Nocardia seriolae bacteriophage with high thermostability and composition and application thereof

Abstract

The present application relates to the field of microbial technology, in particular to a Nocardia seriolae bacteriophage with high thermal stability and a composition and application thereof. The Nocardia seriolae bacteriophage is named RDP-NS004, which was preserved in the China General Microbiological Culture Collection Center on August 25, 2025, and the preservation number is CGMCC No. 46675. A composition comprises the above-mentioned bacteriophage. The above-mentioned bacteriophage or composition is used in (1) preparation of a product for killing Nocardia seriolae; (2) preparation of a product for inhibiting Nocardia seriolae; (3) preparation of a product for preventing and / or treating fish Nocardiosis caused by Nocardia seriolae. The bacteriophage of the present application can maintain a titer of 10 6 PFU / mL at 50 DEG C for 60 min, and has the characteristics of high thermal stability. The isolation of the Nocardia seriolae bacteriophage with high thermal stability can effectively solve the problem that the conventional bacteriophage is limited in effect in a high-temperature environment, and better prevent and control the bacterial disease problem faced in the high-incidence period of Nocardiosis in summer.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a heat-stable Nocardia amberjack bacteriophage, its composition, and its applications. Background Technology

[0002] Nocardiosis is an infectious disease in humans and animals caused by various bacteria of the genus Nocardia. It is a systemic disease. Nocardiosis has been found in many fish species worldwide, causing large-scale mortality in economically valuable farmed fish such as king salmon, largemouth bass, and large yellow croaker, resulting in significant economic losses to both marine and freshwater aquaculture.

[0003] Nocardia bacteria are Gram-positive, aerobic, partially acid-resistant, and non-motile. Nocardiosis in fish is primarily caused by three species of Nocardia: *Nocardia asteroides*, *Nocardia seriolae*, and *Nocardia salmonicida*. *Nocardia seriolae* has become the leading cause of nocardiosis in fish. Nocardia bacteria can enter fish through wounds, gills, or food. Infected fish exhibit typical symptoms such as pinpoint hemorrhages, ulcerative lesions, redness around the mouth and nose, white nodular lesions on the gills, kidneys, head, and spleen, and granulomatous lesions in organ tissues. Nocardia bacteria are characterized by long disease cycles, high infection rates, and high mortality rates; the natural morbidity rate ranges from 20% to 60%, and cases with mortality rates as high as 100% have been reported.

[0004] Nocardiosis is a chronic, granulomatous, systemic disease that is often asymptomatic in its early stages. As the disease progresses, granulomatous tissue gradually forms, and its presence reflects the degree of tissue damage. This disease can also infect other aquatic animals and humans, making it a zoonotic disease in a broad sense. The largemouth bass is a freshwater fish of significant economic value in China's aquaculture industry, with Zhejiang Province being one of its major farming bases. However, the adoption of intensive or semi-intensive farming methods significantly increases the risk of infectious diseases. Nocardiosis caused by *Nocardia amberjack* severely hinders the development of largemouth bass farming.

[0005] Nocardiasis in fish, caused by *Nocardia* spp., has become a major challenge in aquaculture, and currently there are no commercially available effective treatments for this infection. Previously, treatment for nocardiasis primarily relied on chemical agents and drug therapies, but the use of these compounds can lead to water pollution, antibiotic accumulation in fish, and the development of drug-resistant bacteria. This serious situation has prompted the exploration of sustainable and environmentally friendly alternative treatments for nocardiasis. Phage therapy, an innovative method that utilizes bacteriophages to combat bacterial pathogens, is one of the most promising antibiotic alternatives currently under investigation. Phage therapy has been widely used to treat various bacterial diseases in fish and shellfish with significant efficacy.

[0006] Every year from April to October, outbreaks of infectious diseases characterized by skin ulcers and nodular lesions of internal organs occur in almost every region. Nocardiosis is particularly prevalent in summer and autumn, with water temperatures between 30 and 35°C favoring the reproduction of Nocardia. Sustained high temperatures cause heat stress in fish, leading to weakened immunity and increased susceptibility to pathogens. Phages with poor heat stability are rapidly inactivated in high water temperatures, affecting their bactericidal effect. In contrast, Nocardia bacteriophages with high heat stability can tolerate fluctuations in ambient temperature, ensuring a sufficiently long window for finding a host bacterium and successfully infecting and replicating, thus achieving a long-lasting and highly effective bactericidal effect and avoiding the need for frequent reapplication. Furthermore, highly heat-stable bacteriophages can be stored and transported for extended periods at room temperature or even higher, significantly reducing logistics costs, energy consumption, and complexity.

[0007] Therefore, isolating and identifying a bacteriophage that is effective against Nocardia (a fish pathogen) and has high thermal stability is a pressing problem that needs to be solved in the treatment of Nocardia disease.

