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Isolation of Lytic Bacteriophages infected Indonesian-strain Vibrio parahaemolyticus and its Protective Effects on Brine Shrimp (Artemia sp.)
Corresponding Author(s) : Dinamella Wahjuningrum
Jurnal Ilmiah Perikanan dan Kelautan, 2025: IN PRESS ISSUE (JUST ACCEPTED MANUSCRIPT, 2025)
Abstract
Graphical Abstract
Highlight Research
- vB_Vp_TSK01 and vB_Vp_JKT02 a specific parahaemolyticus bacteriophage were isolated from the shrimp aquaculture water.
- Compared to control vB_Vp_TSK01 reduced 11.46±1.35% of bacterial growth, and by 9.86±5.92% for vB_Vp_JKT02 treatment.
- Their cocktail had the highest parahemolyticus growth inhibition by 30.92±3.89%.
- The phage treatment increased the survival of the Artemia by 28.57% compared to infection control.
Abstract
Acute Hepatopancreatic Necrosis Disease (AHPND) caused by V. parahaemolyticus infection was one of the major diseases in shrimp culture in recent years. The Vibrio could also affect the survival of Artemia as the shrimp’s main live feed in the hatchery and they become the possible carrier for the AHPND. Phage therapy in shrimp aquaculture could reduce the application of the antibiotic as an antibacterial agent for the AHPND. The present study aimed to isolate the specific lytic phage for the Indonesian strain of V. parahaemolyticus (Vp) and evaluate the phage therapy for the brine shrimp Artemia infected with the Vp. The Vp-specific phage was isolated from the shrimp farm’s water at Tasikmalaya, and North Jakarta City, Indonesia. After isolation and plaque assay, brine shrimp were used as a model to evaluate the phages’ anti-Vibrio activity The Vp-lytic phage was successfully isolated from shrimp culture water at North Jakarta and Tasikmalaya (Vb_Vp_TSK01 and Vb_Vp_JKT01, respectively) and the results showed that both isolated phages and their cocktails were capable to inhibit the growth of Vp with the highest inhibition shown at the cocktail treatment (p<0.05). The survival of Artemia was higher in the phage treatments (p<0.005) compared to the infected control. Infected control had 68.33% of brine shrimp survival, and the Vb_Vp_TSK01, Vb_Vp_JKT01, and their cocktail had similar average brine shrimp survival of 91.11%. In conclusion, phage therapy proved effective in preventing vibriosis in brine shrimp under the conditions tested.
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- Abdel-Latif, H. M. R., Yilmaz, E., Dawood, M. A. O., Ringø, E., Ahmadifar, E., & Yilmaz, S. (2022). Shrimp vibriosis and possible control measures using probiotics, postbiotics, prebiotics, and synbiotics: A review. Aquaculture, 551:737951.
- Bondad-Reantaso, M. G., & Arthur, R. (2018). FAO Technical assistance efforts to deal with Acute Hepatopancreatic Necrosis Disease (AHPND) of cultured shrimp. Asian Fisheries Science, 31S:1-14.
- Cao, Y., Zhang, Y., Lan, W., & Sun, X. (2021). Characterization of vB_VpaP_MGD2, a newly isolated bacteriophage with biocontrol potential against multidrug-resistant Vibrio parahaemolyticus. Archives of Virology, 166(2):413-426.
- Chen, L., Fan, J., Yan, T., Liu, Q., Yuan, S., Zhang, H., Yang, J., Deng, D., Huang, S., & Ma, Y. (2019). Isolation and characterization of specific phages to prepare a cocktail preventing Vibrio sp. Va-F3 infections in shrimp (Litopenaeus vannamei). Frontiers in Microbiology, 10(2337):1-11.
- Food and Agriculture Organization of the United Nations (FAO). (2020). The state of world fisheries and aquaculture 2020. Sustainability in action. Rome: FAO.
- Gazeev, S. (2018). Applications of phage therapy in veterinary medicine applications of phage therapy in veterinary medicine. Uppsala: Swedish University of Agricultural Sciences.
- Hamsah, H., Widanarni, W., Alimuddin, A., Yuhana, M., Junior, M. Z., & Hidayatullah, D. (2019). Immune response and resistance of Pacific white shrimp larvae administered probiotic, prebiotic, and synbiotic through the bio-encapsulation of Artemia sp. Aquaculture International, 27(2):567-580.
