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January | February 2013 Novel additives to reduce the economic impact of disease on shrimp production

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FEATURE

Novel additives to reduce the economic impact of disease on shrimp production

by Peter Coutteau PhD, Business Unit Manager Aquaculture, and Tim Goossens PhD, R&D Engineer Gut Support, Nutriad International NV, Belgium

enaeid shrimp production is under continuous threat from bacterial and particularly viral infections which have caused disastrous collapses of the industry in all major shrimp producing countries. Disease problems in shrimp production are complex and often still poorly understood. Regulations, consumer demands and sustainable management strategies restrict the number of drugs available to treat pathogens. Vaccines are likely to be ineffective in crustaceans, which lack a specific immune system similar to that of vertebrates. Therefore, shrimp producers must consider the seed stock quality, husbandry procedures and healthy nutrition as the major tools to control disease. The current article reports on recent progress in the development of feed additives capable of reducing the impact of diseases on productivity and profitability in shrimp farming.

an average annual growth rate of 18 percent diseases consists of increasing the level of key over the period 1970-2008, which by far nutrients affecting the health and immunology exceeds growth for all other aquaculture spe- of shrimp, including vitamin C and E, phoscies (FAO, 2010). World shrimp aquaculture pholipids, essential fatty acids, trace minerals is producing now well over four million mt of and carotenoids. These booster feeds are shrimp (Valderrama, 2011). This rapid increase often supplemented with immunostimulants, in crustacean production largely reflects the dramatic increase in white leg shrimp culture in China, Thailand, Vietnam and Indonesia since 2000. Despite this apparent success story in terms of production expansion, shrimp production in many regions continues to suffer important economic losses due to the impact of a wide variety of diseases. Recent events illustrate the impact of disease Figure 2: The hepatopancreas is the main organ of the shrimps digestive system outbreaks on shrimp production in responsible for digestion, absorption major producing countries. and storage of nutrients. Esophagus (E), The white spot syndrome virus gastric mill (GM), hepatopancreas (HP), (WSSV), one of the main causes mid gut (MG), hind gut (HG), and anus of stagnation in the shrimp indus(A). try in the nineties, is significantly affecting shrimp production in recent Diseases are number one threat The production of crustaceans has shown years in Mexico and Brazil. Early Mortality mostly derived from the cell envelope of Syndrome (Acute micro-organisms, such as polysaccharides, Hepatopancreatic lipoproteins, and lipopolysaccharides. Figure 1 - Table: Effect of booster feed on production parameters in a farm in NE Necrosis Syndrome, The continuous use of immunostimuBrazil during episode of increased disease incidence due to a combination of intensive rains and increased incidence of infectious myonecrosis virus (IMNV) AHPNS), for which the lants is generally discouraged due to the and necrotising hepatopancreatitis (NHP). Booster feed based on enhanced causative agent has not risks for over-stimulation of the immune nutritional specifications and supplementation of an immunomodulator (AQUASTIM S, Nutriad) versus standard feed. been identified so far defense system. Alternating on/off regimes (Flegel, 2012), is affect- for feed additives is often impractical in farm Control feed Boosted feed % change ing shrimp production operations. Encouraging results to improve in China, Vietnam, disease resistance have been obtained by the Malaysia, and to a continuous use of health enhancing booster total pond area (ha) 25 25 lesser extent, Thailand. feeds based on the selection of the appropriCulture period (days) 107 111 ate immunostimulants in combination with a Survival (%) 77.1 80.7 +5% balanced nutritional supply of key nutrients Traditional Final weight (g) 12.77 14.01 +10% to support the enhancement of the immune approaches to Harvest yield (kg/ha) 1771 2034 +15% system (Table 1). boost shrimp However, the efficacy of various commerhealth through FCr 1.86 1.85 -1% cially available immunostimulants to improve the feed avg weekly growth (g) 0.84 0.88 +6% A traditional stress and/or disease resistance of fish and relative production cost 100% 100% approach to reduce shrimp strongly depends on the type of the relative crop value 100% 119% +19% the impact of shrimp product and on the supply of adjuvant nutri44 | InternatIonal AquAFeed | January-February 2013

