New phage to combat Vibrio parahaemolyticus and AHPND in shrimp aquaculture

Shrimp aquaculture faces a persistent threat: bacterial infections. Among the most notorious culprits is Vibrio parahaemolyticus, a bacterium responsible not only for seafood-borne illnesses in humans but also for havoc on shrimp farms. This bacterium is the causative agent of the devastating Acute Hepatopancreatic Necrosis Disease (AHPND), also known as Early Mortality Syndrome (EMS), capable of causing massive mortalities and multi-million dollar economic losses.

The widespread use of antibiotics to combat these infections has led to another serious problem: the emergence of resistant strains, making the search for effective and sustainable alternatives urgent. In this context, bacteriophages (or phages), viruses that infect and destroy specific bacteria, emerge as a promising solution.

A group of scientists from the Yellow Sea Fisheries Research Institute, Fudan University, Kangwon National University, and Zhejiang University have isolated and characterized a new phage with great potential to control V. parahaemolyticus in shrimp.

Seeking a Natural “Predator”: The Isolation of Phage P20211219001-1

Researchers collected 61 water samples from Litopenaeus vannamei shrimp farms affected by an AHPND outbreak. From these samples, they managed to isolate strains of V. parahaemolyticus and, crucially, 32 different types of bacteriophages. The goal was to find “specialist” phages capable of specifically attacking V. parahaemolyticus without affecting other potentially beneficial bacteria.

Among the phages found, one in particular, named P20211219001-1, showed high effectiveness against a specific strain of V. parahaemolyticus (strain 20211219001-1) isolated from the same farm. This phage was selected for a more detailed analysis of its characteristics and potential.

Getting to Know Vibrio’s Enemy: Characteristics of Phage P20211219001-1

To evaluate the practical potential of a phage, it is essential to understand how it behaves and under what conditions it is most effective.

Specificity and Lytic Efficiency

Phage P20211219001-1 proved to be highly specific: it only formed “clear spots” (lysis plaques) in cultures of its host bacterium (V. parahaemolyticus 20211219001-1), without affecting other tested Vibrio strains. This is a great advantage, as it minimizes the impact on the natural microbiota of the shrimp or the pond.

Scientists determined that the optimal ratio for infection (Multiplicity of Infection – MOI) was low (0.001), achieving maximum production of new phages. This contrasts with other studies that required higher phage concentrations but could be advantageous in reducing the emergence of bacterial resistance.

Rapid Life Cycle

The lytic cycle (the process from when the phage infects the bacterium until it destroys it to release new phages) was relatively fast. It had a “latent period” (time before releasing new phages) of only 20 minutes and a “burst period” (during which new phages are released) of 30 minutes. Each infected bacterium released an average of 23 new phage particles (“burst size”). This rapid replication is key to effectively controlling a bacterial population.

Environmental Resistance

Phage P20211219001-1 showed good stability over a wide range of environmental conditions. It maintained its viability at temperatures between 4°C and 50°C and across a broad pH range (from 3 to 11). This robustness is important for its potential application in the diverse conditions of aquaculture farms and for its storage.

Morphology and Genome

Observed via electron microscopy, the phage has an icosahedron-shaped head (a polyhedron with 20 faces) about 83 nm in diameter and a short tail of 24 nm, classifying it within the order Caudoviricetes. Its genetic material is circular double-stranded DNA, with a size of 35,961 base pairs.

Genomic analysis identified 41 protein-coding genes. Among them were key genes for the phage structure (capsid, tail), the replication of its DNA within the bacterium, and, fundamentally, for the lysis (destruction) of the host bacterial cell. Lytic proteins such as holins and N-acetylmuramoyl-L-alanine amidase, responsible for breaking the bacterial cell wall from within, were identified.

Efficacy Tests: Does the Phage Work in Practice?

Characterization is important, but the crucial test is its application in living organisms. Trials were conducted using Artemia salina (a small crustacean used as a model) and juveniles of Litopenaeus vannamei (whiteleg shrimp).

