November | December 2011 Feature title: Evaluation of probiotic bacteria in tilapia production

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F: Probiotic bacteria
Figure 1: de Man, Rogosa & Sharpe (MRS) plates of digesta from fish fed a control diet (top plates) and a P. acidilactici supplemented diet (bottom plates) Ferguson et al. (2010) used more accurate and reliable molecular techniques to demonstrate that diets supplemented with Pediococcus acidilactici exhibited alterations in GI microbiota. PCR-DGGE revealed direct antagonism of P. acidilactici with an uncultured bacterium (closest known relative was a bacterial clone isolated from the intestine of Atlantic salmon) during a period of reverting to nonsupplemented feeding. Recent work conducted at the Aquaculture and Fish Nutrition Research Aquarium, University of Plymouth supports this (see Figure 1). Here fish fed a P. acidilactici supplemented diet exhibited considerably higher LAB populations in their digesta, which, were identified as P. acidilactici. This colonisation of the GI tract (at least during continual supplementation) is thought to be a major advantage for potential probionts.

F: Probiotic bacteria
Lactobacillus plantarum, Bacillus pumilus, Lactobacillus acidophilus and Streptococcus faecium as well as various mixtures of these candidates. However, other studies have failed to show a difference in growth parameters with the use of various probiotics. Contradictory results may reflect the differences in rearing conditions and diet where fish reared under near optimal conditions are unlikely to benefit from probiotic applications. Probiotics can improve growth performance by increasing nutrient utilisation and uptake, production of enzymes, amino acids, short-chain fatty acids and vitamins. However, the specific mechanisms in scientific evaluations are often hard to elucidate, due in part to the ethical and methodological limitations of animal studies, together with complex relationships between possible modes of action. Bacteria commonly found in the gut, including Aeromonads, are known to produce proteases and other gut microbes produce amino acids which could be used by the host. This helps to explain the findings of Newsome et al. (2011) who showed that tilapia can obtain their essential amino acid requirements from GI microbiota alone when dietary sources are low or absent. Other authors have isolated gut microbes that can produce other enzymes involved in digestion (carbohydrases, esterases, lipases, phosphatases, peptidases, cellulases), some of which are being assessed as potential probiotics. Anaerobic microbes can produce shortchain fatty acids (which can elevate gut motility and be used for energy purposes or further lipid synthesis) by fermenting dietary carbohydrates. Obligate anaerobes, primarily Cetobacterium somereae and Chlostridium spp. can produce large amounts of vitamin B12, thus tilapia do not require a dietary source of this vitamin because of the microbial production capability. Another mechanism which may improve digestive function is the enhancement of the morphology of the GI tract.

Evaluation of probiotic bacteria in tilapia production

by Benedict Standen & Ali Abid, Aquatic Animal Nutrition and Health Research Group, The University of Plymouth, United Kingdom

Growth performance and effect on digestion and nutrient utilisation

when administered in adequate amounts can enhance the growth and health of the host, as well as the quality of its ambient environment beyond inherent basic nutrition (Fuller, 1989; Merrifield et al., 2010a). These measures help facilitate consumer perceptions of bio-security and eco-friendly aquaculture. Aquatic animals are constantly in contact with the composition and changes in their surrounding environment. Potential pathogens are able to maintain themselves in the external medium and proliferate independently of the host causing disease or rendering aquatic animals immunocompromised. The gastrointestinal (GI) tract is one of the key sites of interaction with the external world and is considered one of the major portals for pathogenic invasion in fish (Ring et al., 2007). The GI microbiota provide a number of functions that benefit the health of the host by promoting nutrient and enzyme supply, enhancing innate immune function, preventing colonisation of pathogenic microbes (by either competition or production of inhibitory compounds), energy homeostasis and are also involved in localised morphological development and maturation of the intestine. Culture dependent and independent studies indicate that bacteria are the main colonisers of the GI tract with early studies recording the predominance of anaerobes in the GI tract of tilapia (Sugita et al., 1989). We now know that in freshwater fish, Vibrio spp., Aeromonas spp., Pseudomonas spp. and Cetobacterium somereae are major colonisers of the intestine followed by Pleisomonas spp., Enterobacteriaceae, Micrococcus spp., Actinobacter spp., Clostridium spp., Bacteroides spp. and Fusarium spp. . Also present in tilapia intestine are Burkholderia spp., Chromobacterium spp., Citrobacter spp. and Flavimonas spp. (Molinari et al., 2003). Despite being prevalent in mammalian and avian intestines lactic acid bacteria (LAB) are present, but generally sub-dominant, in fish. Improved growth performance has been observed in tilapia fed diets supplemented with a number of probiotics including S. cerevisiae, Micrococcus luteus, B. subtilis,