FEATURE Nevertheless, the existence of small Discourage the use of non-sustainable could be effective ways of providing the companies willing to take part in sources of MBt libraries research arm for partnering with large ents that are essential to support the buildup both as a digestive gland biodiscovery de-risks this activity Commercial context investment, regupharma as well as a storage depot of the immune system. for big companies and justifies the lation, IP Technology Centers for marine for energy. Therefore, perceived need for entrepreneurial The need for long-term stability means biotechnology, with specialised infrafarmers routinely look at Novel approaches (1): boosting companies to supply into bigger that VC and short-term investment structure might provide one-stop squash preparates to evaluthe nutritional status and lipid pharma (and equivalent big compastrategies are not appropriate either shops for chain connection ate the nutritional status of reserves of the hepatopancreas nies food/nutraceutical, cosmetics/ Science, technology and infrastructures:of the at set-up or for with hepatopancreas, longer survival of Shrimps do not tolerate high levels cosmeceutical) new businesses; encouraging business Marine biotech A number of welldietary fat very well. is not being studies ample lipid reserves being Better and more efficient recogniangel groups and raising awareness served by lack of knowledge amongst show reduced growth at levels above 10 per- an indicator of better resisttion, development and transfer of and stress and disease technology-transfer offices cent of dietary lipid. Nevertheless, quality and ance to knowledge-levels amongst these academic IP in this area is needed would be fruitful More fora for meeting of scientists quantity of dietary lipids play a primordial role challenges. The attributes and benefits of MBt Lipid digestion in shrimp Smaller players in innovation should and industrial players would generate in growth and health of shrimp. Shrimp have could be better communicated. In consider more collaborations between better understanding and sharing of no or very limited capacity to biosynthesize a occurs for a big proportion terms of giving MBt a different image, them, and selling skills and knowledge, needs and possibilities number of lipid molecules which are essential intracellular in the hepatothe higher hit rate could be a starting rather than pushing A global source of soft funding for normal growth, including cholesterol, high- pancreas epithelium from molecules at point big pharma or trying would promote the transition from where it is transported to to go too far ly unsaturated fatty acids and phospholipids. Some of the points raised above were proof of concept are often the most the down the value-chain; it is usually too target organs via the Fishmeal and fish oil to demonstration Figure 3. Effect of the supplementation of a discussed additive (AquagestS, difficult for small companies to handle and commercial-scale for innovations important sources of cholesterol and HUFA haemolymphe under the digestibility-enhancing further in Think Tank 5 in the the cost and stresses Specific incubator programmes marine form of lipoproteins (Fig. 2). of regulatory on context of of lipid vacuolization in and the Nutriad) the degree Marine Biotechnology in the diet. Increasing cost of these could the hepatopancreas of shrimp fed the different Environment. processes be recommended ingredients has forced formulators to reduce The formation and absorpfeeds during 30 days (van de Braak et al., 2012). dietary specifications for these essential lipids. tion of lipid micelles from Although these nutrient levels may not show the lumen of the hepatosignificant differences on growth performance pancreas tubuli is therefore a limiting step to use fats as essential components and as in Companies attending this Think Tank: the lipid digestive process. Digestibility source of energy for growth and surviving feeding trials under controlled conditions, in Aqua Bio Technology ASA - A-Spark Good Ventures - Algal Bioenergy Special Interest Group - Bioalvo - BioNova - BioTech North - they may become critical for maintaining enhancers based on natural emulsifying episodes of stress or disease pressure. The Bretagne Developpement Innovation - CCMar - EMPA - European Marine Biological Resource Centre (EMBRC) - Government of Portugal health and immune defenses under disease agents, selected for their compatibility with enhancement of the lipid reserves in the - Grette Law - innoVactiv - Innovation Norway - JPI Oceans - The Research Council of Norway - Kiel Center for marine natural products challenges and fluctuating ambient conditions the shrimps digestive system, have shown to hepatopancreas of white shrimp Penaeus - Marealis - Marine Biotechnology Programme of Ireland - Max Planck Institute - National Research Council of Canada - Nautilus Biosciences be capable of complementing the process of indicus as a result of the supplementation encountered in production. Canada - Novagraaf Technologies - Novus International - Soliance - Marine Bio-Technologies Center of Innovation - National University of of Furthermore, the energy status of shrimp is emulsification and absorption of dietary fats in a digestibility-enhancing additive was demonIreland, Galway - OceanGate, Inc. - Oceanomics project, Roscoff - Polaris - Polytechnic Institute of Leiria - PwC - Roscoff Marine Biological largely determined by its lipid reserves depos- the hepatopancreas (Coutteau et al., 2012). strated recently by van de Braak et al. (2012). Station - Saint Malo Agglomeration - ScandiDerma AS - Univeristy College Cork - University of Aveiro ited in the hepatopancreas which functions This in turn improves the efficiency of shrimp Histological analyses showed a three-fold

