synergistic effect between these immunostimulants (Devaraja et al., 1998). Haemolymph from prawns immunized by injection or spray techniques had haemocyte kinetic factor(s) that activated haemocytes to migrate through a membrane in a Boyden chamber (Itami et al., 1989).

Besides substances such as bactericidins which directly contribute to the killing of the pathogens, crustaceans may have humoral factors which function as recognition molecules to recognise non-self molecules and interact with cellular factors. Such molecules generally recognise cell wall components of microorganisms and are referred to as Pattern Recognition Proteins (PRPs). Proteins which recognise and bind ? 1,3 glucans and lipopolysaccharides have been identified in a number of crustacean species. Following binding of the PRPs, these proteins activate pro-phenol oxidase (proPO), clotting cascade and expression of genes coding for antibacterial proteins (Sritunyalucksana et al., 1999; 2002; Roux et al., 2002). RNA interference mediated suppression gene coding for proPO in P. monodon increases susceptibility to V. harveyi infection (Charoensapsri et al. 2009). In the freshwater crayfish, Pacifastacus Ieniusculus, a ? 1,3 glucan binding protein has been characterized, its gene cloned and the haemocyte receptor to which it bind after reacting with glucans has been partially characterized (S?derhäll et al., 1996). A protein which binds to ? 1,3 glucans has been purified and characterized in the shrimp P.californiensis (Vargas-Albores et al., 1996) and P.vannamei, P.stylirostris and P. monodon (Vargas – Albores et al., 1997, Sritunyalucksana et al., 2002). Enhanced phenoloxidase activities have been demonstrated in P. monodon treated with immunostimulants such as ? 1,3 glucan by immersion (Sung et al., 1996) or through feed (Devaraja et al., 1998) and in kuruma prawn, P. japonicus fed with diet containing lipopolysaccharide (Takahashi et al 2000).

1.3 Cellular activities and their induction by immunostimulation

Haemocytes play a very important role in defence of crustaceans against pathogens. Using morphological and biochemical criteria, three types of haemocytes: hyaline, semi-granular and granular are recognised in crustaceans (S?derhäll and Cerenius, 1992). Enhanced phagocytic activity is seen in granulocytes of P.monodon fed with ? glucan and in P. japonicus fed with peptidoglycan derived from Bifidobacterium thermophilum (Itami et al., 1994; 1998a). Foreign particles such as bacterial cells can be removed by phagocytosis or by haemocyte encapsulation which is initiated by the pro-PO system (S?derhäll and Cerenius, 1992). Sung et al., (1996) noted that cells of Vibrio spp. are largely eliminated from shrimp haemolymph within 12 h following invasion and are completely undetectable at 24 h. In the vertebrate system, both oxygen dependent reactions (generation of reactive oxygen species such as superoxide anions, hydrogen peroxide, hydroxide ions, singlet oxygen, myeloperoxidase – catalysed hypochlorite) and non oxygen dependent substances such as digestive enzymes are involved in destruction of phagocytosed microorganisms. Techniques using nitroblue tetrazolium (NBT) staining to detect the presence of superoxide anions, have shown that in P.monodon treated with ? glucans, 41% of cells were stained and treatment with zymosan and phorbol 12-myristate 13 acetate (PMA) resulted in staining of 31% and 9% cells compared to 5% in untreated animals (Song and Hsieh 1994). In P.monodon with glucan, zymosan and Vibrio bacterin by immersion the production of reactive oxygen species (ROS) was significantly greater in glucan, Vibrio bacterin and zymosan – treated shrimp compared to control shrimp (Sung et al., 1996) Enhanced ROS production could be noted in shrimp treated with glucan and Vibrio bacterin through feed and in these treatments, peak activity was recorded 48 h after feeding, treatment with glucan and bacterin together resulting in much higher activity compared to the two components used individually (Devaraja et al., 1998).

