Evaluation of probiotic bacteria in tilapia production

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November | December 2011

Feature title: Evaluation of probiotic bacteria in tilapia production

The International magazine for the aquaculture feed industry

Freshwater 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 infec-tions which frequently causes mass mor-talities, 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

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 nutri-tion (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 caus-ing 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 (Ringø et al., 2007). The GI microbiota provide a number of functions that benefit the health of the host by promoting nutri-ent 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.

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 supplementa-tion 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).

16 | InternatIonal AquAFeed | november-December 2011 november-December 2011 | InternatIonal AquAFeed | 17

F: Probiotic bacteria

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

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)

IAF11.06.indd 16 04/11/2011 08:41

Ferguson et al. (2010) used more accu-rate and reliable molecular techniques to demonstrate that diets supplemented with Pediococcus acidilactici exhibited altera-tions in GI microbiota. PCR-DGGE revealed direct antagonism of P. acidilactici with an uncultured bacterium (closest known rela-tive 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. acidilac-tici supplemented diet exhibited consider-ably 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.

Growth performance and effect on digestion and nutrient utilisation

Improved growth performance has been observed in tilapia fed diets supplemented with a number of probiotics including S. cerevisiae, Micrococcus luteus, B. subtilis,

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 perform-ance 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 stud-ies, together with complex relationships between possible modes of action.

Bacteria commonly found in the gut, including Aeromonads, are known to pro-duce 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 short-chain 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 mor-phology of the GI tract.

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)

16 | InternatIonal AquAFeed | november-December 2011 november-December 2011 | InternatIonal AquAFeed | 17

F: Probiotic bacteria

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18 | InternatIonal AquAFeed | november-December 2011

HeadquartersEvonik Industries AGHealth & Nutritionfeed additives Rodenbacher Chaussee 4 63457 Hanau-Wolfgang, Germanyphone +49 6181 59-2256 fax +49 6181 59-6734

Europe & Middle East Africa +49 6181 59-6766Latin America +49 6181 59-6761North America +1 678 797-4300Asia North +86 10 85 27-6400Asia South +65 6890-6861

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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.

ReferencesFAO (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.

Gómez GD, Balcázar JL (2008) A review on the interactions between gut microbiota and innate immunity of fish. FEMS Immunol Med Mic 52: 145-154.

Magnadóttir 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, Ringø, 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).

Ringø 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.

response of Nile tilapia were positively affected by P. acidilactici. The authors found that the serum lysozyme activity was sig-nificantly higher in the fish fed the probiotic diet in comparison with the control fish; furthermore, the total number of circula-tory leucocytes was also elevated. In addi-tion, 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 innate immune parameters can be affected by several external and internal aspects, including handling, crowding stress and temperature changes.

The dietary supplementa-tion of probiotics can enhance both systematic and localised immunity in a wide range of fish including tilapia, rainbow trout, gilthead seabream, European seabass and grouper.

The effectiveness of pro-biotics in terms of protection against infectious pathogens is often attributed to elevated

immunity. In tilapia, numerous probiotics have shown increased protection against Edwardsiella tarda and Aeromonas hydrophi-la infections. In other aquaculture species, protection against enteric red mouth disease, edwardsiellosis, furunculosis, lactococcosis and several other diseases have been documented with probiotic feeding.

Furthermore, probiotic treatment may provide ‘herd immunity’ from multiple dis-eases. Protection against viral and protozoan infections has also shown some successes in the fight to control Ichthyophthiriasis in rainbow trout and iridovirus in grouper.

ConclusionThe intensification of tilapia production

has caused an increase in bacterial infec-tions and issues associated with the over usage of antibiotics has highlighted the need to fight disease using more robust and sustainable methods, namely the administra-tion of probiotics.

Current literature clearly demonstrates that probiotics can benefit tilapia production.

Contrary results may stem from differ-ences in the species and strain of probiont, dosage, species and age/size of host fish, mode of application, feeding management, 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

comparison with the control group of fish. However, a study with rainbow trout

showed that dietary P. acidilactici may improve microvilli length and enterocyte endocytic activity (Merrifield et al. 2010b), which has yet to be studied in tilapia.

Stimulating the innate immune response

It is well documented that the modula-tion of the nonspecific immune system is one of the most important modes of action for probiotics.

In general, growth inhibitors, natural antibodies, cytokines, antibacterial peptides, chemokines, various lytic enzymes and components of the complement pathways are considered to be components of humoral parameters of the innate immune system whereas nonspecific cytotoxic cells and phagocytes constitute innate cellular components (Magnadóttir, 2006, Gómez and Balcázar, 2008).

Previous studies, have generally dem-onstrated some visible benefits on either the immune function, disease resistance, or both.

In order to explore an improvement of the immune system due to probiotic appli-cation, Ferguson et al (2010) confirmed that some aspects of the nonspecific immune

18 | InternatIonal AquAFeed | november-December 2011

F: Probiotic bacteria

"The intensification of tilapia

production has caused an increase

in bacterial infections and issues

associated with the over usage of

antibiotics has highlighted the need

to fight disease using more robust

and sustainable methods, namely

the administration of probiotics"

HeadquartersEvonik Industries AGHealth & Nutritionfeed additives Rodenbacher Chaussee 4 63457 Hanau-Wolfgang, Germanyphone +49 6181 59-2256 fax +49 6181 59-6734

Europe & Middle East Africa +49 6181 59-6766Latin America +49 6181 59-6761North America +1 678 797-4300Asia North +86 10 85 27-6400Asia South +65 6890-6861

Amino AcidsChoosing the right nutrients for your Aquafeed

Our amino acids help to•  replace costly and scarce raw materials •  improve protein balance and production efficiency •  reduce environmental pollution

[email protected] | www.evonik.com/feed-additives

11-01-377 vND AZ Aquaculture A4 us.indd 1 27.10.11 16:58IAF11.06.indd 18 04/11/2011 08:41

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