Objective 1:  Five Bacillus strains from the collection of Probiotics Internationial were tested by the Liles laboratory, Auburn University.

Samples provided were pure Bacillus cultures, shipped either as active cultures or as spore preparations. Each culture was grown on Tryptic Soy Agar (TSA) medium and a glycerol stock was prepared and maintained as a cryostock at -80oC for each strain to maintain this culture for the duration of the study.

Objective 2: Each of the 5 Bacillus strains was evaluated for their ability to inhibit the growth and/or viability of bacterial and fungal pathogens important in aquaculture farmed fish. 

Specifically, each Bacillus strain was tested for its ability to produce antibiotic(s) that inhibit the growth of:

  • Aeromonas hydrophila epidemic strain ML09-119 (this is the epidemic strain that all of the current research studies are using, that is highly virulent in catfish, tilapia and other fish species)

  • Aeromonas hydrophila strain AL06-06 (bacterial pathogen, this strain is representative of the typical Aeromonas hydrophila found to infect catfish prior to the start of the epidemic)

  • Edwardsiellaictaluri (bacterial pathogen)

  • Saprolegniaferax (Oomycete “water mold” pathogen)

  • Yersinia ruckeri (bacterial pathogen)

Method: Water agar plates were prepared (16 g agar/liter) and with a sterilized bore, (2) holes were punched in the water agar plates.  Each hole was filled with TSA up to the same level as the water agar.  Each agar disc was spotted with 10ml of the Bacillus strain from a spore or broth preparation with a cell concentration > 108 CFU/ml.  Plates were then incubated at 30oC for 48 hours.  After 48 hours, plates were then exposed to UV (1000 x 100 μJ/cm2) to kill Bacillus vegetative cells to reduce outgrowth of the Bacillus in the soft agar overlay. To conduct the overlay, the respective pathogen culture was grown in an appropriate medium for 24 hours and 1 ml of the pathogen culture was added to 100 ml of soft agar that has been cooled to 37oC and gently swirled, and 7-10 ml of the soft agar was added to each plate. Agar plates were then incubated at 30oC for 24 hours and the zone of inhibition was measured in mm for each respective Bacillus strain and pathogen combination.

Results: The application of Probiotic International's bacillus strains were observed to control common diseases and bacterial pathogens in aquaculture settings. The mechanisms used by the bacillus strains to improve disease resistance include exclusion of pathogens by producing antimicrobial substances and competing with pathogens for nutrients and space. This can enhance a hosts (fish or shrimp) ability to fight off infection by improving specific immune response in defense against pathogens.