The proPO system participates in the defence in a number of ways. This may lead to production of microbicidal compounds such as quinines and melanin (Bachere et al., (1995). They may also interact with other proteins and mediate haemocyte adherence and stimulate encapsulation of large particles by granular cells (S?derhäll and Cerenius, 1992). Enhanced PO activity following treatment with immunostimulants has been reported. Sung et al., (1994) noted that in Vibrio, ? glucan treatment enhanced PO in shrimp haemocytes. When P.monodon was treated with ? glucan by immersion, enhanced PO activity was observed from 5 min to 24 h after treatment with peak activity at 3 h and the activity returned to basal levels on day 3 (Sung et al., (1996). Devaraja et al., (1998) studied PO activity in haemocytes of P. monodon treated with Vibrio bacterin suggesting synergistic effect between the two immunostimulants. Litopenaeus vannamei fed for 98 days with a diet containing 108 Bacillus subtilis E20 showed enhanced phenol oxidase activity, phagocytic activity and clearance efficiency against Vibrio alginolyticus (Tseng et al., 2009).

These reports indicate that immunostimulation using microbial cell wall components enhance the cellular component of non-specific defence in shrimp in vitro.

1.4 Enhanced survival of shrimp treated with immunostimulants.

Numerous papers have been published describing that shrimp treated with immunostimulants have shown enhanced survival when challenged with bacterial or viral pathogens (Table 1). Cultured kuruma prawns treated with Vibrio bacterin by injection, immersion or spray showed reduced mortalities compared in untreated controls, when challenged by Vibrio injection 30 days later (Itami et al., 1989). P .monodon treated with ? glucan by immersion showed better survival when challenged with Vibrio vulnificus cells and protective effect lasted till day 18 following immersion (Sung et al., 1994). Kuruma prawn P. japonicus treated orally with ? 1,3 glucan from Schizophyllum commune or fed with peptidoglycan (PG) derived from Bifidobacterium thermophilum survived challenge by Vibrio or by WSSV better than untreated control (Itami et al., 1994, 1998a; 1998b; Chang et al., 1999). Similarly, treatment of kuruma prawn with fucoidan, a sulphated polysaccharide also improved survival after challenge with WSSV (Takahashi et al., 1998). P. monodon broodstock fed with diet containing ? 1,3 glucan from Schizophyllum commune showed higher survival under both indoor and outdoor rearing conditions compared to untreated controls (Chang et al 2000). Larvae from glucan treated spawners were better protected against challenge with WSSV by immersion (Huang and Song, 1999).

The application of dietary immunostimulants as management tool for viral diseases such as Taura syndrome (Brock, et al., 1997; Lightner and Redman, 1998) and WSSV (Karunasagar and Karunasagar, 1999) has been suggested. In case of Taura syndrome, results varied from slight improvement in survival (Klesius and Shoemaker, 1997; Dixon and Dorado, 1997) to survival rates of TSV- challenged P.vannamei comparable to unchallenged control groups (Lightner and Redman, 1998). In India, field trials of immunostimulants were conducted and the results suggest that a regular once a week application of immunostimulants resulted in over 80% survival in ponds where WSSV could be detected by histological methods (Karunasagar and Karunasagar, 1999).

However, wide spread adoption of the use of immunostimulants by the industry has not occurred. Despite the abundant scientific data published and the large number of commercial products, shrimp farmers, in general, have not felt that the cost of these products was worth their benefit. This might be due to the variability of the quality of commercial products or it could also be due to the fact that the field application does not reflect the experimental application. One possibility is that shimp in a pond, may obtain the so called immunostimulants in a natural way, from the pond environment, while shrimp under experimental conditions do not have access to them, at least in the same amount in an aquaria set up and therefore their dietary addition shows an improvement under challenge conditions while it is not so clearly proven under production conditions.

