What if the same feed could help farmed tilapia grow faster and survive a serious bacterial challenge? In this study, one Bacillus strain cut mortality to five percent while improving growth. Fish global demand has been growing in the last decades, and to satisfy present requirements, the aquaculture sector has developed several alternatives to improve its production.

Intensive systems are capable of increasing fish production, but also increase fish susceptibility to bacterial infections due to stress induced on animals. Aeromonas hydrophila is one of the most important pathogens in freshwater fish production, and this bacterium is responsible for disease outbreaks and economical losses in aquaculture systems.

A. hydrophila could produce toxins, hemolysins, and proteases, which are responsible for inducing systemic damage and fish death. Antibiotics and other chemicals are the main compounds used for disease control in aquaculture, but this strategy has induced pathogen resistance and food safety problems.

Functional feeds formulated with probiotics and high levels of vegetable ingredients have been a successful alternative to improve the health status and growth performance of animals. Bacillus strains used in this research were obtained from the laboratory collection at the Ensenada Center for Scientific Research and Higher Education in Ensenada, Baja California, Mexico.

Three strains named sp1, sp2, and sp3 were selected for their capacity to grow in soy-based media and inhibit A. hydrophila development in agar plates. Strain identity was determined by the NCBI database using 16S rDNA gene and Basic Local Alignment Search Tool, or BLAST, and phylogenetic identification analysis was performed using the MEGA X program.

Table 2 shows the ingredients used in the control and experimental diets formulated in this experiment. Diets were prepared, dried at sixty degrees Celsius, and stored. Bacillus sp1, sp2, and sp3 strains were added to feed formulation at two times ten to the sixth colony-forming units per gram.

Juveniles, one hundred eighty Nile tilapia weighing thirty-three point forty-four plus or minus zero point zero five grams, were obtained from a local fish farm and acclimated for two weeks. Fish were randomly distributed in twelve tanks with two-hundred-liter water capacity and fed twice a day until satiation with the corresponding diet.

After forty days, growth performance was measured using the next equations. To evaluate the capacity of A. hydrophila to produce virulence factors, six pairs of oligonucleotides were designed.

PCR results showed that this strain contained hylA and ahpB virulence genes, reported with hemolytic and protease activity, respectively. To evaluate virulence factor production, samples were taken every two hours throughout the growth curve. Hemolysin activity was observed only at the early exponential growth phase and did not appear again.

Protease activity began just after hemolysin activity ended, lasted several hours, and showed its highest proteolytic activity at six hours of culture initiation. Figure five measures the inhibition zone produced by three Bacillus strains against Aeromonas hydrophila, a pathogen associated with tissue damage and mortality in Nile tilapia.

The chart reports approximately thirty percent inhibition for Bacillus sp1, about ninety-five percent for sp2, and roughly sixty-five percent for sp3; the caption summarizes sp2’s activity as threefold and twofold greater than sp1 and sp3, respectively. This in-vitro result helps explain why sp2 was selected for further growth and disease-protection experiments.

Figure 5 represents the antimicrobial activity produced by Bacillus sp1, sp2, and sp3 strains against A. hydrophila. The sp2 strain produced the highest inhibition activity against the pathogen bacteria, producing three- and twofold more activity than the sp1 and sp3 strains, respectively.

Figure seven compares four growth measures in Nile tilapia after forty days on a control diet or diets containing Bacillus strains one, two, or three. Across gained weight, specific growth rate, thermal growth coefficient, and initial weight increment, the Bacillus strain two group shows the largest plotted values, with statistical brackets marking several significant differences, while some comparisons are labeled not significant.

The authors therefore use this figure to show that Bacillus supplementation, particularly strain two, was associated with improved growth performance alongside its potential protective role against Aeromonas hydrophila. After forty days of feeding, the fish were measured, and the results were recorded.

Table 5 shows the weight gained, specific growth rate, and other growth parameters that were analyzed in the experiment. Figure 7 shows that most of these parameters were significantly improved in the Bacillus sp2 group with respect to the control, sp1, and sp3 experimental groups.

Figure eight tracks cumulative mortality in Nile tilapia for fifteen days after challenge with Aeromonas hydrophila, comparing the control diet with diets containing Bacillus strains one, two, and three. The control group reached fifty percent mortality on day thirteen, while the reported mortality values for Bacillus strains one, two, and three were twenty-five percent, five percent, and fifteen percent on day thirteen.

This matters because it shows the functional diets were associated with reduced mortality during the infection period, particularly for strain two. Fish infected with Aeromonas hydrophila after forty days of feeding were followed for an additional fifteen days.

The control group reached lethal dose fifty after thirteen days of initial infection. On the same day, cumulative mortality of Bacillus sp1, sp2, and sp3 reached twenty-five percent, five percent, and fifteen percent, respectively.

Bacillus sp2 was the most effective in inhibiting A. hydrophila development, producing sixty percent and thirty percent more antimicrobial activity than sp1 and sp3 strains, respectively. Groups fed with Bacillus sp1, sp2, or sp3 showed less cumulative mortality than the control group, which had fifty percent mortality.

Therefore, Bacillus sp2 was the most effective strain in protecting Nile tilapia from A. hydrophila infections. The mortality of the sp2 group was five percent, the lowest rate reported until then.

Diets supplemented with Bacillus sp1, sp2, and sp3 showed better growth performance than the control group without Bacillus, especially the Bacillus sp2 group, which improved all parameters. The Bacillus sp2 group produced better results as it duplicated the weight of the tilapia in comparison with the control group.

The sp2 strain induced better growth performance than the other reported Bacillus strains. The growth performance of Nile tilapia obtained in this work suggests that Bacillus strains, especially Bacillus sp2, could alleviate antinutritional factor effects produced by soybean products.

Gobi and colleagues found a similar growth rate using even more soybean meal; however, they used almost three times more fishmeal than was used in this experiment. In this work, hylA and ahpB gene products seem to be the virulence factors responsible for inducing tissue damage and death in cultured fish.

The functional feed was formulated with soybean meal and the Bacillus sp2 strain, capable of increasing the health status of Nile tilapia by inhibiting A. hydrophila strain development and virulence factor production. This feed enhanced growth performance in Nile tilapia as the Bacillus sp2 strain had the capacity to grow in a great variety of plant-based substrates such as soybean meal and other leguminous plants.

The sp2 strain was identified as part of the B. subtilis group and therefore can be considered generally recognized as safe for further commercial feed development. Among the three strains tested, Bacillus sp2 gave the strongest combined result: lower A.

hydrophila mortality and better growth in a soybean-meal functional feed. That makes it a promising candidate for tilapia aquaculture.