Decreased Severity of Acute Hepatopancreatic Necrosis Disease in White Shrimp (Litopenaeus vannamei) by Mixed Culture of Bacillus subtilis, Bacillus licheniformis and Bacillus megaterium

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Saowapha Surawut
Kunyarut Suntara
Winyou Puckdee
Chutapa Kunsook
Pornpimon Kanjanavas
Anchalee Kompatiparn
Prachuab Leeruksakiat


The objective of this study was to investigate the mixed culture of Bacillus subtilis, B. licheniformis and B. megaterium to control acute hepatopancreatic necrosis disease (AHPND) or EMS (Early Mortality Syndrome) in white shrimp Litopenaeus vannamei as a model. The infected shrimps with Vibrio parahaemolyticus AHPND strain were divided into tanks and different feeding of either B. subtilis, B. licheniformis, B. megaterium or all Bacillus strains. The infected shrimps that were fed with a mixed culture of Bacillus showed significantly highest survival rate and revealed lower percent detection of V. parahaemolyticus AHPND strain by Polymerase Chain Reaction (PCR) (57.14%) with a small amount of viability count in their hepatopancreas. In contrast, the infected shrimps that were fed with each of B. subtilis, B. licheniformis or B. megaterium, revealed the spread of V. parahaemolyticus AHPND strain in all tissue by PCR detection (86.67%–100%) with a large amount of viability count (3.53 – 4.24 × 103 CFU/g). This study indicated that the mixed culture of Bacillus subtilis, B. licheniformis and B. megaterium could control the dissemination of V. parahaemolyticus in shrimps, especially in hepatopancreatic that is the target tissue of AHPND in white shrimp (L. vannamei). The result of this study revealed the efficiency and mechanism of the mixed culture of B. subtilis, B. licheniformis and B. megaterium to control the virulence of AHPND and support the application of this mixed culture in aquaculture of shrimp farms to avoid chemical and antibiotic treatment by using it as a biological control.

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Saowapha Surawut, Kunyarut Suntara, Winyou Puckdee, Chutapa Kunsook, Pornpimon Kanjanavas, Anchalee Kompatiparn, & Prachuab Leeruksakiat. (2023). Decreased Severity of Acute Hepatopancreatic Necrosis Disease in White Shrimp (Litopenaeus vannamei) by Mixed Culture of Bacillus subtilis, Bacillus licheniformis and Bacillus megaterium . Tropical Life Sciences Research, 34(1), 85–98.
Original Article


Cha J H, Samad R, Si Y Y, Kang W K and Kyeong J L. (2013). Evaluations of Bacillus spp. as dietary additives on growth performance, innate immunity and disease resistance of olive flounder (Paralichthys olivaceus) against Streptococcus iniae and as water additives. Aquaculture 402–403: 50–57.

De Schryver P, Defoirdt T and Sorgeloos P. (2014). Early mortality syndrome outbreaks: A microbial management issue in shrimp farming? PLOS Pathogens 10(4):e1003919.

Elsabagh M, Radi M, Eman M M, Ahmad H, Foad F, Decamp O, Mahmoud A O D and Mahmoud E. (2018). Assessing the impact of Bacillus strains mixture probiotic on water quality, growth performance, blood profile and intestinal morphology of Nile tilapia, Oreochromis niloticus. Aquaculture Nutrition 24(6): 1613–1622.

Fan Y, Xiaolu W, Youhong W, Haibin Y, Xiaoqing Y, Shuxian W, Jing D, La X, Chunlei G, Hongjun L and Danping M. (2021). Effect of dietary Bacillus licheniformis on growth, intestinal health, and resistance to nitrite stress in Pacific white shrimp Litopenaeus vannamei. Aquaculture International 29(6): 2555–2573.

Hlordzi V, Kuebutornye F K A, Afriyie G, Abarike E D, Lu Y, Chi S and Anokyewaa M A. (2020). The use of Bacillus species in maintenance of water quality in aquaculture: A review. Aquaculture Reports 18: 100503.

Khimmakthong U and Sukkarun P. (2017). The spread of Vibrio parahaemolyticus in tissues of the Pacific white shrimp Litopenaeus vannamei was analyzed by PCR and histopathology. Microbial Pathogenesis 113: 107–112.