2. Probiotics:

The term "probiotic" has been traditionally used to refer to live microbial feed supplements that beneficially affect the host by improving its intestinal balance (Fueller, 1989). However, the term has been more broadly used in aquaculture to refer to microbial agents that have beneficial effects on cultured animals in a number of ways (Gatesoupe 1999). Most of the aquaculture probiotics are thought to be modifying the microbial community around the animals in favour of beneficial microorganisms, that may improve the water or sediment quality, suppress pathogenic bacteria or stimulate immune system of the host or improve digestion (Gatesoupe, 1999, Verschuere et al 2000). Suggested mode of action for probiotic bacteria in aquaculture include production of inhibitory compounds; competion for nutrients, iron, adhesion sites; enhancement of host immune response and degradation of harmful wastes like ammonia (Verschuere et al 2000).

In contrast to lactic acid bacteria that have been used as probiotics in terrestrial animals, a range of gram positive and gram negative bacteria have been used as biocontrol agents in aquaculture (Table 2). Bacillus spp are widely used as probiotics in shrimp aquaculture, but gram negative bacteria like V. alginolyticus and Pseudomonas spp have been shown to be effective as well (Verschuere et al., 2000). Probiotic bacteria have been shown to enhance survival, molting rate and growth of black tiger shrimp, P. monodon (Rengpipat et al 1998), L. vanamei (Garriques and Arevalo, 1995); to reduce population of pathogenic Vibrio spp (Moriarty 1998; Chythanya et al 2002; Karunasagar et al 2005) improve digestibility of food (Liu et al 2009) and stimulate immune system of P. monodon (Rengpipat et al 2000). In various studies, application of probiotics has been either to larvarl rearing tank water, pond water or added to the feed. Feed supplementation has been preferred in field applications and has been found to be more effective than direct addition to rearing water (Hai et al 2009), but this may depend on the intended purpose in using the probiotic. If the purpose is to improve water quality, then application to rearing water would be more useful. However, though bioremediation potential of probiotic bacteria has been proposed (Gatesoupe 1999), some studies failed to confirm this in shrimp ponds (Rengpipat et al 1999). Field studies in Indonesia and Thailand show improved shrimp health, better production and economic returns to the farmers (Moriarty, 1998). However, there is not much information on the colonization and establishment of probiotic bacteria in hatchery or pond environment following initial application. In some hatchery studies, daily application of Bacilus probiotics was involved, but probiotic application resulted in larval survival rates similar to that obtained with antibiotic application (Decamp et al 2008). Despite of some commercial claims that probiotics can colonize the digestive tract, this has not been demonstrated yet. Actually, contrary to terrestrial animals, it is believe that bacterial populations of the digestive tract of aquatic species are transient. In the case of shrimp, the periotrophic membrane lining the midgut, isolate the intestinal content from the intestinal epithelium, impeding any chance of colonization of that space. Moreover, electron microscopy studies has shown that that space is sterile (Martin and Hose, 2010 this volumen).

Regulatory approval for use of probiotics as feed supplements has been documented in some regions. European Union regulation EC/710/2009 permits use of authorized probiotics for disease control in organic aquaculture.

One of the major difficulties to find effective commercial probiotics is the wide range of environmental condition where shrimp is grown. This means that commercial products need to be adapted to a range of conditions and that would probably mean that different products will need to be developed for specific culture conditions as the growth and properties of some of the probiotic strains may not work as expected in all environmental conditions. Often, the best probiotics are the ones produced with local isolates but this implies that the know-how of their selection and production needs to be available to . Also, the amount of probiotics that need to be added to positively alter the microbial balance needs to be evaluated for cost efficiency before large scale application.

