According to the estimation of the Food and Agriculture Organization of the United Nations (FAO), aquaculture is one of the fastest growing food production sectors in the world. The latest statistics show that the world's total production of aquatic organisms reached a record 210.9 million tons in 2018, with aquaculture accounting for 54.3% of the total (Figure 1).
Figure 1: Global catch and aquaculture percentage in 2018
Bacterial diseases are still the biggest threat to aquaculture!
Vibrio products recognized by most shrimp farmers
The rapid growth of aquaculture production has raised concerns about the health quality and safety of aquatic animals. Like other livestock production sectors, aquaculture also adopts intensive and semi intensive practices, resulting in higher animal density in small waters and significantly increasing the risk of infectious diseases. Bacterial diseases of fish and crustaceans will not only cause huge economic losses to farmers, but also lead to a large number of deaths of fish and shrimp.
In aquaculture, bacterial diseases often encountered and causing death are mainly caused by gram-negative bacteria, such as Salmonella salmonella, Edwardsiella tarda, flavobacterium acidophilus, Pseudomonas fluorescens and various vibrios. Few diseases are caused by gram-positive bacteria. Most of the bacterial pathogens causing aquaculture problems naturally exist in the aquatic environment. Under the influence of external pressure factors (including transportation, high aquaculture density, poor water quality and insufficient nutrition), aquatic animals are more prone to diseases.
Due to incomplete statistical information, it is difficult to estimate the actual amount of antibiotics used in aquatic animals. With the "post antibiotic era" announced by the World Health Organization, people are increasingly interested in alternative products of antibiotics. One of the solutions for environmental alternatives and antibiotic resistance is the use of bacteriophages. The use of bacteriophages is not a new concept. Its use results have been confirmed in human medicine, veterinary medicine, agriculture and food industry, but its use in aquatic animal husbandry has attracted more attention recently.  
The analysis of the number of records containing the words "bacteriophage" and "aquaculture" in the Web of Science All database from 2000 to 2021 shows that the number of scientific reports on bacteriophages related to the aquatic environment has increased rapidly, indicating a significant increase in interest in studying the subject (Figure 2)
Figure 2: Number of records of "bacteriophage" and "aquaculture" in the database from 2000 to 2021
In aquaculture, antibiotics are mainly used for prevention and treatment purposes. After the antibiotics are ingested by fish and shrimp, about 80% of them turn into feces that are not absorbed, or turn into urine and other secretions into the environment after absorption. In addition, if the fish and shrimp are sick and anorexic, most of the antibiotics in the feed remain in the pond sediment, which changes the composition of the sediment microbiota, thus producing antibiotic resistant bacteria, which ultimately makes the fish and shrimp disease more difficult to cure, and may also affect the health of consumers.
Phage, the most likely method to solve bacterial diseases in the future
Vibrio products recognized by most shrimp farmers
Phage is the most abundant microorganism on the earth, and they are almost everywhere, including extreme environments, as well as the niche of almost all human and animal organisms. They were discovered by Frederick W. Twot in England in 1915 and Felix d'Herelle in Paris Ranch Research Institute in 1917. These are viruses infecting bacteria, and they vary greatly in size, morphology and genome organization. However, almost all phages currently classified are divided into three families: Myoviridae, Siphoviridae and Podoviridae.
Life cycle of bacteriophage
Vibrio products recognized by most shrimp farmers
Like other viruses, bacteriophages must infect host cells to reproduce. They are very specific to the host. Usually, a bacteriophage only infects one bacterium, or even some strains of a species. Phage can recognize several components of bacterial cell structure as their receptors. These include outer membrane protein, peptidoglycan (PG), teichoic acid, oligosaccharides, lipopolysaccharide (LPS), flagella and fimbriae. This means they can only infect the bacteria of the target molecule.
The first step of phage infection is to produce polysaccharide degrading enzyme, also known as polysaccharide depolymerase. These can be released into the environment, combined with the tail or capsid of bacteriophages, and used for enzymatic degradation of envelope or structural polysaccharides, including extracellular polysaccharides as the main components of bacterial biofilms. In the final stage of the cycle, lysine is produced, which is responsible for the decomposition of bacteria and the release of offspring viruses.
1. Cracking cycle
Although technically they are not living organisms, bacteriophages must be dynamic entities. During the lytic replication cycle, phages attach to sensitive host cells. It then introduces its genome into the bacterial cytoplasm and uses its ribosomes to produce proteins. Bacterial resources are rapidly transformed in capsid proteins and viral genomes, which are composed of multiple copies of original bacteriophages. By reproducing in bacterial cells, it destroys bacteria. When the host cell dies, it usually releases new phages with the participation of lysine, and then infects another bacterial cell. Such phages are called toxic lysis or lysis cycles.