Protection in Artemia salina

Researchers infected groups of Artemia with V. parahaemolyticus and applied the phage at different times: 6 hours before infection (Pre-PTS), at the same time as infection (PTS), and 12 hours after infection (Post-PTS). The results showed that both the pre-treatment (Pre-PTS) and simultaneous treatment (PTS) significantly increased the survival of Artemia compared to the group only infected (VIS), which had the highest mortality. The post-treatment (Post-PTS) was not as effective, suggesting that early or preventive intervention is more beneficial.

Success in Litopenaeus vannamei

A similar experiment was conducted with L. vannamei shrimp. The groups included a control (untreated), a group treated only with phages (Phage), a group infected with Vibrio (VIS), a group pre-treated with phages before infection (Pre-PTS), and a group post-treated with phages after infection (Post-PTS). After 7 days, the Control and Phage groups showed survival rates close to 97%. The Pre-PTS and Post-PTS groups maintained survival rates above 75%, while the group only infected (VIS) experienced a drastic drop, with only 47% survival. Again, pre-treatment (Pre-PTS) offered the best protection.

Shrimp from the phage-treated groups (Pre-PTS and Post-PTS) and the control group looked healthy externally, whereas shrimp from the VIS group showed signs of disease such as body whitening.

Histological Evidence

Microscopic analysis of tissues (hepatopancreas and muscle) confirmed the findings. Shrimp from the VIS group showed severe damage, such as cellular disruption and inflammation in the hepatopancreas and muscle. In contrast, pre-treated shrimp (Pre-PTS) showed much less tissue damage, and post-treated shrimp (Post-PTS) showed moderate improvement compared to the infected group. This indicates that the phage not only increases survival but also reduces the internal damage caused by the bacterium.

Implications for the Shrimp Industry

The results of this study are encouraging. Phage P20211219001-1 proves to be a tool with great potential for combating V. parahaemolyticus infections in shrimp aquaculture.

Advantages: Phage therapy offers specificity (attacks only the target bacteria), is environmentally safe (phages are natural and do not pollute), and represents a viable alternative to the growing antibiotic resistance. Furthermore, phages are already considered safe (GRAS) for certain food applications.

Preventive Application: Experiments suggest that preventive application of the phage (before infection occurs) is the most effective strategy, which could be integrated into biosecurity protocols on farms.

Future Challenges: While the results are promising, more research is needed. It is crucial to evaluate the phage’s efficacy against a broader range of V. parahaemolyticus strains and other pathogenic Vibrio. Additionally, for commercial-scale implementation, challenges such as mass and cost-effective phage production, development of efficient istration methods (e.g., in feed or water), and evaluation of long-term effects, including the possible emergence of phage-resistant bacteria and the impact on the pond ecosystem, must be addressed.

Conclusion: A Future with Phages for Shrimp

Bacteriophage P20211219001-1 represents a beacon of hope in the fight against Vibrio parahaemolyticus and AHPND in shrimp farming. Its specificity, stability, and efficacy demonstrated in the laboratory and animal models position it as a strong candidate for developing new health control strategies. Although challenges remain for large-scale application, phage therapy is emerging as a sustainable and ecological alternative to antibiotics, contributing to shrimp health, food safety, and the economic viability of the sector. Continued research and the development of application technologies will be key to turning the potential of phages into a reality on shrimp farms.

Juhee Ahn
Department of Biomedical Science, Kangwon National University
Chuncheon, Gangwon 24341, Republic of Korea.
Email: [email protected]

Reference
Xu, H., Li, W., Zhang, Y., Li, M., Dong, X., Zhang, S., Huang, J., & Ahn, J. (2025). Characterization of novel bacteriophage as a promising alternative for controlling Vibrio infections in shrimp. Aquaculture, 606, 742579. https://doi.org/10.1016/j.aquaculture.2025.742579