3. Phage therapy

Though bacteriophages were discovered during 1915-1917 and their potential application in treatment of bacterial diseases was recognized soon thereafter, the discovery of antibiotics in 1941 and their effectivity in treatment of wounds in soldiers during the World War II led to a decline in the interest in bacteriophages as therapeutic agents. But the emergence of multi-drug resistance in several bacteria pathogens has led to a renewed interest in phage therapy. Bacteriophages are widely distributed in the environment and in the aquatic environment, there are tenfold more phages compared to bacteria in aquatic environment (Skurnik and Strauch 2006). An additional advantage of their use is that they increase and decrease in numbers following the target population, so they are self-regulated. The term bacteriophage literally means bacteria eater and lysis of the bacterial host occurs after the replication of bacteriophages in their hosts. Such phages are called "lytic phages". Some bacteriophages have a "lysogenic phase" in which the bacteriophage genome gets integrated with the host genome and replicates along with it during bacterial replication. Bacteriophages that are often found in lysogenic state are referred to as temperate phages and the phage genome inside the host is termed "prophage" (Wilson and Mann, 1997). Lysogenic phages are not suitable for phage therapy.

Application of bacteriophages in therapy against fish pathogens was investigated by Nakai and coworkers ( Nakai et al 1999; Park et al 2000; Nakai and Park 2002). Bacteriophages are host specific, they can be not only genus and species specific but strain specific. Hence they lyse only the target bacteria, unlike antibiotics that can have a wide spectrum. Thus bacteriophage therapy would not suppress useful commensal flora that are required for the health of the animals or would alter the bacterial environmental balance. Oral administration of bacteriophages against Lactococcus garvieae to young yellowtails (Seliora quinqueradiata) resulted in 100% survival following intraperitoneal challenge with the pathogen compared to 10% survival in control (Nakai et al 1999). Oral administration of phage impregnated feed to ayu (Plecoglossus altivelis) brought down cumulative mortality to 22.5% from 65% in control following oral challenge with Pseudomonas plecoglossicida (Park et al 2000).

Bacteriophages against the shrimp pathogen V. harveyi may belong to the family Siphoviridae or Myoviridae (Oakey and Owens 2000, Shivu et al 2007, Crothers-Stomps et al 2009). Generally members of Siphoviridae have been reported to be lytic phages (Vinod et al 2006, Shivu et al 2007, Karunasagar et al 2007, Crothers-Stomps et al 2009). A V. harveyi myovirus like phage (VHML) has been reported to be temperate and confer virulence to the host strains (Pasarawipas et al 2005).

Shivu et al (2007) tested the host range of a collection of V. harveyi phages against 180 isolates from different geographical regions. Three strains were able to lyse 65-70% of the strains indicating broad host range. Vinod et al (2006) tested bacteriophage therapy of shrimp (P. monodon) larvae and postlarvae in both laboratory microcosms as well as in hatchery during natural outbreak of luminous bacteria disease. In microcosms, larval survival was 25% in control and 85% with treatment. In hatchery trial, the survival was 86% with bacteriophages, 40% with antibiotics and 17% in control (Vinod et al 2006). Bacteriophage treatment brought down counts of luminous bacteria in the tanks. In another hatchery trial during a natural outbreak of luminous bacteria disease, 86-88% survival was obtained with bacteriophage treatment compared to 65-68% with antibiotics (Karunasagar et al 2007). A common problem in shrimp larval health management is the persistence of V. harveyi in hatchery environment by forming biofilm that is resistant to antibiotic and sanitizer treatment (Karunasagar et al 1996). The ability of bacteriophage to bring about 3 log reduction in biofilm cells of V. harveyi on high density polyethylene (HDPE) surfaces was demonstrated by Karunasagar et al (2007). This provides an additional advantage for bacteriophages in shrimp larval health management. These studies show the potential for bacteriophages to be effective alternatives to antibiotics in shrimp larval health management. Bacteriophages used by Vinod et al (2006) and Karunasagar et al (2007) lacked the putative virulence gene carried by VHML and hence the concern regarding carriage of virulence gene could be minimal.