2. Lysogen cycle
In the lysogenic replication cycle, bacteriophages also attach to susceptible bacterial cells and introduce their genomes into bacterial cytoplasm. The phage genome is then integrated into the chromosomes of bacterial cells. In both cases, it will be replicated and transferred to the bacterial cells of future generations, but the phage genome that will not crack their integration is called a prophage. The prophage can return to the crack replication cycle that causes the host to crack. The most common is to respond to changing environmental conditions.
3. Pseudolysogen cycle
In the process of pseudolysogens, phages enter cells, but do not replicate in cells, nor can they integrate stably with the host genome. Pseudolysogenicity seems to play an important role in the survival of bacterial viruses. When host cells encounter adverse growth conditions (such as lack of sufficient nutrients), they can preserve their genomes. Pseudolysogens are not permanent. After changing the conditions that cause it, phages usually enter the cleavage or lysogen pathway.
4. Chronic infection
In the case of chronic infection, new phage particles will be produced continuously for a long time. However, host cell lysis does not occur. Virus particles are released or exported out of cells through protein complexes. It is related to high energy consumption and may have a negative impact on the ability of bacteria to compete for niche. Examples of bacteriophages that can cause chronic infections include archaeoviruses, filamentous bacteriophages (ssDNA bacteriophages), and viruses that infect mycoplasmas.
5. Abortion infection
In the face of frequent contact with bacteriophages, bacteria have developed many mechanisms to resist infection, including abortion infection, also known as phage exclusion. The phage genome enters the host cell, but the infected cell destroys itself before the phage completes its replication cycle. This reduces the number of progeny particles and limits their spread to other cells, allowing bacterial populations to survive. Abortive infection is manifested in a variety of bacterial defense systems. One example is Lactococcus. In uninfected bacteria, the role of the two components of the system is balanced. After virus infection, bacterial death occurs due to the increase of toxin antitoxin ratio.
Bacteriophage therapy
Vibrio products recognized by most shrimp farmers
For targeted therapy, strictly dissolved bacteriophages are preferred, which are characterized by rapid proliferation leading to bacterial cell dissolution, and their number increases exponentially. Lysogenic bacteriophages are avoided because they have the inherent ability to mediate gene transfer between bacteria, which can increase the virulence of bacteria. At present, advances in sequencing technology and synthetic biology have also created new possibilities for the use of these bacteriophages to treat bacterial infections. In addition to the ability to lyse bacterial cells, several important characteristics determine the antibacterial efficacy of bacteriophages.  
The first is the time of phage production, including effective adhesion, incubation period and the release of virus particles from offspring.  
The second aspect is the growth rate of phage population, that is, the number of phage particles formed in a life cycle. High adsorption rate, large outbreak rate and short formation time for specific bacteria are the decisive factors for strong antibacterial efficacy.
Methods of managing bacteriophages
Vibrio products recognized by most shrimp farmers
The effectiveness of phage application depends on their ability to reach the host, the location of infection in vivo, the effectiveness of penetration and the ability to maintain the highest phage transmission.
In aquaculture, the most commonly used bacteriophage method is to put it into water or take it orally, such as mixing it with feed.  
Oral administration of bacteriophages has been proved to be effective in the treatment of gastrointestinal tract infections, and it has also been proved that oral administration of bacteriophages can easily be absorbed into the systemic circulation, which makes this route of administration available for the treatment of systemic infections. The passage of bacteriophages is determined by several factors, including their concentration, the presence of specific sequences in capsid proteins that interact with intestinal epithelial cell receptors, and the interaction between bacteriophages and intestinal immune cells.
The appropriate method of phage administration depends on many factors and should be considered according to the specific situation. It is impractical to inject very small fish or crustaceans. Similarly, it is difficult to soak with high concentrations of bacteriophages in large ponds. The method of immersion will depend on the environment, nature of infection or bacteriophage characteristics. Each method of administration has its advantages and disadvantages. The selection of methods depends largely on the nature of bacterial pathogens, the species and size of animals.
There are two forms of treatment - active and passive. In active treatment, the dosage of bacteriophage can reduce the host population through multiple reproduction cycles. In passive therapy, such reproduction is not required to ensure effective treatment, because so many phages are applied that the whole host population is cracked without one or more phage reproduction cycles. However, passive methods are more expensive, but can bypass bacterial defense mechanisms, such as abortion infection.