[0008] The information disclosed in this background section is only intended to enhance the understanding of the background technology of this application and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a heat-stable Nocardia seriolae bacteriophage, its composition, and its applications. This application isolates a highly virulent Nocardia seriolae bacteriophage NS004 from a sea bass farm, and uses this Nocardia seriolae bacteriophage as a host to isolate a Nocardia seriolae bacteriophage RDP-NS004. This bacteriophage retains lytic activity (>10) even under high-temperature conditions (50°C). 6 (PFU / mL), which has the characteristics of high pyrolysis rate and high thermal stability.

[0010] The technical solution of this invention is as follows:

[0011] In a first aspect, the present invention provides a thermostable strain of Nocardiaseriolae bacteriophage, named RDP-NS004, which was deposited on August 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46675.

[0012] Further electron microscopy revealed that the Nocardia amberjack bacteriophage had a polyhedral, three-dimensionally symmetrical head encapsulating nucleic acid, with a diameter of 39 nm and a tail length of approximately 164 nm.

[0013] Furthermore, the heat-stable Nocardia amberjack bacteriophage exhibits high activity and maintains a titer of 10 after being exposed to 20-37°C for 60 minutes. 8 ~10 9 PFU / mL. When the temperature was further increased to 50℃ and incubated for 60 min, the phage titer remained at 10. 6 The activity of the bacteriophage decreased significantly and eventually became completely inactivated when the temperature was raised to above 60°C (PFU / mL). This indicates that the bacteriophage strain has good stability at 20–50°C, a wide temperature tolerance range, and strong thermal stability, enabling it to withstand high-temperature environments.

[0014] Furthermore, the thermally stable Nocardia amberjack phage exhibits similar lysis kinetics at 40°C as at 28°C, indicating that this Nocardia amberjack phage maintains high lysis activity even at 40°C.

[0015] Furthermore, when the multiplicity of infection was 0.1, the thermostable Nocardia amberjack phage exhibited the highest titer, at 9.3 × 10⁻⁶. 9 PFU / mL.

[0016] Furthermore, the heat-stable Nocardia amberjack phage still exhibits strong lytic ability and maintains a titer of 10 in the pH range of 6 to 8. 9 High PFU / mL levels. Phage activity rapidly decreases until complete inactivation under strongly acidic or alkaline conditions (pH below 4 or above 10). The thermostable Nocardia amberjack phage has an optimal pH of 7.0, and this phage strain exhibits moderate tolerance to strong acids and alkalis, surviving more readily in neutral environments.

[0017] Furthermore, the heat-stable *Nocardia amberjack* phage showed no significant increase in phage numbers within 50 minutes of infecting the host bacteria, indicating a phage latency period of 50 minutes. Between 50 and 140 minutes after host bacteria infection, the phage numbers rapidly increased; this period is the phage burst phase, approximately 90 minutes, with a burst dose of approximately 127 PFU / cell. The phage numbers remained constant for the subsequent 40 minutes, reaching the stationary growth phase. The *Nocardia amberjack* phage exhibits a relatively long latency and lysis period, a large burst dose, and good lysis efficiency, making it suitable for phage therapy.

[0018] On the other hand, the present invention provides a composition comprising the above-mentioned heat-stable Nocardia amberjack bacteriophage.

[0019] On the other hand, the above-mentioned heat-stable Nocardia amberjack bacteriophage or the above-mentioned composition is used in at least one of the following (1) to (3):

[0020] (1) Prepare a product that kills Nocardia amberjack;

[0021] (2) Preparation of products that inhibit Nocardia amberjack;

[0022] (3) Prepare products for the prevention and / or treatment of nocardiac disease in fish caused by Nocardia amberjack.

[0023] Furthermore, the product is a veterinary drug preparation, feed additive, or disinfectant.

[0024] Furthermore, the veterinary drug preparation also includes a pharmaceutically acceptable carrier, and its dosage form is a powder, solution, emulsion, gel, granule or lyophilized agent.

[0025] Furthermore, the product is a feed additive treated at temperatures below 50°C.

[0026] Furthermore, the product is a preparation for soaking fish medicine.

[0027] Furthermore, the fish species are perch, mandarin fish, or large yellow croaker.

[0028] The beneficial effects achieved by this invention are as follows:

[0029] (1) The present invention isolated a highly virulent strain of Nocardia amberjack NS004, and used this strain of Nocardia amberjack as a host to isolate a strain of Nocardia amberjack phage RDP-NS004 with high heat stability. This phage has strong lytic activity against Nocardia amberjack and can treat symptoms such as ulcers, tissue nodules, organ granulomatous lesions and acute death caused by Nocardia amberjack;

[0030] (2) The Nocardia amberjack bacteriophage RDP-NS004 in this invention remains stable for 10 years after continuous passage. 9 The PFU / mL titer level was maintained. Furthermore, it retained its cleavage activity (>10) even under high temperature conditions (50℃). 6 The PFU / mL phage exhibits high lysis rate and thermal stability. The isolation of this heat-resistant Nocardia amberjack phage effectively addresses the limitations of conventional phages in high-temperature environments, providing better control over bacterial diseases during the peak summer season for Nocardiac infection.

