Summary
202 strains of lactic acid bacteria (LAB) were isolated from the digestive tracts of cultured and wild shrimp, including the species Litopenaeus vannamei, Metapenaeus brevicornis and Penaeus merguiensis. These bacterial strains were challenged to determine their antibacterial activity against Vibrio harveyi. The results showed that all lactic acid bacterial strains had strong antagonistic activity against V. harveyi. The highest activity belonged to the strain Lactobacillus plantarum. When co-cultured, Vibrio harveyi and Lactobacillus plantarum showed that Vibrio harveyi was completely eliminated after 24 hours, under both aerobic and anaerobic conditions. The experiment also compared the survival rate and growth performance of cultured shrimp fed with and without LAB for 6 weeks. The results showed the remarkable effectiveness of LAB: the survival rate, growth rate, and feed conversion ratio of the group supplemented with LAB in their diet were significantly higher than those of the control group, especially the survival rate (98.89% in the LAB-fed group and 68.89% in the control group). And after being challenged with Vibrio harveyi, after 10 days, the shrimp group supplemented with LAB achieved a survival rate of 77%, while the control group only reached 67%.
Introduction
Vibrio is known as one of the bacteria present in the normal microbiota of shrimp (accounting for 38-81% of the bacterial population). Vibrio spp is one of the causes of bacterial infections in cultured shrimp, invading various parts such as appendages, hepatopancreas, intestines, etc. Vibrio harveyi causes luminous vibriosis in post-larval shrimp, and recent findings show that it also causes bright-red syndrome (BRS) in whiteleg shrimp.
The use of probiotics in intensive shrimp farming is an effective measure to prevent diseases caused by V. anguillarum , V. vulnificus , V . alginolyticus and V. harveyi. Many probiotics containing Lactobacillus spp, Bacillus spp, Saccharomyces spp, and other types have been proven effective in inhibiting Vibrio in shrimp farming. Among these, LAB, with its ability to produce antibacterial substances including lactic acid, acetic acid, hydrogen peroxide, and bacteriocin, effectively inhibits competing bacteria.
In this study, the inhibitory effect of LAB on V. harveyi isolated from the digestive tracts of cultured and wild shrimp, and its competitive efficacy, were evaluated both in vitro (in test tubes) and in vivo (on cultured shrimp). The results reported on the growth rate, survival rate, feed conversion ratio, and bacterial density in the digestive tract of whiteleg shrimp.
Materials and Methods
V. harveyi was isolated from diseased shrimp and cultured on TSB medium.
LAB was isolated from intensively cultured shrimp and wild shrimp, and cultured on MRS medium.
To evaluate the inhibitory capacity of V. harveyi, a LAB strain, Lactobacillus plantarum, was co-cultured with V. harveyi in 3 media: TSB, MRS, and a TSB-MRS mixture. The cell counts of both species were monitored after 0, 12, 18, 24, and 48 hours.
In another experiment, healthy 30-day-old shrimp were stocked into 500-liter tanks at a density of 30 individuals/tank. Shrimp were fed 4 times a day, and one tank was supplemented with LAB in all feedings for 6 weeks, with each feeding containing 2 – 4 x1010 cells/150g of feed.
Finally, at the end of the 6-week feeding trial, 20 individuals were taken from the tanks to be challenged with V. harveyi (10-day LD50 = 5.7 – 6 log CFU/ml). Subsequently, each individual was divided into 2 groups: one fed with LAB supplementation and a control group. The survival rate, feed conversion ratio, and growth rate of each group were monitored. Body fluid was extracted from shrimp to isolate and determine the cell count of V. harveyi.
Results
Among the 202 LAB strains (124 isolated from wild shrimp and 78 from cultured shrimp), Lactobacillus plantarum was found to have the strongest inhibitory effect on V. harveyi.
In the co-culture experiment of V. harveyi and Lactobacillus plantarum in a mixed medium, after 12 hours, L. plantarum began to produce antibacterial compounds, started to act against V. harveyi after 18 hours, and completely inhibited them after 24 hours.
Regarding the experiment with LAB supplementation in feed, after 6 weeks, the growth rate of the LAB-supplemented group was significantly higher than that of the control group (953% growth compared to 942%). There was also a significant difference in feed conversion ratio: 0.42 in the LAB-supplemented group and 0.45 in the control group. More notably, the survival rate of shrimp supplemented with LAB reached 98.89%, while the control group only achieved 68.89%.
In the experiment challenging shrimp with V. harveyi (by injection and immersion), the survival rates of the LAB-supplemented group and the control group were 80% and 66.7%, respectively, after 9 hours of challenge, decreasing to 63.3% and 43.3% after 18 hours of challenge. After the challenge, shrimp were again divided into two groups: one with LAB supplementation and one without. After 10 days, the mortality rate of the group not fed LAB was 90%, whereas the group supplemented with LAB to inhibit V. harveyi only had a 37% mortality rate.
Discussion
In nature, LAB is not the dominant bacterial type in the digestive tract of shrimp. Therefore, to combat the pathogenicity of Vibrio, the supplementation of lactic acid bacteria into shrimp feed is of great interest. Not only V. harveyi, but other Vibrio strains also show inhibited growth in environments with abundant LAB, as demonstrated by other studies, for example, Pediococcus acidilactici has been shown to strongly suppress V. nigripulchritudo. This solution has been proven to help shrimp increase growth rate, enhance immune capacity, and improve feed digestibility while ensuring biosafety.