- Holmström, K., Gräslund, S., Wahlström, A., Poungshompoo, S., Bengtsson, B. E., & Kautsky, N. (2003). Antibiotic use in shrimp farming and implications for environmental impacts and human health. International Journal of Food Science and Technology, 38(3):255-266.
- Hong, N. T. X., Linh, N. T. H., Baruah, K., Thuy, D. T. B., & Phuoc, N. N. (2022). The combined use of Pediococcus pentosaceus and Fructooligosaccharide improves growth performance, immune response, and resistance of whiteleg shrimp Litopenaeus vannamei against Vibrio parahaemolyticus. Frontiers in Microbiology, 13:1-10.
- Hudzicki, J. (2012). Kirby-Bauer disk diffusion susceptibility test protocol author information. American Society For Microbiology, 1-13.
- Javahery, S., Noori, A., & Hoseinifar, S. H. (2019). Growth performance, immune response, and digestive enzyme activity in Pacific white shrimp, Penaeus vannamei Boone, 1931, fed dietary microbial lysozyme. Fish & Shellfish Immunology, 92:528-535.
- Jun, J. W., Han, J. E., Giri, S. S., Tang, K. F. J., Zhou, X., Aranguren, L. F., Kim, H. J., Yun, S., Chi, C., Kim, S. G., & Park, S. C. (2018). Phage application for the protection from Acute Hepatopancreatic Necrosis Disease (AHPND) in Penaeus vannamei. Indian Journal of Microbiology, 58(1):114-117.
- Kokkari, C., Sarropoulou, E., Bastias, R., Mandalakis, M., & Katharios, P. (2018). Isolation and characterization of a novel bacteriophage infecting Vibrio alginolyticus. Archives of Microbiology, 200(5):707–718.
- Kumar, V., Nguyen, D. V., Baruah, K., & Bossier, P. (2019). Probing the mechanism of VPAHPND extracellular proteins toxicity purified from Vibrio parahaemolyticus AHPND strain in germ-free Artemia test system. Aquaculture, 504:414-419.
- Kurniawinata, M. I., Sukenda, S., Wahjuningrum, D., Widanarni, W., & Hidayatullah, D. (2021). White faeces disease and abundance of bacteria and phytoplankton in intensive pacific white shrimp farming. Aquaculture Research, 52(11):5730-5738.
- Martínez-Díaz, S. F., & Hipólito-Morales, A. (2013). Efficacy of phage therapy to prevent mortality during the vibriosis of brine shrimp. Aquaculture, 400–401:120-124.
- Morshedi, V., Mozanzadeh, M. T., Hamedi, S., Naserifard, I., Ebrahimi, H., Agh, N., Nafisi, M., Azodi, M., & Rashidian, G. (2022). Enrichment of livefeed with very low level of docosahexaenoic acid (DHA) is enough for yellowtail sea bream (Acanthopagrus latus) larvae. Aquaculture Reports, 26:101310.
- Munaeni, W., Widanarni, Yuhana, M., Setiawati, M., & Wahyudi, A. T. (2020). Effect in white shrimp Litopenaeus vannamei of Eleutherine bulbosa (Mill.) urb. powder on immune genes expression and resistance against Vibrio parahaemolyticus infection. Fish & Shellfish Immunology, 102:218-227.
- Nakamura, R., Pedrosa-Gerasmio, I. R., Alenton, R. R. R., Nozaki, R., Kondo, H., & Hirono, I. (2019). Anti-PirA-like toxin immunoglobulin (IgY) in feeds passively immunizes shrimp against acute hepatopancreatic necrosis disease. Journal of Fish Diseases, 42(8):1125-1132).
- Nikapitiya, C., Dananjaya, S. H. S., Edirisinghe, S. L., Chandrarathna, H. P. S. U., Lee, J., & De Zoysa, M. (2020). Development of phage delivery by bioencapsulation of artemia nauplii with Edwardsiella tarda phage (ETP-1). Brazilian Journal of Microbiology, 51(4):2153-2162.
- OIE. (2019). Acute Hepatopancreatic Necrosis Disease (Chapter 2.2.1). Paris: OIE.
- Panah, A. H., Rafiee, G., Rezaei Tavabe, K., Bozorgi, S., & Mirvaghefi, A. (2021). Effects of utilization of Lactococcus lactis and Pediococcus pentosaseus as probiotic to improve quality of west white leg shrimp (Litopenaeus vannamei) postlarvae. Aquaculture Research, 52(4):1724-1732.