[0031] (3) The Nocardia amberjack phage RDP-NS004 in this invention remains stable within a wide temperature range of 20-50℃, meaning that in real aquaculture scenarios, it will not rapidly become ineffective due to daily fluctuations in water temperature, thus ensuring the therapeutic effect. During cold chain transportation, even if there is a brief failure of insulation (e.g., the temperature rises to 30-40℃), the phage preparation will not be easily discarded, reducing logistics costs and risks. The phage RDP-NS004 does not require strict low-temperature refrigeration (4℃) and can maintain high activity when stored at room temperature (20-25℃), which greatly reduces the storage threshold and cost, making it very suitable for aquaculture farms with potentially inadequate infrastructure.

[0032] (4) The Nocardia phage RDP-NS004 of this invention can be used for the prevention and treatment of bacterial diseases in fish such as bass and yellowtail amberjack caused by Nocardia. Its high-temperature adaptability, high specificity, and environmental safety can overcome temperature limitations to adapt to complex environments and accurately control Nocardia infection, providing an efficient and sustainable new solution for the prevention and treatment of Nocardia-related diseases. Furthermore, the rational application of phages can avoid the overuse of antibiotics, thereby reducing the development of bacterial resistance and solving problems such as water pollution and ecological imbalance caused by the extensive use of antibiotics and chemical drugs.

[0033] (5) The Nocardia scutellariae bacteriophage RDP-NS004 of this invention has a significant therapeutic effect on largemouth bass infection caused by NS004 strain, can greatly reduce the mortality rate of infected fish, and effectively eliminate pathogens in the spleen of fish. This fully demonstrates that bacteriophage RDP-NS004, as a biological agent, has an excellent therapeutic effect on largemouth bass NS004 infection and is a very promising means of fish disease prevention and control;

[0034] (6) The Nocardia amberjack phage RDP-NS004 of this invention exhibits lytic activity against 17 strains of Nocardia, with a lysis rate of 68%. This demonstrates that the Nocardia amberjack phage RDP-NS004 possesses strong specificity and a relatively broad lytic spectrum against Nocardia species. It also indicates that the Nocardia amberjack phage RDP-NS004 has the potential to treat most (more than half) infections caused by Nocardia, providing a solid foundation for its development as a commercial aquatic therapeutic agent. Attached Figure Description

[0035] Figure 1 This describes the colony morphology of Nocardia amberjack NS004 on BHI plates in this invention.

[0036] Figure 2 This refers to the plaques of the host bacteriophage RDP-NS004 of Nocardia amberjack in this invention.

[0037] Figure 3 This is an electron microscope image of the Nocardia amberjack bacteriophage RDP-NS004 used in this invention.

[0038] Figure 4 The titer of Nocardia amberjack phage RDP-NS004 under different infection multiplicity conditions.

[0039] Figure 5 This is a one-step growth curve of the Nocardia amberjack phage RDP-NS004 in this invention.

[0040] Figure 6 This is the temperature stability curve of the Nocardia amberjack phage RDP-NS004 in this invention.

[0041] Figure 7 This is the pH stability curve of the Nocardia amberjack phage RDP-NS004 in this invention.

[0042] Figure 8 This is the genetic stability curve of the Nocardia amberjack phage RDP-NS004 in this invention.

[0043] Figure 9 This is the time-kill curve of the Nocardia amberjack bacteriophage RDP-NS004 in this invention. Detailed Implementation

[0044] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In this invention, *Nocardia amberjack* NS004 is referred to simply as "host bacterium" or "host bacterium NS004" or "NS004," and *Nocardia amberjack* bacteriophage RDP-NS004 is referred to simply as "bacteriophage RDP-NS004" or "RDP-NS004" or "bacteriophage."

[0046] Example 1: Screening and identification of Nocardia amberjack phage RDP-NS004

[0047] 1.1 Isolation and Identification of Nocardia amberjack NS004

[0048] (1) Collect sea bass samples with typical symptoms from the fish farm, and inoculate the lesions (ulcerated skin and white nodules in the internal organs) into brain heart broth (BHI) solid blood agar plates. Incubate the plates upside down in a 28°C incubator for 5 days; observe the colony morphology, such as... Figure 1 As shown, yellowish-white dried colonies were selected for strain purification. After purification three times, colonies with uniform morphology were obtained. The colony was scraped and inoculated into 5 mL of BHI liquid medium. The test tubes were placed in a shaker at 28℃ and shaken at 160 rpm for 4 days.