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FEATURE proteases, invasion factors or other virulence factors (Defoirdt, et al., 2011). In recent years, research focusing on ways to disturb QS signaling (also called quorum quenching) is therefore gaining particular interest (Figure 5). Feeding manually from a boat and harvesting shrimp at the CAMACO farm, Panama (with courtesy of Jorge This is especially true in Cullar-Anjel) the field of human medicine, where QS-inhibitors are investigated as potential alternatives to increase of the percentage of shrimp with a high degree of lipid vacuolization in the antibiotics in tackling pathogenic bacterial hepatopancreas after supplementing the feed infections (Sintim et al., 2010). Interestingly, additive during one month (Figures 3, 4). The chances that bacteria build up resistance results of a parallel pond study indicated two against QS disruptors are predicted to be low, percent higher average body weight (ABW), giving that the selective pressure against these four percent higher survival, and six percent in se non-lethal molecules is limited. This higher biomass for the treatment ponds. stands in stark contrast with what is seen with cific bacterial biosensors and model organisms. Synergistic blends of different natural compounds resulted to be extremely efficient in QS quenching activity against signaling by Vibrio harveyi, an pathogen causing vibriosis in penaeid shrimp (Figure 6).

Putting QS inhibition into practice: effect of optimising gut health on productivity and economics of semiintensive shrimp farming
Shrimp are actively grazing on the substrate present in the pond bottom and water column, and therefore highly exposed to exchanges of microflora between the environment and the digestive system. This increases the risk for the proliferation of an unfavorable gut microflora or frequent destabilization of the microflora, which can affect the optimal functioning of the digestive system. Furthermore, the digestive system of shrimp is the main entry port for bacterial and viral infections, which remain a major risk for the profitability of shrimp production. Sustainable approaches to modulate the gut microflora in farmed animals include the use of selected bacteria to inoculate the gut (probiotics), specific nutrients promoting the development of selected bacterial strains (prebiotics), and specific natural compounds (mostly derived from yeast and herbal extracts called phytobiotics) capable of modulating the microflora towards a favorable composition, favoring the development of beneficial bacteria and inhibiting potentially pathogenic micro-organisms. The latter strategies have the advantage of being easily applicable at the feedmill on large volumes of feed and avoiding major adaptations of the production protocols at the farm. A synergistic blend of botanical extracts (Sanacore GM, Nutriad) was originally selected for its bacteriostatic and bactericidal properties against pathogenic and potentially pathogenic bacteria in vitro using the disk diffusion method.

Figure 4: Histological determination of the degree of lipid vacuolization of the hepatopancreas in shrimp Penaeus indicus, showing a high (left picture) and low (right picture) level of lipid vacuolization (100x magnification; van de Braak et al., 2012)

However, removal of outliers for survival from control and treatment set showed eight percent higher ABW, 12 percent higher survival, and 23 percent higher biomass.

Novel approaches (2): Quorum Sensing technology

Quorum Sensing (QS) is a form of bacterial communication. Over the last decade, many bacterial species have been documented to be able to produce and secrete small signaling molecules, such as acyl homoserine lactones or certain oligopeptides, which can be detected by adjacent bacteria of the same or of distinct species. When population density rises, these molecules will accumulate in the extracellular environment, thereby providing a means for bacteria to quantitatively monitor the presence of other bacteria. These signaling molecules will, upon reaching a certain threshold concentration, initiate intrabacterial signaling that culminates in the activation of specific genes. QS communication is therefore used by bacteria to synchronize gene expression alterations and coordinate biochemical responses within the entire population. In most pathogenic bacteria from which the QS system has been studied, QS has been associated with pathogenicity, such as biofilm formation and the production of

conventional antibiotics (Defoirdt et al., 2010). Initial studies of quorum sensing in aquaculture organisms are very limited but point out exciting results. Halogenated furanones isolated from red marine algae, for example, have been demonstrated to reduce QS-regulated gene expression in Vibrio and to protect fish and shrimp from vibriosis (Rasch et al., 2004; Defoirdt et al., 2006). At the Nutriad Technology Center, QS technology is being applied in a novel generation of natural feed additives capable of modulating gut micro flora. Compounds are tested for their capacity to inhibit QS-signaling using an array of genetically modified bacterial biosensors and QS-dependent infection protocols in simple model organisms. Using these sensitive assays, potent QS modulators, able to shut down QS signaling at concentrations far below the minimum inhibitory concentration, are being identified. Different QS quenching activities are selected for agriculture and Figure 5: Quorum Sensing (QS), an innovative mechanism to tackle pathogenicity aquaculture species based on screening work using spe-

46 | InternatIonal AquAFeed | January-February 2013

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FEATURE Furthermore, this synergistic blend has proven to be a powerful interrupter of bacterial QS signaling at concentrations well below minimal inhibitory concentrations, allowing it to effectively modulate the gut flora towards a more favorable composition. The supplementation of Sanacore GM promoted growth significantly in healthy shrimp growing under controlled lab conditions; showing a remarkable 20 percent increase of weekly weight gain and four percent improvement on food conversion (Coutteau et al., 2010). The effect of this botanical product showing combined activities in QS inhibition and bactericidal action against a wide range of pathogenic bacteria was verified in a semiintensive shrimp farm in Panama.The second production season in Panama, stocked between August-September, is characterised by unstable

Figure 6: Dose response of a synergistic blend of botanical compounds (SANACORE GM, Nutriad) on Quorum Sensing signaling activity of Vibrio harveyi. Graphs show signaling activity in QS biosensor system Vibrio harveyi BB170, relative to control, exposed to for different dilutions of the product extract (Nutriad Technology Center, in-house results).