- Quiroz-Guzmán, E., Peña-Rodriguez, A., Vázquez-Juárez, R., Barajas-Sandoval, D. R., Balcázar, J. L., & Martínez-Díaz, S. F. (2018). Bacteriophage cocktails as an environmentally-friendly approach to prevent Vibrio parahaemolyticus and Vibrio harveyi infections in brine shrimp (Artemia franciscana) production. Aquaculture, 492:273-279.
- Raja, R. A., Sridhar, R., Balachandran, C., Palanisammi, A., Ramesh, S., & Nagarajan, K. (2017). Pathogenicity profile of Vibrio parahaemolyticus in farmed Pacific white shrimp, Penaeus vannamei. Fish & Shellfish Immunology, 67:368-381.
- Ramadhani, D. E., Widanarni, W., & Sukenda, S. (2019). Microencapsulation of probiotics and its applications with prebiotic in Pacific white shrimp larvae through Artemia sp. Jurnal Akuakultur Indonesia, 18(2):130-140.
- Sarjito, Haditomo, A. H. C., Desrina, Djunaedi, A., & Prayitno, S. B. (2018). The diversity of vibrios associated with vibriosis in Pacific white shrimp (Litopenaeus vannamei) from extensive shrimp pond in Kendal District, Indonesia. IOP Conference Series: Earth and Environmental Science, 116(1):1-7.
- Sorgeloos, P., & Roubach, R. (2021). Past, present and future scenarios for SDG-aligned brine shrimp artemia aquaculture. FAO Aquaculture Newsletter, 63:56-57.
- Srinivasan, R., Chaitanyakumar, A., Subramanian, P., Mageswari, A., Gomathi, A., Aswini, V., Sankar, A. M., Ramya, M., & Gothandam, K. M. (2020). Recombinant engineered phage-derived enzybiotic in Pichia pastoris X-33 as whole cell biocatalyst for effective biocontrol of Vibrio parahaemolyticus in aquaculture. International Journal of Biological Macromolecules, 154:1576-1585.
- Thornber, K., Verner-Jeffreys, D., Hinchliffe, S., Rahman, M. M., Bass, D., & Tyler, C. R. (2020). Evaluating antimicrobial resistance in the global shrimp industry. Reviews in Aquaculture, 12(2):966-986.
- Tian, F., Li, J., Hu, Y., Zhao, F., Ren, H., Pan, Q., Nazir, A., Li, F., & Tong, Y. (2022). Characterization and complete genome sequence analysis of a newly isolated phage against Vibrio parahaemolyticus from sick shrimp in Qingdao, China. PLoS ONE, 17:1-15.
- Xue, T., Liu, Y., Cao, M., Li, J., Tian, M., Zhang, L., Wang, B., Liu, X., & Li, C. (2021). Transcriptome analysis reveals deep insights into the early immune response of turbot (Scophthalmus maximus) induced by inactivated Aeromonas salmonicida vaccine. Fish & Shellfish Immunology, 119:163-172.
- Yasin, A., Begum, K., Eshik, M. E., Punom, N. J., Ahmmed, S., & Rahman, M. S. (2022). Molecular identification and antibiotic resistance patterns of diverse bacteria associated with shrimp PL nurseries of Bangladesh: Suspecting Acinetobacter venetianus as future threat. PeerJ, 10:1-24.
References
Abdel-Latif, H. M. R., Yilmaz, E., Dawood, M. A. O., Ringø, E., Ahmadifar, E., & Yilmaz, S. (2022). Shrimp vibriosis and possible control measures using probiotics, postbiotics, prebiotics, and synbiotics: A review. Aquaculture, 551:737951.
Bondad-Reantaso, M. G., & Arthur, R. (2018). FAO Technical assistance efforts to deal with Acute Hepatopancreatic Necrosis Disease (AHPND) of cultured shrimp. Asian Fisheries Science, 31S:1-14.
Cao, Y., Zhang, Y., Lan, W., & Sun, X. (2021). Characterization of vB_VpaP_MGD2, a newly isolated bacteriophage with biocontrol potential against multidrug-resistant Vibrio parahaemolyticus. Archives of Virology, 166(2):413-426.