[0049] (2) DNA extraction from the prepared bacterial suspension was performed using a bacterial DNA extraction kit from Tiangen Biotech Co., Ltd. Then, using the bacterial DNA as a template, PCR amplification was performed using universal 16S rDNA primers. After identification by 16S rRNA gene sequencing, it was identified as *Nocardia amberjack*, named NS004, and the bacteria were preserved. The *Nocardia amberjack* NS004 bacterial suspension was densely streaked onto BHI solid medium plates and incubated upside down at 28°C for 5 days. The bacterial growth was then scraped and placed in BHI liquid medium, mixed thoroughly, and then mixed with 60% glycerol broth at a 1:1 ratio and stored at -80°C.

[0050] 1.2 Isolation and purification of Nocardia amberjack phage RDP-NS004

[0051] (1) Wastewater treatment: Mix the sea bass farming water, sludge and organ tissue grinding liquid and filter it. Centrifuge at 12,000 rpm for 5 min. Take the supernatant and filter it through a membrane (0.22 μmol). Store the filtrate ① at 4℃ for later use.

[0052] (2) Phage enrichment: Take 0.1 mL of logarithmic growth phase NS004 (10 6 Add 1 mL of filtrate ① (CFU / mL) and 1 mL of filtrate ① to 5 mL of BHI liquid medium, and incubate at 160 rpm and 28 °C on a shaker for 2 days. Then centrifuge at 12,000 rpm for 10 min, and filter the supernatant through a 0.22 μm filter to obtain filtrate ②. Repeat the above operation to enrich the phage again, and store filtrate ③ at 4 °C for later use.

[0053] (3) Phage isolation: Phage isolation was performed using the double plate method. 0.1 mL of logarithmic growth phase NS004 bacterial suspension was mixed with 0.6% BHI soft agar and spread on a BHI solid plate. After the soft agar solidified, 40 μL of filtrate ③ was spotted onto the plate and allowed to stand until the mixture was absorbed. The plate was then incubated in a 28℃ incubator with a bag for 5 days. Transparent phage plaques were picked up with an inoculation loop and placed in 1 mL of SM buffer. The plate was shaken for 15 s to allow the phage to be completely released, thus obtaining the initial Nocardia amberjack phage.

[0054] (4) Phage purification: Phage purification was performed using the double-plate method. A 1:1 mixture of phage extract and host bacterial suspension was added to BHI soft agar cooled to approximately 50°C, then spread onto BHI solid plates and incubated at 28°C for 5 days. A single clear plaque with smooth edges was picked and placed in 1 mL of SM buffer. This process was repeated three times until uniformly sized, smooth-edged phage plaques were purified. Figure 2 As shown, the purification of the bacteriophage is now complete;

[0055] (5) Phage proliferation: Phage proliferation was carried out using the double plate method. Phage extract and host bacterial suspension were mixed 1:1 and added to 5 mL of BHI liquid medium. The mixture was then incubated in a shaker at 160 rpm and 28 °C for 2 days. The proliferation solution was then centrifuged at 12,000 rpm for 10 min and filtered through a 0.22 μm filter to complete the phage proliferation. The phage was stored at 4 °C for later use.

[0056] (6) Preparation of bacteriophage: Take 0.1 mL of Nocardia amberjack bacterial suspension and 1 mL of bacteriophage proliferation solution and add them to 35 mL of BHI liquid medium. Shake and culture at 160 rpm and 28 ℃ for 2 days. Then centrifuge at 12,000 rpm for 10 min. Filter the supernatant through a membrane (0.22 μmol) and store it at 4 ℃ for later use.

[0057] (7) Preservation of bacteriophage: One strain of Nocardia amberjack bacteriophage was isolated and named RDP-NS004. The bacteriophage suspension was mixed with 60% glycerol at a ratio of 1:1 and stored at -80℃. It was deposited at the China General Microbiological Culture Collection Center on August 25, 2025, with the accession number CGMCC No. 46675.

[0058] 1.3 Electron microscopic observation of Nocardia amberjack phage RDP-NS004

[0059] 1.3.1 Experimental Methods:

[0060] Take 20 μL of liquid containing crude phage RDP-NS004 particles and drop it onto a copper grid. Allow it to settle naturally for 15 min. Then, use filter paper to absorb the excess liquid from the side. Add one drop of 2% phosphotungstic acid (PTA) to the copper grid to stain the phage for 10 min. Then, use filter paper to absorb the staining solution from the side. After the sample dries, observe the phage morphology using an electron microscope.

[0061] 1.3.2 Experimental Results:

[0062] Electron microscopy observation results as follows Figure 3 As shown: Bacteriophage RDP-NS004 has a polyhedral head with three-dimensional symmetry, which encapsulates nucleic acid, with a diameter of 39 nm and a tail length of about 164 nm.