There could be different mechanisms of host resistance to bacteriophages. Bacteria carrying prophages are generally resistant to infection by another phage. Vibrio harveyi strains resistant to four phages (Vinod et al., 2006) were analyzed for the possible presence of prophages (unpublished). In order to identify the possible presence of a prophage in the resistant isolates, these were treated with mitomicin C which induces the lytic phase in a prophage (Levine, 1961). Only 2 of the isolates were infected with a prophage, which suggests that the mechanism of resistance could be different in these bacterial strains. It should also be pointed out that phage resistant variants are not necessarily altered in virulence and Merril et al (2006) suggested that selection for resistance could select against virulence. Many phage resistant variants have been shown to have alterations in receptos used by the bacteriophage (Skurnik and Strauch, 2006).

?-propiolactone (BPL) was used to inactivate the phage cultures. BPL is widely used for the inactivation of viruses (DNA and RNA viruses) and often used for vaccine production (Lawrence, 2000). To investigate whether inactivated phage suspension can be used instead of live bacteriophages for therapy against bacteria, a high titre suspension of Vibrio harveyi phages was treated with ?-propiolactone to give a final concentration of 0.25 %. The suspension was then incubated for 8 hours at 30°C and the reaction was stopped by adding sodium thiosulphate to a final concentration of 35mM. Inactivation of the phages was confirmed by passage of a cleared zone into a new lawn of a susceptible strain where no lytic effect was observed, suggesting no further phage replication. The preparation was added (1 or 0,1ml) to a susceptible bacterial culture in TSB (1L) and bacteria were quantified by spectrophotometer (OD600).

Results (table 4) showed that the starting bacteria culture had a concentration of 105cfu. After 48h treatment with 1ml of inactivated phage, the counts had gone down to 103 or 104, The culture treated with 0.1ml had a count of 104 to 105.

During the exposure of the bacterial culture to the phage culture, it was noticed that in some isolates, bacterial colonies appeared in the cleared area after 24h suggesting induction of resistant variants from the isolate. In order to understand the lytic effect of the inactivated phage, possibly through enzymatic mechanism, colonies of nine sensitive V. harveyi strains (wild type) and their possible resistant variants were selected for further studies. Full strength and four tenfold dilutions were applied (10?l) on a lawn of the selection of host bacteria and growth inhibition was recorded after 8h.

All wild type strains were lysed by each of the four phage preparations up to the highest dilution tested (10-4). Resistant variants were all sensitive of lysis by each of the four full strength inactivated phage preparations but varied with the dilutions depending on the variant and the phage tested (Table 5). Such results suggest that it is possible to use the enzymatic mechanism of the phages to achieve the lytic effect in the absence of the living phage. Lysins are the major group of enzymes produced by the bacteriophages to lyse the host cells and some investigators have advocated the use of purified lysins rather than live phages for therapy (Loessner, 2005).

It would be necessary to test if the resistant strains to the live phage were susceptible to the inactivated phage. However, in general, these enzymes were able to kill only the species/strains from which they were produced. So these lysins would only kill the pathogens with little or no effect on the normal bacterial flora. Another advantage if that apparently there is low chance of the bacteria developing resistance to the lysins (Loessner, 2005). But most investigations on lysins have been done on gram positive bacteria._

In conclusion, the authors believe that the use of the lytic enzymes could be an effective mechanism for the control of V. harveyi population and the treatment of disease outbreak caused by this agent.

Application of bacteriophages in biocontrol of pathogens has been permitted by regulatory authorities in some situations. Use of commercial product "AgriPhage" from Omilytics Inc against plant pathogenic bacteria has been permitted by the US Environmental Protection Agency and use of Listeria LMP 102 from Intralytix Inc for control of Listeria monocytogenes in ready to eat meats and poultry products has been permitted by the US Food and drug Administration (Garcia et al 2008). These show that bacteriophage application in agriculture and aquaculture would be safe from consumer protection point of view. As a part of safety evaluation before licencing of bacteriophages, it would be important to ensure that they do not carry putative virulence genes that may be transmitted to the host bacterium.