Various phage therapy methods have been tested. In fact, single phage cell therapy refers to the use of a type of bacteriophage for a certain pathogen, but it requires an accurate match between pathogens and bacteriophages.
In aquaculture, the actual situation is often much more complex. Fishes and shrimps may suffer from infections caused by multiple strains or bacterial species at the same time. These bacteria will affect the results of the disease, which poses a great challenge to bacteriophage therapy. Unlike single bacteriophage therapy, multi bacteriophage therapy targets one bacterial species or many strains of many bacterial species. At present, more and more bacteriophage mixtures containing two or more kinds of bacteriophages are tested and used in aquaculture.  
One of the advantages of using multiple bacteriophages is that they can treat infection more thoroughly, because they can attack a wide range of pathogenic bacteria strains, with better and faster effects. In addition, the use of bacteriophage mixtures targeting different receptors of the same bacteria helps to reduce the development rate of drug resistance.
In vivo use of bacteriophages
Vibrio products recognized by most shrimp farmers
In recent years, some in vivo experiments have been conducted to evaluate the potential of bacteriophages against bacterial infection in aquaculture. Their effectiveness has been tested on various animal models, including a variety of fish, crustaceans and molluscs. The results also show encouraging results and reveal that some bacteriophages have the ability to significantly reduce the concentration of pathogens and improve the survival rate of aquaculture animals.
The study of Le et al. (2018) showed that the survival rate of catfish increased by 68% when using different quantities of bacteriophages in catfish treatment, which showed that determining the correct dosage of bacteriophages was very important for treatment. The study also found that the administration time was a very important factor. When the pathogen is infected and then treated with bacteriophage, the mortality usually increases significantly. The best choice is prophylactic use of bacteriophages before infection.
The research of Jun et al. (2018) showed that in the water body, the survival rate of white shrimp increased by 75% due to the preventive use of bacteriophages, and the survival rate of white shrimp increased by 50% due to the preventive use of bacteriophages in feed.  
Compared with traditional antibiotic therapy, bacteriophage therapy has some advantages
Vibrio products recognized by most shrimp farmers
Phage isolation is relatively fast, simple and inexpensive. Phage resistance develops about ten times slower than antibiotic resistance, because phages can evolve to produce new genotypes that can re infect a given bacterial strain. In particular, bacteriophages are still infectious under very harsh environmental conditions and continue to replicate until the host bacterial population density decreases significantly.  
These characteristics indicate that, unlike traditional therapies, bacteriophage therapy requires fewer doses, but is more effective than traditional therapies.
-Bacteriophage infection process-
Seven Advantages of Shanghai Norry Arc Hunting Agent
Broad pyrolysis spectrum
For the common Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, Vibrio cholerae and other pathogenic bacteria, the cracking rate can be as high as 98%;
Precision treatment
It can precisely treat the early death syndrome (EMS) of shrimp caused by Vibrio parahaemolyticus, namely, acute liver gland pancreatic necrosis;
Safe without residue
It is safe without residue, does not affect the activity of beneficial microorganisms in the water, is non-toxic, harmless and non irritating, and is naturally discharged out of the body after splitting, without damage to animal cells;
Effective
The curative effect is definite. Improve the survival rate of shrimp, enhance the vitality and immunity of diseased shrimp;
modern techniques
It has the world's most advanced phage anti-theft technology and resistance prevention and control technology;
High potency
High potency, bacteriophage content ≥ 1012pfu/ml, regular use can reduce vibrio outbreak, reduce breeding risk, improve shrimp seedling survival rate, and improve breeding efficiency;
Can be stored at room temperature
The "heat resistant protective agent" is embedded into the product, so that the bacteriophage can be stored at room temperature.
Comparison with traditional therapeutic products
Research on bacteriophages of Shanghai Norry in the field of aquaculture
Top
In the past three years, the R&D team of Shanghai Norris Phage Research Institute has continued to collect water samples from Guangdong, Guangxi, Fujian, Hainan, Vietnam, Thailand and other places where premature shrimp death syndrome is highly prevalent. More than 180 species of virulent pathogens have been isolated from them, and more than 100 bacteriophages have been isolated from water samples sent from all over the country every year, A cocktail preparation containing several bacteriophages was developed based on the actual curative effect. At present, China has a phage virus library with the largest number of drug-resistant strains, the most advanced phage anti-theft technology and resistance prevention and control technology in the world, and is at the forefront of phage research.