[0063] 1.4 Whole genome sequencing of bacteriophage RDP-NS004

[0064] 1.4.1 Experimental Methods:

[0065] The library was constructed using the Illumina TruSeq™ Nano DNA Sample Prep Kit method; the specific steps are as follows:

[0066] 1) Construct a library starting with 1 μg of phage genomic DNA;

[0067] 2) Covaris M220 ultrasonically breaks down DNA to 300-500 bp;

[0068] 3) Fill in the 3' end with an A and connect the index adapter (TruSeq™ Nano DNA Sample Prep Kit);

[0069] 4) Library enrichment, PCR amplification for 8 cycles;

[0070] 5) 2% agarose gel recovery target band (Certified Low Range Ultra Agarose);

[0071] 6) TBS380 (Picogreen) quantitative analysis: mix according to data ratio and run on the machine;

[0072] 7) Bridged PCR amplification was performed on the cBot solid-phase vector to generate clusters;

[0073] 8) Illumina Hiseq sequencing platform, performing 2×150bp sequencing.

[0074] 1.4.2 Experimental Results:

[0075] The complete genome of bacteriophage RDP-NS004, as shown in SEQ ID NO.1, is 42275 bp in size. Predictive analysis indicates that the genome of bacteriophage RDP-NS004 contains 55 coding sequences. It is characterized as a strictly lytic bacteriophage, and its genome does not contain integrase genes, repressor protein genes, or any known bacterial virulence genes or antibiotic resistance genes.

[0076] Example 2: Characterization of Nocardia amberjack phage RDP-NS004

[0077] 2.1 Determination of the titer of bacteriophage RDP-NS004

[0078] 2.1.1 Experimental Methods:

[0079] Take 0.1 mL of logarithmic growth phase NS004 bacterial suspension and phage RDP-NS004 concentrate, respectively, and add them to 10 mL of BHI soft agar (dissolved at high temperature and cooled to room temperature). Mix well and pour onto BHI solid plates (completely covering a 9 cm plate). Incubate at 28°C upside down for 2 days. Then, collect a single plaque and place it in 1 mL of SM buffer, shake thoroughly, and filter through a 0.22 μm filter membrane. Take 0.1 mL of the filtrate and serially dilute it 10-fold with sterile water. Then, take 0.1 mL of the bacterial suspension and the diluted phage solution, respectively, and add them to 10 mL of BHI soft agar. Mix well and spread on BHI solid plates. Incubate at 28°C in a bagged incubator for 2 days. Select countable plates and count the phage plaques.

[0080] Calculate phage titer (PFU / mL) = number of plaques × dilution factor / sample volume (mL).

[0081] 2.1.2 Experimental Results:

[0082] The titer of bacteriophage RDP-NS004 was determined to be 9.3 × 10⁻⁶. 9 PFU / mL.

[0083] 2.2 Determination of the optimal multiple of infection (MOI) of Nocardia amberjack phage RDP-NS004

[0084] 2.2.1 Experimental Methods:

[0085] The concentration of the host bacteria cultured to the stable phase was adjusted to 10. 8 CFU / mL. MOIs were set at 0.001, 0.01, 0.1, 1, 10, and 100. Phage solutions with determined titers were added to the host bacterial culture in the specified proportions, mixed thoroughly, and incubated at 160 rpm and 28°C with shaking for 6 h. The culture was then centrifuged at 12,000 rpm for 5 min, and the supernatant was used to determine the phage titer using the double-plate method. The highest possible multiplicity of infection (MOI) is the optimal MOI.

[0086] 2.2.2 Experimental Results:

[0087] The results are as follows Figure 4 As shown, when the multiplicity of infection (MCI) was 0.1, the phage RDP-NS004 exhibited the highest titer, 9.3 × 10⁻⁶. 9 PFU / mL.

[0088] 2.3 One-step growth curve of Nocardia amberjack phage RDP-NS004

[0089] 2.3.1 Experimental Methods:

[0090] Phage fluid and host bacterial fluid (7.4 × 10⁻⁶) were collected separately. 6 Mix 0.5 mL of BHI (CFU / mL) at the ratio required for the optimal multiple of infection (MOI=0.1), incubate at room temperature for 10 min, then centrifuge at 12,000 rpm for 10 min, discard the supernatant, wash with BHI liquid medium, and centrifuge again at 12,000 rpm for 1 min. Repeat the above operation, washing the precipitate 3 times, then add 10 mL of BHI liquid medium preheated to 28 °C, mix thoroughly, and quickly place in a shaker at 28 °C. Take a sample every 10 min initially and thereafter, aspirating 0.2 mL, then centrifuge at 12,000 rpm for 2 min, and aspirate 0.1 mL of the supernatant for phage titer determination. Finally, plot a one-step growth curve with infection time on the x-axis and the logarithm of the phage titer in the infection system on the y-axis.