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Table 1
Immunostimulants Method of application Effects Reference
Vibrio bacterin Injection, immersion spray Improved survival on challenge on day 30 Reference
Vibrio alginolyticus bacterin Injection Induction of bactericidines lasting till day 5 Adams, 1991
Lactobacillus pantarum Oral Increase in PO, Superoxide dismutase activity, survival after challenge with V. alginolyticus Chiu et al., 2007
Bacillus subtilis Oral Increase in PO, phagocytic activity, efficiency of clearance of V. alginolyticus Tseng et al., 2009
? 1,3 glucan Immersion Improved survival on challenge with Vibrio valnificus upto day 18. Sung et al., 1994.
? 1,3 glucan Oral Increased phagocytic index, improved survival on challenge with Vibrio, WSSV Itami et al., 1994, Chang et al., 1999
? 1,3 glucan, zymosan, Vibrio ctivity Immersion Enhanced PO activity, oxygen brust activity Sung et al., 1996
Peptidoglycan Oral Enhanced phagocytic index, improved survival on challenge with WSSV Itami et al., 1998ª, 1998b
? 1,3 glucan, Vibrio bacterin Oral Enhanced bactericidal, oxygen burst, PO activity, improved survival on challenge with WSSV Devaraja et al., 1998 Karunasagar and Karunasagar 1999
Fucoidan Oral Improved survival on challenge with WSSV Takahashi et al., 1998
Table 2
Bacterial strain Method of application Effect Reference
Bacterial strain PM-4 (later identified as Thalassobacter utilis) Addition to P. monodon larval rearing water Improved survival and molting rate Maeda and Liao 1992; Nogami et al 1997
Vibrio alginolyticus Addition to rearing water Improved growth and survival of L. vannamei postlarvae Garriques and Arevalo, 1995
Bacillus Addition to pond water Improved survival in P. monodon, decreased levels of luminous Vibrio in water and sediment Moriarty 1998
Bacillus strain S11 Through feed Increase in mean weight, survival of P. monodon larvae and postlarvae, activation of cellular and humoral defenses Rengpipat et al 1998; 2000
Pseudomonas I-2 Addition to water Reduction in V. harveyi levels Chythanya et al 2002
Bacillus subtlis BT23 Addition to water Reduction in mortality of P. monodon juveniles challenged with V. harveyi Vaseeharan and Ramasamy 2003
Bacillus subtilis UTM 126 Through feed Reduction in mortality of L. vannamei juveniles challenged with V. harvei Balcazar and Rojas-Luna, 2007
Bacillus spp Addition to larval rearing tanks Improved survival of P. monodon and L. vannamei larvae and postlarvae Decamp et al 2008
Pseudomonas synxantha and P. aeruginosa Through feed Penaeus latisulcatus Hai et al 2009
Bacillus subtlis E20 Through feed Improved growth performance of L. vannamei Liu et al 2009
Table 3: Examples of bacteriophages with potencial for application in shrimp aquaculture.
Bacteriophage Source Lytic activity and trials Reference
Siphoviridae shrimp farm water In mesocosm studies, brought about 80% survival in larvae challenged with V. harveyi compared to 25% in control; in hatchery trials during natural outbreak of luminous bacteria disease, 86% survival in treated and 17% in control Vinod et al 2006

Siphoviridae

shrimp hatchery water Three phages lysed 65-75% of the 183 isolates ofV. harveyi obtained from different geographical regions Shivu et al 2007
Siphoviridae

oyster tissue and shrimp farm water Lysis of 55-70% of 100 isolates of V. harveyi tested; reduction in V. harveyi biofilm cells, 86-88% survival in natural luminous bacterial disease outbreak in hatchery compared to 65-68% with antibiotics Karunasagar et al 2007

Table 4. Bacterial counts after addition of BPL inactivated phage (1ml or 0.1ml suspension) to TSB bacterial culture (1L.) at 0, 24 and 48h post inoculation



Table 5. Growth inhibition after 8h of nine sensitive V. harveyi strains (wild type) and their possible resistant mutant (resistant variant) at full strength and four tenfold dilutions of inactivated phage culture

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