Testing the efficacy of probiotics for disease control in aquaculture

Infectious diseases have been estimated to cost the global aquaculture industry billions of dollars annually^1,2. With concerns over emerging resistance and residues of antibiotics in food³ many such chemicals are now being banned and environmentally friendly alternatives are being sought. Probiotics influence the composition of the gut microbiota and confer health benefits to their host^4,5 and are one of several alternative approaches gaining significant popularity in aquaculture. Whilst primarily used to manage bacterial disease, there is also some evidence that probiotics can provide protection against parasites⁴ and viruses⁶. Probiotics can inhibit the growth of pathogens in the gut through the excretion of antagonistic substances including bacterocins^6,7; prevent pathogen adhesion in the gut through competition of space and nutrients³ and by modulating the immune system⁵. Some probiotics have been reported to improve growth and feed utilisation efficiency⁵ and others can also improve water quality^2,8,9, which confers indirect benefits to host health⁶.

It has been estimated that 50 000 tonnes of probiotics are used annually in the aquaculture industry¹⁰ yet analysis of the literature reveals a great deal of equivocal data on their efficacy. This is likely due, at least in part, to the wide diversity of both hosts and probiotic species within this industry and the fact that probiotic efficacy against particular pathogens is often both host specific and probiotic strain specific⁵. These factors demonstrate the need for testing to ensure probiotics are fit for purpose. Aquaculture species span many phyla and their probionts are far more diverse than the typical lactic acid bacteria (LABS) used in terrestrial animals⁶ and which do not dominate in the normal gut flora of aquatic animals². The diversity of probiotics used in aquaculture is highlighted by Newaj-Fyzul et al. who reviewed 18 genera of Gram-negative and 19 genera of Gram-positive bacteria that have been used in aquaculture⁴. This diversity reflects that of the aquatic habits where aquaculture species live and closely interact with these microbes^3,7.

Despite this diversity, Bacillus subtilis and B. licheniformis remain the most commonly used commercial probiotics in aquaculture⁵, due to their spore forming nature and subsequent proclivity for long term storage and stability in formulated feeds^6,11. Evidence exists, however, that probiotics isolated from host gut or their environment are more likely to outperform commercial products because they are more likely to colonise the host gut and effectively compete with pathogens^6,12. The first stage of screening potential probiotics usually occurs in vitro and seeks to measure antagonism against pathogens of interest using methods including well diffusion, cross streaking and disc diffusion and the co-culture method^3,13. These methods can also be used to test antagonism of putative probiotics against pathogens of interest. Whilst such methods allow cost effective, simultaneous screening of many probiotic candidates, several authors have pointed out the limitations of relying on in-vitro tests^3,7. Given the many different modes of action of probiotics, a lack of in vitro antagonism does not necessarily exclude the bacteria as a probiotic. Conversely, it has also been demonstrated that the expression of antagonism in vitro does not guarantee that a candidate will perform effectively in vivo^2,6.

Administration of the probiotic to the host and measuring in vivo performance is therefore critical and indeed Kesarcodi-Watson et al. advocates for the use of in vivo testing in the preliminary screening phase to prevent exclusion of those probiotics which do not exhibit antagonism but which may still be effective based on other modes of action³. Whilst probiotic administration for such in vivo testing is usually achieved in dry feed, in hatchery applications probiotics can be delivered using live feeds as delivery vectors to the larval host or via the culture water. Application via culture water can also be effective in ponds⁸.

Measurement of effectiveness in vivo can be achieved in several ways. As effective probiotics should colonise and thrive in the host gut^5,7, the first step can therefore simply involve confirming such colonisation and indeed not all commercial strains of probiotics originally developed for terrestrial animals thrive in the gut of aquatic species⁵. Gatesoupe, however, points out that colonisation is not essential and that transient bacteria may also make effective probiotics if they can be provided continuously at high doses⁶.

Challenging the host with a live pathogen of interest following probiotic administration is the most effective method of determining a probiotic’s efficacy. Whilst such tests do not elucidate the mode of action of the probiotics, they are the most relevant in proving efficacy against pathogen-induced mortality. Appropriate pathogen dosages must be selected to ensure the probiotic effect can be detected without completely overwhelming the host. Preliminary testing should therefore be conducted to determine an appropriate LD₅₀ and an appropriate route of administration for the pathogen of interest (for example bath immersion versus injection). Care must also be taken to ensure the virulence factors of the pathogen are not lost in culture prior to administration. When biosecurity or animal ethical considerations prevent challenge trials using live pathogens, administration of inactivated pathogens or pathogen-associated molecular patterns (PAMPS) may elicit measurable immune responses that indicate probiotic efficacy. Conversely, live pathogens can be used in vitro to measure immune response in certain host tissues. Many options exist for measuring the immune response in the host following probiotic application. These range from simple assessments of basic haematological parameters¹¹ to the measurement of factors such as lysozyme and serum bactericidal activity, immunoglobulin levels, phagocytosis and respiratory burst activity to more complex methods such as quantifying gene expression profiles for inflammatory markers such as cytokines⁵.

Determining host-specific and strain-specific optimum probiotic dose and treatment duration are also key elements that requires in vivo testing to ensure optimum efficacy^12,14. Effective in-feed dose rates generally range from 10^6–10 CFU/gram of feed and in water treatments from 10⁴ to 10⁵ CFU/mL, with excessive doses shown to cause deleterious effects in the host^5,8. For most species for which probiotics have proven effective, improved immunity has been detected 1 to 10 weeks following commencement of treatment, however, extended use can also be detrimental³.

Finally, testing probiotics for safety, both for the host and the human consumer, is also very important. This is particularly relevant in the field of aquaculture where many probiotics being considered for use have closely related strains that are known pathogens. Testing for safety should therefore include confirmation of non-transmission of antimicrobial resistance genes or virulence plasmids, as well as using molecular techniques to confirm with certainty the identity of the species under investigation¹⁵.