[0091] 2.3.2 Experimental Results:

[0092] The one-step growth curve of phage RDP-NS004 is as follows: Figure 5As shown, within 50 minutes of phage infection of the host bacteria, the number of phages did not increase significantly, indicating a phage latency period of 50 minutes. From 50 to 140 minutes after infection, the number of phages increased rapidly; this period is the phage burst phase, approximately 90 minutes, with a burst dose of approximately 127 PFU / cell. The phage number remained constant for the following 40 minutes, reaching the stationary growth phase. Phage RDP-NS004 has a relatively long latency and lysis period, a large burst dose, and good lysis efficiency, making it suitable for phage therapy.

[0093] 2.4 Determination of the optimal growth temperature for Nocardia amberjack phage RDP-NS004

[0094] 2.4.1 Experimental Methods:

[0095] Take a sterile 2mL centrifuge tube and add 1mL of phage stock solution (initial titer 9.3×10⁻⁶). 9 The phage (PFU / mL) was incubated at 20℃, 28℃, 37℃, 45℃, 50℃, 60℃, and 70℃ for 60 min. After the incubation period, the sample tubes were removed, diluted to an appropriate concentration, and 0.1 mL of the treated phage solution and the host bacterial solution were taken respectively. The phage titer was determined using the double plate method.

[0096] 2.4.2 Experimental Results:

[0097] The results are as follows Figure 6 As shown, phage RDP-NS004 exhibits high activity at 20–37°C, maintaining a titer of 10. 8 ~10 9 PFU / mL. This range covers standard laboratory operating temperatures (~25°C), common temperatures in fish farming water, and mammalian body temperature (37°C). Within this range, phage RDP-NS004 is very stable with almost no inactivation. This indicates its high reliability under normal handling and storage conditions.

[0098] When the temperature continues to rise to 50℃, the phage titer can still be maintained at 10. 6 The activity of the bacteriophage decreased significantly and eventually became completely inactivated when the temperature was raised to above 60°C (PFU / mL). This indicates that the bacteriophage strain has good stability at 20–50°C, a wide temperature tolerance range, and strong thermal stability, enabling it to withstand high-temperature environments.

[0099] The water temperature in aquaculture ponds or cages varies with day and night and seasons. For example, in summer, the surface water temperature can easily reach 30°C or even higher. The phage RDP-NS004 remains stable over a wide range of 20-50°C, meaning that in real-world aquaculture scenarios, it will not rapidly lose its effectiveness due to daily temperature fluctuations, ensuring therapeutic efficacy. Even during cold chain transportation, if there is a temporary loss of insulation (e.g., the temperature rises to 30-40°C), the phage preparation will not easily become unusable, reducing logistics costs and risks. The phage RDP-NS004 does not require strict low-temperature refrigeration (4°C) and maintains high activity when stored at room temperature (20-25°C), significantly lowering storage barriers and costs, making it ideal for aquaculture farms with potentially inadequate infrastructure.

[0100] 2.5 pH stability determination of Nocardia amberjack phage RDP-NS004

[0101] 2.5.1 Experimental Methods:

[0102] Add 3 mL of BHI liquid culture medium to each 5 mL sterile test tube, and adjust the pH to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 respectively. Then place them in a 28°C constant temperature water bath. After temperature equilibration, add 0.1 mL of phage stock solution (9.3 × 10⁻⁶) to each tube. 9 The phage titer was determined by incubating the phage culture (PFU / mL) and host bacterial culture at 28°C for 1 hour using the double-layer plate method. The pH was adjusted to approximately 7.0 using hydrochloric acid or sodium hydroxide solution before the assay.

[0103] 2.5.2 Experimental Results:

[0104] like Figure 7 As shown, phage RDP-NS004 maintains strong lytic activity and a titer of 10 within a pH range of 6 to 8. 9 High PFU / mL levels. Phage activity rapidly decreases until complete inactivation occurs under strongly acidic or alkaline conditions with pH below 4 or above 10. Figure 7 It is known that the optimal pH for phage RDP-NS004 is 7.0, and this strain of phage RDP-NS004 has moderate tolerance to strong acids and alkalis, and is more likely to survive in a neutral environment.

[0105] 2.6 Genetic stability assay of Nocardia amberjack phage RDP-NS004

[0106] 2.6.1 Experimental Methods:

[0107] Take 0.5 mL of phage fluid and host bacterial fluid respectively, mix them at the ratio required for the optimal multiple of infection (MOI=0.1), add 10 mL of BHI liquid medium, mix thoroughly, and then quickly place in a shaker at 28℃ and 160 rpm for 5 days. Then centrifuge at 12,000 rpm for 10 min to remove bacterial fragments, and collect the phage supernatant by 0.22 μm filtration (denoted as P1). Repeat the above steps to infect fresh host bacteria with the previous generation of phage, and continue passage for 30 generations (P1-P30). The phage titer is determined using the double plate method.