Kumar V, Roy S, Meena D K and Sarkar U K. (2016). Application of probiotics in shrimp aquaculture: importance, mechanisms of action, and methods of administration. Reviews in Fisheries Science and Aquaculture 24(4): 342–368.

Kyeong-Jun L, Jong S E, Jae W K, Sung H K, Ji E K, Jee E H and Chorong L. (2019). Effect of dietary supplementation of Bacillus spp. on growth performance, and resistance of Pacific white shrimp (Litopenaeus vannamei) to acute hepatopancreatic necrosis disease. Israeli Journal of Aquaculture – Bamidgeh 71(January): 1–10.

Lai H C, Ng T H, Ando M, Lee C T, Chen I T, Chuang J C, Mavichak R, Chang S H, Yeh M D, Chiang Y A, Takeyama H, Hamaguchi H O, Lo C F, Aoki T and Wang H C. (2015). Pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimp. Fish and Shellfish Immunology 47(2): 1006–1014.

Li K, Zheng T, Tian Y, Xi F, Yuan J, Zhang G and Hong H. (2007). Beneficial effects of Bacillus licheniformis on the intestinal microflora and immunity of the white shrimp, Litopenaeus vannamei. Biotechnology Letters 29: 525–530.

Liu C H, Chiu C S, Ho P L and Wang S W. (2009). Improvement in the growth performance of white shrimp, Litopenaeus vannamei, by a protease-producing probiotic, Bacillus subtilis E20, from natto. Journal of Applied Microbiology 107(3): 1031–1041.

Nakayama T, Lu H and Nomura N. (2009). Inhibitory effects of Bacillus probionts on growth and toxin production of Vibrio harveyi pathogens of shrimp. Letters in Applied Microbiology 49(6): 679–684.

Nguyen N D, Pande G S J, Kashem M A, Baruah K and Bossier P. (2021). Acute hepatopancreatic necrosis disease (AHPND) toxin degradation by Bacillus subtilis DSM33018. Aquaculture 540: 736634.

Prachumwat A, Taengchaiyaphum S, Mungkongwongsiri N, Aldama-Cano D J, Flegel T W and Sritunyalucksana K. (2019). Update on early mortality syndrome/acute hepatopancreatic necrosis disease by April 2018. Journal of the World Aquaculture Society 50(1): 5–17.

Reddy, V K, Vamshi K R A, Babu B S and Lakshmi T V. (2018). Applications of Bacillus sp. in aquaculture wastewater treatment. International Journal of Scientific Research in Science and Technology 4(5): 1806–1812.

Shen W Y, Fu L L, Li W F and Zhu Y R. (2010). Effect of dietary supplementation with Bacillus subtilis on the growth, performance, immune response and antioxidant activities of the shrimp (Litopenaeus vannamei). Aquaculture Research 41(11): 1691–1698.

Tinwongger S, Proespraiwong P, Thawonsuwan J, Sriwanayos P, Kongkumnerd J, Chaweepack T, Mavichak R, Unajak S, Nozaki R, Kondo H and Hirono I. (2014). Development of PCR diagnosis for shrimp acute hepatopancreatic necrosis disease (AHPND) strain of Vibrio parahaemolyticus. Fish Pathology 49(4): 159–164.

Vaseeharan B and Ramasamy P. (2003). Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon. Letters in Applied Microbiology 36(2): 83–87.

Velázquez-Lizárraga A E, Juárez-Morales J L, Racotta I S, Villarreal-Colmenares H, Valdes-Lopez O, Luna-González A, Rodríguez-Jaramillo C, Estrada N and Ascencio F. (2019). Transcriptomic analysis of Pacific white shrimp (Litopenaeus vannamei, Boone 1931) in response to acute hepatopancreatic necrosis disease caused by Vibrio parahaemolyticus. PLOS ONE 14 (8): e0220993.

Yuniarti A, Guntoro D A, Maftuch and Hariati A M. (2013). Response of indigenous Bacillus megaterium supplementation on the growth of Litopenaeus vannamei (Boone), a new target species for shrimp culture in East Java of Indonesia. Journal of Basic and Applied Scientific Research 3(1): 747–754.