reshwater fish culture has traditionally been dominated by various carp species but recently tilapia production has increased significantly; as a result tilapia are currently being cultured in over 70 countries with an estimated annual output of over three million tonnes (FAO, 2010). This rapid growth suggests that tilapia may become one of the most important cultured fish species in the future. Although tilapia are relatively resistant to infection and disease compared to other finfish, intensification of tilapia production has resulted in an increase of bacterial infections which frequently causes mass mortalities, leading to severe economic losses in major fish producing countries. Traditional practices involve using antimicrobial compounds to treat disease outbreaks and control infections in aquatic animals. However, a number of issues exist with these measures, namely the increasing problem of emerging antibiotic resistant pathogens with the threat of resistance transfer to human pathogens as well as food and environmental contamination. Consequently, in recent years there has been considerable effort to evaluate the feasibility of using alternative methods, particularly probiotics to enhance growth, stimulate the innate immune system and/or prevent disease in the host. Probiotics are live microorganisms, including many yeasts and bacteria, which

Morphological effects on the intestine

It has been reported that probiotics can affect fish GI function and morphology. In this respect, a study by Pirarat et al (2011)

Modification of microbial composition

The GI tract is available to microbial colonisers upon opening of the mouth of larval fish. In this sense the commensal microbiota reflects the microbiota of the eggs, rearing water and the microbiota of any dietary intake during the first-feeding. It has been proposed that any probiotic supplementation at this stage has a significant advantage, whereby good microbes get there first. However, at any developmental stage the microbiota can shift as a result of farming practices, rearing environment, seasonality and diet. Furthermore, the administration of a probiotic may also cause a shift in the microbial composition. For example, Oreochromis niloticus fed a diet supplemented with a mixture of Saccharomyces cerevisiae and Bacillus subtillus showed an altered composition, lacking Escherichia coli, Salmonella spp., Klebsiella spp. and Pseudomonas fluorescens which were present in control fish (Marzouk et al., 2008).

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16 | InternatIonal AquAFeed | november-December 2011