Chen, L., Fan, J., Yan, T., Liu, Q., Yuan, S., Zhang, H., Yang, J., Deng, D., Huang, S., & Ma, Y. (2019). Isolation and characterization of specific phages to prepare a cocktail preventing Vibrio sp. Va-F3 infections in shrimp (Litopenaeus vannamei). Frontiers in Microbiology, 10(2337):1-11.
Food and Agriculture Organization of the United Nations (FAO). (2020). The state of world fisheries and aquaculture 2020. Sustainability in action. Rome: FAO.
Gazeev, S. (2018). Applications of phage therapy in veterinary medicine applications of phage therapy in veterinary medicine. Uppsala: Swedish University of Agricultural Sciences.
Hamsah, H., Widanarni, W., Alimuddin, A., Yuhana, M., Junior, M. Z., & Hidayatullah, D. (2019). Immune response and resistance of Pacific white shrimp larvae administered probiotic, prebiotic, and synbiotic through the bio-encapsulation of Artemia sp. Aquaculture International, 27(2):567-580.
Holmström, K., Gräslund, S., Wahlström, A., Poungshompoo, S., Bengtsson, B. E., & Kautsky, N. (2003). Antibiotic use in shrimp farming and implications for environmental impacts and human health. International Journal of Food Science and Technology, 38(3):255-266.
Hong, N. T. X., Linh, N. T. H., Baruah, K., Thuy, D. T. B., & Phuoc, N. N. (2022). The combined use of Pediococcus pentosaceus and Fructooligosaccharide improves growth performance, immune response, and resistance of whiteleg shrimp Litopenaeus vannamei against Vibrio parahaemolyticus. Frontiers in Microbiology, 13:1-10.
Hudzicki, J. (2012). Kirby-Bauer disk diffusion susceptibility test protocol author information. American Society For Microbiology, 1-13.
Javahery, S., Noori, A., & Hoseinifar, S. H. (2019). Growth performance, immune response, and digestive enzyme activity in Pacific white shrimp, Penaeus vannamei Boone, 1931, fed dietary microbial lysozyme. Fish & Shellfish Immunology, 92:528-535.
Jun, J. W., Han, J. E., Giri, S. S., Tang, K. F. J., Zhou, X., Aranguren, L. F., Kim, H. J., Yun, S., Chi, C., Kim, S. G., & Park, S. C. (2018). Phage application for the protection from Acute Hepatopancreatic Necrosis Disease (AHPND) in Penaeus vannamei. Indian Journal of Microbiology, 58(1):114-117.
Kokkari, C., Sarropoulou, E., Bastias, R., Mandalakis, M., & Katharios, P. (2018). Isolation and characterization of a novel bacteriophage infecting Vibrio alginolyticus. Archives of Microbiology, 200(5):707–718.
Kumar, V., Nguyen, D. V., Baruah, K., & Bossier, P. (2019). Probing the mechanism of VPAHPND extracellular proteins toxicity purified from Vibrio parahaemolyticus AHPND strain in germ-free Artemia test system. Aquaculture, 504:414-419.
Kurniawinata, M. I., Sukenda, S., Wahjuningrum, D., Widanarni, W., & Hidayatullah, D. (2021). White faeces disease and abundance of bacteria and phytoplankton in intensive pacific white shrimp farming. Aquaculture Research, 52(11):5730-5738.
Martínez-Díaz, S. F., & Hipólito-Morales, A. (2013). Efficacy of phage therapy to prevent mortality during the vibriosis of brine shrimp. Aquaculture, 400–401:120-124.
Morshedi, V., Mozanzadeh, M. T., Hamedi, S., Naserifard, I., Ebrahimi, H., Agh, N., Nafisi, M., Azodi, M., & Rashidian, G. (2022). Enrichment of livefeed with very low level of docosahexaenoic acid (DHA) is enough for yellowtail sea bream (Acanthopagrus latus) larvae. Aquaculture Reports, 26:101310.
Munaeni, W., Widanarni, Yuhana, M., Setiawati, M., & Wahyudi, A. T. (2020). Effect in white shrimp Litopenaeus vannamei of Eleutherine bulbosa (Mill.) urb. powder on immune genes expression and resistance against Vibrio parahaemolyticus infection. Fish & Shellfish Immunology, 102:218-227.