Table 2: Production results for P. vannamei in Panama during the second production cycle for control ponds and treatment ponds receiving a phytobiotic supplement after 141 days of culture (average and standard deviation of eight replicate ponds of three ha per treatment).

treatment

Survival

Shrimp size

Crop Yield (kg/ha)

Feed (kg/pond 3ha)

FCr

Weekly Growth(g/wk)

average coefficient of variation for parameters listed (CV%) 10% 18% -41% ---

Sanacore GM Control % change Sanacore vs Control P Value

55.5 7.1 a 44.6 10.6 b +24.4% 0.0304

16.6 1.5 a 15.7 2.9 a +5.8% 0.4395

735 78 a 543 90 b +35.2% 0.0004

4,170 338 a 3,464 396 b +20.4% 0.0018

1.91 0.23 a 2.17 0.39 a -12.1% 0.7130

0.825 0.075 a 0.776 0.137 a +6.3% 0.3876

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FEATURE

About the authors

Tim Goossens got a Masters degree in Biotechnology at Ghent University, and subsequently worked as an academic researcher in the Laboratory of Molecular Biotechnology at the University of Antwerp. After working for four years on the characterisation of a gene family involved in bone development, he moved to the KU Leuven, where he took up a doctoral project on neurodevelopment in the Laboratory of Developmental Genetics. After obtaining his PhD. in Biomedical Sciences, he joined Nutriad to work as an R&D Engineer, focusing on the development and technical support of the Gut Support range of feed additives. Peter Coutteau, currently Business Unit Manager Aquaculture for Nutriad, obtained in 1992 a PhD. in Biological Sciences at the Laboratory of Aquaculture & Artemia Reference Center, University of Gent on the filter feeding biology of Artemia and bivalves. He continued academic research at postdoc level till 1997 on lipid nutrition of bivalves, fish and shrimp, publishing over 40 refereed papers in scientific journals. In 1997, joined the INVE group, as head of research and product development in the aquaculture division. During 2002-2008, he was responsible for global product development and customer service for feed concentrates and additives as product manager farm nutrition for INVEs Business Unit Aquaculture. Following restructuration of the INVE group in January 2009, the support team, research activities and product lines for aquaculture additives were reorganised under Nutriads Business Unit Aquaculture.

was confirmed by immuno-chromatography and nested-PCR tests. The presence of a synergistic blend of phytobiotics provided an array of antimicrobial activities, including quorum sensing inhibition capabilities, in the shrimps digestive system. This offers additional protection against co-infections with opporFigure 7: Survival percentage at harvest for tunistic bacteria such as control ponds and treatment ponds receiving the vibriosis, often the major phytobiotic supplement in two production cycles cause of mortality in in semi-intensive production of white shrimp L. WSSV-infected shrimp vannamei (average and standard deviation of eight and five replicate ponds of three ha per (Phuoc et al., 2009). treatment, respectively; data from Vaca et al., The evaluation in 2010, 2011). the second cycle on eight replicates per climatological conditions, resulting in strong tem- treatment allowed a good evaluation of perature fluctuations which in turn affect shrimp variability among ponds for the different growth and increase the impact of outbreaks of production parameters. The addition of white spot virus (WSSV). The first production the phytobiotic reduced drastically the cycle, seeded between January-April, provides variability of production results among more suitable growth conditions and generally ponds fed the same feed (average coefficient of variation between ponds for results in better survival and productivity. Two treatments were compared which the six production parameters: control only differed with regard to the supplementa- 18 percent versus Sanacore group 10 tion or not of the phytobiotic growth pro- percent; Table 2). This further indicated moter (Sanacore GM) to the standard feed the importance of increased control of used at the farm. The supplementation of the gut microflora on the reproducibility of botanical feed additive drastically improved production in semi-intensive pond envisurvival, amounting to a relative increase with ronments. References available on request 24 percent and 18 percent compared to the control group for the second and first cycle, respectively (Figure 7). More InforMatIon: Natural White Spot Disease outbreaks were Email: p.coutteau@nutriad.com observed during shrimp farming in both treatments Website: www.nutriad.com under similar frequency and severity; WSSV virus

48 | InternatIonal AquAFeed | January-February 2013

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