Using Bacillus to Inhibit Pathogenic Vibrio Harveyi Bacteria in Farmed Shrimp

Study on the effect of lyophilized Bacillus in the form of freeze-dried\r\nV. harveyi against pathogenic V. harveyi.

Shrimp farming in Southeast Asia is developing very fast,\r\nincluding Vietnam. The highest production belongs to white leg shrimp, so\r\ninvestment in farming systems is expanding and becoming more active. However,\r\nalong with the rapid development is the great threat of an increasingly serious\r\ndisease caused by bacteria and viruses. Therefore a large amount of\r\nantibiotics has been used and residues.

However, shrimp farming is now developing in a safe and environmentally\r\nfriendly manner. New techniques that contribute to productivity\r\nimprovement without endangering shrimp farmers, as well as users, are being\r\nincreasingly applied. One of them is the management of the microbial\r\ncommunity in aquaculture systems, through probiotics as an effective disease\r\nprevention tool. Supplementing to Bacillus probiotic diets has been shown\r\nto enhance immune responses, help with rapid growth, while also improving the\r\nquality of culture water and balancing the intestinal microflora in both tiger\r\nshrimp and whiteleg shrimp.

In order to have a high effect on aquaculture systems,\r\nprobiotics must survive in unbalanced living conditions such as salinity,\r\nvariable pH, osmotic pressure, low dissolved oxygen ... Micro-capsule\r\ntechnology has shown that it is possible to protect packaged\r\nmicroorganisms. This study will determine the benefits of Bacillus strain\r\nfor the post-larval stage in Litopenaeus vannamei and resistance to V. harveyi of\r\nshrimp.

Methods and materials

The study was conducted in Thailand. The first is to\r\ncollect postlarval at a hatchery, then carry out rearing for 15 days until\r\nreaching PL30 size. Tested by scientific methods to prove that the shrimp\r\nseed does not carry the pathogen is V.harveyi.

Bacillus strains ( Bacillus thuringiensis, B.\r\nmegaterium, B. licheniformis, B. polymyxa and B. subtilis )\r\nwere isolated from tiger shrimp intestine through culture. Convert to dry\r\nand wrapped Bacillus microorganisms so that the right amount is needed for the\r\nstudy and ensure that only one pure Bacillus in each sample is prepared,\r\nmaintaining the viability of the bacteria enough time for testing. experience.

Stock PL30 shrimp with a density of 52 shrimp / m 2 \r\nwith aeration in each tank (9 tanks in total). Care management carefully,\r\nmonitoring shrimp health daily to have appropriate treatment when abnormalities\r\noccur. The experiment consists of three contents: (a) a normal feeding\r\nregime for control samples (without probiotics), (b) a diet containing\r\nfreeze-dried Bacillus (FB), (c) ) Add to Bacillus microbiota culture media\r\n(MB). In the case of MB the shrimp are normally fed and each tank is a\r\ndifferent species of Bacillus. Add 3 times daily to ensure the final\r\nconcentration is 10 7 CFU / ml.

Preparation of V. harveyi pathogen ,\r\ndetermining LD50 lethal concentration according to a previous study, LD50 =\r\n1x10 7 CFU / ml (Reed and Muench 1983).

Then check the infection, signs of disease occurrence,\r\nsurvival, the mortality of experimental shrimp. Sampling from shrimp then\r\nculturing, isolating bacteria on TCBS medium, then determining their\r\nbiochemical shape and characteristics, sequencing their genes. Also, take\r\nsamples of water and feces and isolate bacteria on the\r\ninoculum. Bacterial DNA extraction, running PCR to amplify the gene\r\nsegments needed for research. Finally, statistical analysis is needed to\r\ndetermine the effectiveness of treatment.

Result

After 30 days of feeding, there was an increase in the weight of\r\nFB and MB compared to the control group. The survival rate of the control\r\ngroup was 86.7% but in the two groups FB and MB up to 96% and\r\n95.3%. Cumulative mortality in the control group was 63.06%, in the remaining\r\n2 groups were 43.24% and 45.05%, respectively. Some manifestations on\r\nshrimp are anorexia, lethargy on the water. The inside of the intestine is\r\nempty and contains lots of mucus, inflammation, and hemorrhage, shrimp liver, and\r\npancreas are gray and swollen.

Bacillus ratio in shrimp samples was very high in FB and MB\r\ngroups, but both dropped sharply after V. harveyi infection and\r\nremained stable after that. Bacillus is dominant in the FB and MB groups,\r\nof which MB is higher than FB. After the experiment, the ratio of V.\r\nharveyi in shrimp in FB and MB groups was much lower than the control\r\ngroup. Bacillus numbers were inversely proportional to the number of V.\r\nharveyi counted in groups. The monitoring of water quality indicators\r\nindicates that conditions are acceptable for shrimp growth.

Discuss

Bacillus feeds and feeds into the surface of the intestinal\r\nmucosa, competing for nutrients and fouling with pathogenic bacteria. Bacillus\r\nhas been studied for its ability to produce extracellular enzymes that enhance\r\ndigestibility and absorption of nutrients, improving feed efficiency. In\r\naddition, Bacillus also has the ability to synthesize vitamins, coenzymes,\r\namino acids, etc. They act as an additional source of nutrition in the\r\nintestinal tract of shrimp. Although there was no difference in the\r\nresults of the lyophilized and microfiltration method in this\r\nstudy. However, the use of microfiltration is somewhat more advantageous\r\nin improving the growth performance and survival rate of shrimp.

Bacillus has the ability to produce inhibitors such as\r\nantibiotics alone or in combination. Previous research has demonstrated\r\nthat Bacillus has an inhibitory effect on V. parahaemolyticus and V.\r\ncholerea that causes disease in the digestive tract of black tiger\r\nshrimp. Bacillus 's V. harveyi protection has been\r\nshown to be effective in black tiger shrimp, giant freshwater shrimp, and other\r\nspecies. Observed that shrimp survived to have a higher phagocytic index,\r\nimmune activity, antibacterial activity, the number of blood cells also\r\nincreased significantly.

Micro-coated microorganisms have superior protection\r\nagainst lyophilized form against V. harveyi causes diseases in white leg shrimp. Although the mechanism of action is not known, the beneficial\r\neffects are evident in helping to balance the microflora in the intestinal\r\ntract of shrimp, reducing mortality from vibrio sp. cause. It also\r\nhelps reduce damage to the liver and pancreas.

Quorum Sensing is a means that V. harveyi uses to\r\nproduce their virulence, hemolysin. However, Bacillus strains have the\r\nability to cut off the signal to produce this virulence of bacteria. And\r\nso a protective film formed by Bacillus prevented V.harveyi 's production of virulence. Microfilm\r\ncontaining Alginate is an immunostimulant to shrimp.

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Some Bacillus species have been shown to prevent the\r\naccumulation of nitrogen-containing wastes (NH3 , NO2 ...)\r\nthrough nitrification or aerobic denitrification processes, thereby improving\r\nquality. country. This study also showed that lyophilized Bacillus and\r\nmicrocapsules were equally effective in terms of promoting growth and improving\r\nwater quality against V. harveyi. However, the stability and survival of\r\nBacillus microflora needs further research.

Source: tepbac.com