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F: Probiotic bacteria
response of Nile tilapia were positively affected by P. acidilactici. The authors found that the serum lysozyme activity was significantly higher in the fish fed the probiotic diet in comparison with the control fish; furthermore, the total number of circulatory leucocytes was also elevated. In addition, another study examined the potential probiotic effect of Lactobacillus rhamnosus GG on tilapia immunity. After being fed a probiotic supplemented diet for 30 days, nonspecific parameters were significantly enhanced in the probiotic group (Pirarat et al., 2011). It is noteworthy that the activity of "The intensification of tilapia innate immune parameters can be affected by several external production has caused an increase and internal aspects, including handling, crowding stress and in bacterial infections and issues temperature changes. associated with the over usage of The dietary supplementation of probiotics can enhance antibiotics has highlighted the need both systematic and localised immunity in a wide range of fish to fight disease using more robust including tilapia, rainbow trout, and sustainable methods, namely gilthead seabream, European seabass and grouper. the administration of probiotics" The effectiveness of probiotics in terms of protection against infectious pathogens is often attributed to elevated immunity. In tilapia, numerous probiotics comparison with the control group of fish. have shown increased protection against However, a study with rainbow trout Edwardsiella tarda and Aeromonas hydrophishowed that dietary P. acidilactici may la infections. In other aquaculture species, improve microvilli length and enterocyte protection against enteric red mouth endocytic activity (Merrifield et al. 2010b), disease, edwardsiellosis, furunculosis, which has yet to be studied in tilapia. lactococcosis and several other diseases have been documented with probiotic Stimulating the innate feeding. immune response Furthermore, probiotic treatment may It is well documented that the modulaprovide herd immunity from multiple distion of the nonspecific immune system is eases. Protection against viral and protozoan one of the most important modes of action infections has also shown some successes in for probiotics. the fight to control Ichthyophthiriasis in In general, growth inhibitors, natural rainbow trout and iridovirus in grouper. antibodies, cytokines, antibacterial peptides, chemokines, various lytic enzymes and components of the complement pathways Conclusion are considered to be components of The intensification of tilapia production humoral parameters of the innate immune has caused an increase in bacterial infecsystem whereas nonspecific cytotoxic cells tions and issues associated with the over and phagocytes constitute innate cellular usage of antibiotics has highlighted the components (Magnadttir, 2006, Gmez need to fight disease using more robust and and Balczar, 2008). sustainable methods, namely the administraPrevious studies, have generally demtion of probiotics. onstrated some visible benefits on either Current literature clearly demonstrates the immune function, disease resistance, that probiotics can benefit tilapia production. or both. Contrary results may stem from differIn order to explore an improvement of ences in the species and strain of probiont, the immune system due to probiotic applidosage, species and age/size of host fish, cation, Ferguson et al (2010) confirmed that mode of application, feeding management, some aspects of the nonspecific immune duration of supplementation, environmental demonstrated that L. rhamnosus application as a dietary supplement encouraged the development of the intestinal structure of Nile tilapia. The authors found the length of villous in the proximal and middle sections of the intestine were greater in the group of fish fed the probiotic. On the contrary, Nile tilapia that were fed P. acidilactici at 107 CFU /g for 32 days (Ferguson et al., 2010) showed no gross morphological differences in the intestine of fish fed the probiotic in conditions, farming practices and stocking densities. Therefore, in order for the probiotics concept to be beneficial and cost-effective, future work must address these issues associated with the application methods and must be used in concert with suitable farm management.

References
FAO (2010) The State of World Fisheries and Aquaculture 2010 (Food and Agricultural Organization of the United Nations, Rome, 2010). Ferguson RM, Merrifield DL, Harper GM, Rawling MD, Mustafa S, et al. (2010) The effect of Pediococcus acidilactici on the gut microbiota and immune status of on-growing red tilapia (Oreochromis niloticus). J Appl Microbiol 109: 851-862. Fuller R (1989) Probiotics in man and animals. J Appl Bacteriol 66: 365-378. Gmez GD, Balczar JL (2008) A review on the interactions between gut microbiota and innate immunity of fish. FEMS Immunol Med Mic 52: 145-154. Magnadttir B (2006) Innate immunity of fish (overview). Fish Shellfish Immun: 20: 137-151. Marzouk MS, Moustafa MM, Mohamed NM (2008) The influence of some probiotics on the growth performance and intestinal microbial flora of Oreochromis niloticus. Proceedings of 8th International Symposium on Tilapia in Aquaculture, Cairo, Egypt, pp. 1059-1071. Merrifield DL, Dimitroglou A, Foey A, Davies SJ, Baker RT et al. (2010a). The current status and future focus of probiotic and prebiotic applications for salmonids. Aquaculture 302: 1-18. Merrifield, DL, Harper, GM, Dimitroglou, A, Ring, E, Davies, SJ (2010b) Possible influence of probiotic adhesion to intestinal mucosa on the activity and morphology of rainbow trout (Oncorhynchus mykiss) enterocytes. Aquacult Res 41: 1268-1272. Molinari LM, Scoaris D, Pedroso RB, Bittencourt N, Nakamura CV, et al. (2003) Bacterial microflora in the gastrointestinal tract of Nile tilapia, Oreochromis niloticus, cultured in a semi-intensive system. Acta Sci Biol Sci Mar 25: 267-271. Newsome SD, Fogel ML, Kelly L, Martinez del Rio C (2011) Contributions of direct incorporation from diet and microbial amino acids to protein synthesis in Nile tilapia. Funct Ecol (in press). Ring E, Myklebust R, Mayhew TM, Olsen RE (2007) Bacterial translocation and pathogenesis in the digestive tract of larvae and fry. Aquaculture 268: 251-264. Sugita H, Oshima K, Tamura M, Deguchi Y (1989) Bacterial flora of gastrointestine of freshwater fishes in river. B Jpn Soc Fish 49: 1387-1395.

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