Nakamura, R., Pedrosa-Gerasmio, I. R., Alenton, R. R. R., Nozaki, R., Kondo, H., & Hirono, I. (2019). Anti-PirA-like toxin immunoglobulin (IgY) in feeds passively immunizes shrimp against acute hepatopancreatic necrosis disease. Journal of Fish Diseases, 42(8):1125-1132).
Nikapitiya, C., Dananjaya, S. H. S., Edirisinghe, S. L., Chandrarathna, H. P. S. U., Lee, J., & De Zoysa, M. (2020). Development of phage delivery by bioencapsulation of artemia nauplii with Edwardsiella tarda phage (ETP-1). Brazilian Journal of Microbiology, 51(4):2153-2162.
OIE. (2019). Acute Hepatopancreatic Necrosis Disease (Chapter 2.2.1). Paris: OIE.
Panah, A. H., Rafiee, G., Rezaei Tavabe, K., Bozorgi, S., & Mirvaghefi, A. (2021). Effects of utilization of Lactococcus lactis and Pediococcus pentosaseus as probiotic to improve quality of west white leg shrimp (Litopenaeus vannamei) postlarvae. Aquaculture Research, 52(4):1724-1732.
Quiroz-Guzmán, E., Peña-Rodriguez, A., Vázquez-Juárez, R., Barajas-Sandoval, D. R., Balcázar, J. L., & Martínez-Díaz, S. F. (2018). Bacteriophage cocktails as an environmentally-friendly approach to prevent Vibrio parahaemolyticus and Vibrio harveyi infections in brine shrimp (Artemia franciscana) production. Aquaculture, 492:273-279.
Raja, R. A., Sridhar, R., Balachandran, C., Palanisammi, A., Ramesh, S., & Nagarajan, K. (2017). Pathogenicity profile of Vibrio parahaemolyticus in farmed Pacific white shrimp, Penaeus vannamei. Fish & Shellfish Immunology, 67:368-381.
Ramadhani, D. E., Widanarni, W., & Sukenda, S. (2019). Microencapsulation of probiotics and its applications with prebiotic in Pacific white shrimp larvae through Artemia sp. Jurnal Akuakultur Indonesia, 18(2):130-140.
Sarjito, Haditomo, A. H. C., Desrina, Djunaedi, A., & Prayitno, S. B. (2018). The diversity of vibrios associated with vibriosis in Pacific white shrimp (Litopenaeus vannamei) from extensive shrimp pond in Kendal District, Indonesia. IOP Conference Series: Earth and Environmental Science, 116(1):1-7.
Sorgeloos, P., & Roubach, R. (2021). Past, present and future scenarios for SDG-aligned brine shrimp artemia aquaculture. FAO Aquaculture Newsletter, 63:56-57.
Srinivasan, R., Chaitanyakumar, A., Subramanian, P., Mageswari, A., Gomathi, A., Aswini, V., Sankar, A. M., Ramya, M., & Gothandam, K. M. (2020). Recombinant engineered phage-derived enzybiotic in Pichia pastoris X-33 as whole cell biocatalyst for effective biocontrol of Vibrio parahaemolyticus in aquaculture. International Journal of Biological Macromolecules, 154:1576-1585.
Thornber, K., Verner-Jeffreys, D., Hinchliffe, S., Rahman, M. M., Bass, D., & Tyler, C. R. (2020). Evaluating antimicrobial resistance in the global shrimp industry. Reviews in Aquaculture, 12(2):966-986.
Tian, F., Li, J., Hu, Y., Zhao, F., Ren, H., Pan, Q., Nazir, A., Li, F., & Tong, Y. (2022). Characterization and complete genome sequence analysis of a newly isolated phage against Vibrio parahaemolyticus from sick shrimp in Qingdao, China. PLoS ONE, 17:1-15.
Xue, T., Liu, Y., Cao, M., Li, J., Tian, M., Zhang, L., Wang, B., Liu, X., & Li, C. (2021). Transcriptome analysis reveals deep insights into the early immune response of turbot (Scophthalmus maximus) induced by inactivated Aeromonas salmonicida vaccine. Fish & Shellfish Immunology, 119:163-172.
Yasin, A., Begum, K., Eshik, M. E., Punom, N. J., Ahmmed, S., & Rahman, M. S. (2022). Molecular identification and antibiotic resistance patterns of diverse bacteria associated with shrimp PL nurseries of Bangladesh: Suspecting Acinetobacter venetianus as future threat. PeerJ, 10:1-24.