[0108] 2.6.2 Experimental Results:

[0109] Depend on Figure 8 It can be seen that the titer of Nocardia amberjack phage RDP-NS004 remained stable throughout the passage process, indicating that the phage has good biological genetic stability and is an excellent choice for the production process.

[0110] Example 3: Lysis spectrum experiment of Nocardia amberjack phage RDP-NS004

[0111] 3.1 Experimental Methods:

[0112] The lytic effect of the Nocardia amberjack phage RDP-NS004 on 25 Nocardia strains preserved in the strain library was determined using the double-layer plate method. The bacterial suspensions from the strain library were revived, and then 0.1 mL of the bacterial suspension and phage suspension were mixed and added to BHI soft agar, mixed thoroughly, and quickly poured into BHI solid medium plates. After cooling and solidification, the plates were incubated upside down at 28°C for 6 hours. The appearance of phage plaques was observed to determine the success of lysis. The 25 Nocardia strains in the strain library were isolated from sea bass, mandarin fish, and large yellow croaker, respectively.

[0113] 3.2 Experimental Results:

[0114] Table 1. Lysis effect of Nocardia amberjack phage RDP-NS004

[0115]

[0116] Note: +: cleavage; -: no cleavage.

[0117] Table 1 shows that the *Nocardia amberjack* phage RDP-NS004 exhibited lytic activity against 17 *Nocardia* strains, with a lysis rate of 68%. This demonstrates that the *Nocardia amberjack* phage RDP-NS004 possesses strong specificity and a relatively broad lytic spectrum against *Nocardia* spp. It also indicates that this *Nocardia amberjack* phage RDP-NS004 has the potential to treat most (more than half) infections caused by *Nocardia*, providing a solid foundation for its development as a commercial aquatic therapeutic agent.

[0118] Example 4: In vitro bactericidal experiment of Nocardia amberjack bacteriophage RDP-NS004

[0119] 4.1 Experimental Methods:

[0120] The in vitro sterilization experiment was divided into the following three groups:

[0121] Group 1: The experimental group was the phage group. Nocardia NS004 in the logarithmic growth phase was mixed with phage fluid RDP-NS004 (MOI=0.1) and added to BHI liquid medium at an inoculum of 2%.

[0122] Group 2: The control group was a bacterial control group, with only Nocardia NS004 added;

[0123] Group 3: The blank group was not treated, but BHI liquid culture medium was added to confirm sterility.

[0124] The three groups were incubated at two temperatures: the optimal growth temperature of 28℃ and a high temperature of 40℃ in a shaker at 160 rpm. Samples were taken at 0, 1, 2, 4, 6, 8, and 24 hours. The samples were serially diluted and plated on BHI agar plates for colony counting (CFU / mL).

[0125] 4.2 Experimental Results:

[0126] like Figure 9As shown, compared to the growth conditions at 28°C, Nocardia NS004 exhibited a similar, or even faster, growth rate at 40°C, resulting in a higher final bacterial concentration. This indicates that the high-temperature environment itself did not inhibit bacterial growth; any antibacterial effect is attributed to the action of the bacteriophage. Under both temperature settings, the bacteriophage exhibited rapid adsorption and lysis. Within 2 hours of exposure, the viable bacterial count decreased sharply by more than three orders of magnitude, demonstrating its short infection cycle and high lysis efficiency. Furthermore, after 4 hours, the bacterial count remained below the detection limit (<2.0), and there was no recurrence until the end of the 24-hour experiment, indicating that the bacteria had been completely eliminated. The experiment found that the lysis kinetics of bacteriophage RDP-NS004 at 40°C were similar to those at 28°C, and its killing curve highly overlapped with the morphology of the 28°C group, indicating that this bacteriophage strain maintained high lysis activity even under high-temperature conditions.

[0127] Example 5: Treatment experiment of Nocardia amberjack phage RDP-NS004 on largemouth bass

[0128] 5.1 Experimental Methods:

[0129] Healthy largemouth bass, weighing approximately 20.0 g ± 2.0 g, were selected. Before the experiment, these fish were temporarily held in a recirculating aquaculture system at 26 ± 1°C for one week and fed commercial feed daily. Nocardia NS004 was inoculated onto BHI blood agar plates for resuscitation culture. Subsequently, bacterial colonies were scraped and inoculated into BHI liquid medium, and cultured for 4 days at 28°C and 160 rpm in a shaker. After centrifugation at 12,000 rpm for 3 minutes, the supernatant was discarded, and the bacterial cells were gently homogenized with phosphate-buffered saline (PBS, pH 7.2). The final bacterial suspension concentration was adjusted to 10-1. 6 CFU / mL, to be used in subsequent experiments.

