Agriculture Business Week

Trials in Shrimp
The Sanolife Bacillus strains, when applied through the feed (top dressed at the farm site or at the feed mill), have been evaluated in shrimp grow out (Litopenaeus vannamei, L. stylirostris and Penaeus monodon ) in Asia, the Pacific region and Latin America. The application of these bacteria (concentration ranging from 1 x 107 to 1.5 x 108 cfu/g feed according to rearing conditions), in association with suitable pond management, has led to marked benefits to the farmers:
1. Faster growth – Scientists at IFREMER showed in a controlled experiment with replicates that there was a very significant increase in growth rate when the Sanolife Bacillus strains were mixed with the feed pellets shortly before feeding the shrimp (Moriarty et al., 2006). Similar improvements in growth rates were recorded with L. vannamei under commercial conditions in Ecuador and Brazil (Figure 1 ).

2. Higher survival – The application of the Sanolife Bacillus led to an increased survival rate in all studied species. In a test carried out with L. vannamei in Ecuador (7 ponds with a total area of 96 ha), the survival rate increased by 62%. In Vietnam (Vinh Hau Aquaculture Co., Ltd., Vinh Loi – Bac Lieu), the same combination of probiotics Sanolife PRO-1 and PRO-2 in feed and PRO-W in water, together with adequate water quality management and follow-up of the health status of the animals, led to 100% higher harvested biomass and 10% higher survival rate compared to the control ponds.

3. Improved Feed Conversion Ratio – For the three species evaluated in the three regions, a marked decrease in FCR was recorded (Figure 2 ).

4. Larger animals at harvest – In a trial carried out with P. monodon in India (Andrah Pradesh, triplicate treatment and control ponds), the application of Sanolife Bacillus led to larger average sized animals at harvest (23g). In the treated ponds, 25% of the biomass was larger animals (34g) fetching a much higher price, however, none of the controls reached this size. A combination of higher survival with larger animals leads to higher biomass and, more importantly, together with a more efficient use of compound feed, higher income for the farmers. In all these trials, the net profit was far greater when the probiotics were used.

Trials in fish
Fish trials were performed in recirculation systems (Tianjin Haifa Seafood Industrial Development Co., Ltd., Tianjin, China). Flounder (60 – 80g) were stocked in concrete tanks and received 2 separate mixtures of Bacillus. One was mixed within the feed pellets and the second was applied directly in the water (once every 2 weeks). The fish were fed feed pellets (30% of diet) and trash fish (70% of diet). The survival and FCR were good in all tanks. A noteworthy result was the marked improvement in weight gain each month due to the combination of water and feed probiotics (Figure 3 ). This led to fish reaching market size much faster when receiving the Sanolife. The performance of these Bacillus strains in shrimp and fish larviculture has also been reported (Decamp et al., 2006, Decamp et al., in press).

Safety of strains
A very important issue is the safety of these strains to the target species, the environment and humans. Strains should be tested for their sensitivity to antibiotics. For some strains, tools are available that will allow the screening of specific toxins, or genes associated with the toxin production, but this is not the case with less common species. Hence, probiotic products should not include Vibrio (risk of transfer of virulence genes) or coliforms. Within Bacillus and their related genera Paenibacillus and Brevibacillus, the B. anthracis group is pathogenic for animals. The species within this group (B. anthracis, B. cereus, B. mycoides and B. thuringiensis) must not be present in any probiotics and, furthermore, Bacillus probiotics in other generally safe groups, like the B. subtilis group, must be checked to ensure they do not carry any genes for toxins from the anthrax group. The strains should also be evaluated in challenge tests and rearing trials in order to confirm their lack of toxicity to the host or other animals found in the production facilities, at doses 10 or 100x higher than the recommended doses (Makridis et al., 2005). For some strains, safety is based on historical use; they have been used in human food for a long period of time and are thus considered as safe. However, their safety to aquaculture organisms should still be investigated. Once the possible probiotic effect has been demonstrated in vivo, the ability of the strain to remain active in the aquaculture environment, to perform the expected function and to maintain itself should be demonstrated. The ability of Bacillus species to colonise (actually live and grow) in the intestinal tracts of shrimp, rather than exist as transient flora during feeding, is now the subject of investigation in a project at the Centre for Marine Studies, University of Queensland. Preliminary results reported at the 2006 WAS conference indicate that B. subtilis, or closely related species, do colonise in the midgut of Penaeus monodon (Hill et al ). There are major technical difficulties, however, methods such as DGGE, RT-PCR or FISH should give us better insight into the microbial dynamics associated with reared aquatic animals.

Conclusion
The potential for probiotic use in aquaculture is immense because farmers must avoid prophylactic use of antibiotics, however, bacterial and fungal pathogens will always be present. The use of safe, natural bacteria (probiotics) to modify the microbial communities in aquatic animals and their environment and thus control the pathogens is, therefore, a sustainable alternative to the use of antimicrobial chemicals. However, one should not consider them as miracle products solving health related problems.