[0130] After the largemouth bass were adapted to the environment, experiments were conducted, and they were challenged with NS004 after treatment.

[0131] Fifteen fish in the research group were anesthetized and then injected intraperitoneally with 100 μL of NS004.

[0132] The control group consisted of 15 fish injected with an equal volume of PBS (pH 7.2).

[0133] The treatment group consisted of 15 fish that were injected with 100 μL of RDP-NS004 phage solution 24 hours after the phage was administered. 8 Treatment was administered using PFU / mL.

[0134] The fish were observed for 15 days. The mortality rate was calculated by counting the number of deaths in each group and observing disease symptoms. Samples were taken from 3 fish in each group (n=3) at 2, 4 and 6 days post-infection. Spleen tissue was collected from the samples to calculate the bacterial load.

[0135] Bacterial load: Approximately 0.1 g of spleen tissue was homogenized in 0.9 mL of PBS. It was then serially diluted (10⁻⁶ ppm). -1 ~ 10 -6 Select 2-3 suitable dilutions for plating, and incubate in a 28℃ incubator with a bag for 2 days. Then, select an appropriate plate count (30-300 CFU / plate). Perform 3 parallel plates for the same dilution and take the average value.

[0136] Bacterial load (CFU / g) = (average CFU count × dilution factor) / tissue weight (g).

[0137] 5.2 Experimental Results:

[0138] Table 2. Treatment experiment of bacteriophage RDP-NS004 on largemouth bass

[0139]

[0140] Table 3 Results of spleen bacterial load test (lg(CFU / g))

[0141]

[0142] Note: "-" indicates not detected.

[0143] Table 3 shows that at 2 days post-infection, both the challenge and treatment groups had high bacterial loads with some overlap, indicating that infection had been established. The treatment group had a slightly lower bacterial load than the challenge group, suggesting that phage RDP-NS004 had just begun to take effect or that individual differences existed, and no decisive difference had yet emerged. At 4 days post-infection, the difference widened dramatically. In the challenge group, bacteria continued to proliferate, and the bacterial load increased; while in the treatment group, the bacterial load decreased significantly by nearly 100-fold (from 10...). ^5 Level 10 ^4 (Grade). This indicates that phage RDP-NS004 rapidly proliferated in vivo and exerted a powerful lytic effect. The effect was extremely significant 6 days post-infection. The bacterial load in the challenge group reached its peak, consistent with its high mortality rate. In contrast, the bacterial load in the treatment group decreased dramatically, nearly 100 times lower than on day 4 (10). ^4 Level 10 ^2 The disease has reached a level close to complete clearance (grade 1). This explains why the survival rate in the treatment group was as high as 80% in Table 2.

[0144] Therefore, according to the results of the challenge experiment, largemouth bass began to die on the 4th day after challenge. Table 2 shows that the mortality rate of infected largemouth bass reached over 70%, but no significant swelling of internal organs was observed. Treatment with bacteriophage RDP-NS004 significantly reduced the mortality rate of largemouth bass, ensuring an 80% survival rate, demonstrating a significant therapeutic effect. The control group showed no morbidity or mortality. Bacterial load testing of the spleen revealed that the Nocardia NS004 content in the spleen was significantly lower in the treated group than in the challenge group, effectively inhibiting the infection and invasion of Nocardia NS004 into largemouth bass. This indicates that bacteriophage RDP-NS004 has a significant therapeutic effect on largemouth bass infection caused by the NS004 strain, significantly reducing the mortality rate of infected fish and effectively clearing pathogens from the spleen. The use of Nocardia bacteriophage RDP-NS004 can effectively alleviate bacterial diseases caused by Nocardia in largemouth bass.

[0145] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A heat-stable Nocardia amberjack bacteriophage ( Nocardia seriolae bacteriophage), characterized by RDP-NS004, which was deposited with the China General Microbiological Culture Collection Center on August 25, 2025, and has the accession number CGMCC No. 46675.

2. A composition characterized in that: The high-heat-stable Nocardia seriolae bacteriophage according to claim 1.

3. The high-heat-stable Nocardia seriolae bacteriophage according to claim 1 or the composition according to claim 2, for use in at least one of the following (1) to (3): (1) preparation of a product for killing Nocardia seriolae; (2) preparation of a product for inhibiting Nocardia seriolae; (3) preparation of a product for preventing and / or treating fish nocardiosis caused by Nocardia seriolae. The product is a veterinary preparation, a feed additive, or a disinfectant. The product is a feed additive treated at a temperature of 50°C or lower. The product is a preparation for soaking a fishery drug.

4. Use according to claim 3, characterized in that: The fish is a sea bass, a mandarin fish, or a large yellow croaker.

5. Use according to claim 3, characterized in that: ​ 6. Use according to claim 3, characterized in that: ​ 7. Use according to claim 3, characterized in that: ​

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