Phage for fish nocardia and application thereof

Abstract

The invention relates to the technical field of bacteriophages, and provides a bacteriophage for fish nocardia and application of the bacteriophage, the bacteriophage is used for splitting fish nocardia, and the fish nocardia comes from a water body of a fish culture pond; the bacteriophage is preserved in China General Microbiological Culture Collection Center (CGMCC) on June 17, 2025, and the preservation number is CGMCC No.46495; the bacteriophage belongs to a tail bacteriophage, and the tail length is 165 + / -2nm. According to the technical scheme, the problem that at present, no bacteriophage for effectively treating and preventing fish nocardia exists is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bacteriophages, in particular to a bacteriophage for fish Nocardia and application thereof. BACKGROUND

[0002] Fish Nocardia belongs to the order Actinomycetales, the family Nocardia and the genus Nocardia, and is a gram-positive bacterium. Fish Nocardia is a conditional pathogen, which can easily infect through the mouth, gills or body surface wounds when the body constitution of the farmed fish is weak and the immunity is low. At the initial stage of infection, there is no obvious abnormality on the body surface of the diseased fish, but the fish may exhibit phenomena such as reduced feeding, slow swimming and swimming alone. As the disease worsens, granulomatous lesions may occur in multiple internal organs of the fish, and white or gray nodules may appear on the body surface of the infected fish. In severe cases, skin ulcers and bleeding may occur. According to literature reports, fish Nocardia can infect more than 40 species of marine and freshwater fish, including small yellow croaker, snakehead, oval pompano, large-mouthed black bass and flower bass.

[0003] At present, the prevention and treatment measures for fish Nocardia disease mainly include the use of various antibiotics, such as florfenicol or doxycycline. However, fish Nocardia is a facultative intracellular parasite that can parasitize in host cells and proliferate and spread by forming nodules in tissues. The nodule serves as a shelter and diffusion site for the bacteria, and antibiotics are difficult to effectively penetrate the nodule structure or enter the host cells, resulting in the inability of the drug to directly act on the intracellular bacteria. In addition, the overuse of antibiotics can easily lead to antibiotic residues and bacterial drug resistance problems, and therefore, it is necessary to find safer and more effective green control agents.

[0004] Bacteriophages are widely distributed in nature and are a class of viruses that specifically lyse bacteria. They have a simple structure and strong host specificity. Bacteriophages can recognize receptors and adsorb to the surface of specific host bacteria, inject genetic material into the bacteria to replicate themselves, and finally release progeny by lysing bacteria to achieve reproduction. One administration can achieve exponential amplification. In this process, bacteriophages will be eliminated with the host, and will not affect humans and animals. More importantly, the lysis effect of bacteriophages on bacteria will not be affected by bacterial drug resistance. Therefore, bacteriophages can be used as green control means for preventing or treating diseases caused by fish Nocardia infection due to their strong specificity, no residues and safe use. However, there is currently no bacteriophage resource for fish Nocardia. SUMMARY

[0005] The present application provides a bacteriophage for fish Nocardia and application thereof, which solves the problem that there is currently no effective bacteriophage for treating and preventing fish Nocardia.

[0006] The technical solution of the present application is as follows:

[0007] A bacteriophage for lysing Nocardia seriolae, the Nocardia seriolae originating from the water of a fish farm; the bacteriophage was deposited on June 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46495 and classified as Nocardiaseriolae phage.

[0008] As a further technical solution, the genome length of the bacteriophage is 48.05 kd.

[0009] As a further technical solution, the titer of the bacteriophage is greater than or equal to 1×10⁻⁶. 6 PFU / mL.

[0010] The present invention also provides the use of the bacteriophage for Nocardia fish as described above in the preparation of Nocardia fish agents.

[0011] The present invention also provides a bacteriophage composition comprising the bacteriophage described above for Nocardia fish.

[0012] The present invention also provides a bacteriophage drug formulation, wherein the active ingredient of the bacteriophage drug formulation includes the bacteriophage for Nocardia fish described above or the bacteriophage composition described above.

[0013] As a further technical solution, the phage drug formulation also includes a pharmaceutically acceptable carrier; the dosage form of the phage drug formulation includes one or more of the following: solution, powder, gel, granule, emulsion, suspension, and lyophilized agent.

[0014] The present invention also provides a feed additive comprising the bacteriophage for Nocardia fish described above or the bacteriophage composition described above.

[0015] The present invention also provides a disinfectant comprising the bacteriophage for Nocardia fish described above or the bacteriophage composition described above.

[0016] As a further technical solution, the disinfectant also includes auxiliary agents.

[0017] The working principle and beneficial effects of this invention are as follows:

[0018] 1. This invention is the first to isolate a bacteriophage for Nocardia spp. This bacteriophage has a strong lytic effect on Nocardia spp., which can effectively prevent or treat diseases caused by Nocardia spp. infection in aquaculture, and greatly reduce the incidence of Nocardia spp. disease in aquaculture. In addition, preparations containing this bacteriophage can also be used for disinfection of aquaculture environment, feed, aquaculture supplies, etc., to reduce the morbidity and mortality of aquatic animals caused by Nocardia spp. from different sources; it can also be used for the detection of Nocardia spp. in fish.

[0019] 2. The bacteriophage for Nocardia in fish provided by this invention was isolated from aquaculture ponds in Zhongshan City, Guangdong Province. The bacteriophage and compositions containing it are safe to use and can effectively solve infections caused by Nocardia amberjack and water problems caused by its proliferation, avoiding antibiotic residues and antibiotic resistance issues. Animal health products, disinfectants, or diagnostic reagents prepared from this bacteriophage also have the advantages of being safe and environmentally friendly. Attached Figure Description

[0020] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0021] Figure 1 For preliminary screening of bacteriophages for use in Nocardia fish;

[0022] Figure 2 The in vitro inhibitory effect of bacteriophages used against Nocardia fish on Nocardia fish was investigated.

[0023] Figure 3 Electron microscopy identification results of bacteriophages used in Nocardia fish;

[0024] Figure 4 The results of genomic analysis of bacteriophages used in Nocardia fish;

[0025] Figure 5 To evaluate the lysis effect of bacteriophages used for Nocardia in fish on different species of Nocardia;

[0026] Figure 6 The effect of a biological agent containing a bacteriophage for Nocardia fish on serum enzyme activity in snakehead fish;

[0027] Figure 7 The effects of a biological agent containing a bacteriophage for Nocardia fish on the pathological changes of the spleen tissue of infected snakehead fish. Detailed Implementation

[0028] 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] This invention provides a bacteriophage for Nocardia spp., which was deposited on June 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46495.

[0030] Transmission electron microscopy revealed that the phage used for Nocardia spp. in fish is a tailed phage with a tail length of 165±2 nm. Genome sequencing showed that the genome length of the phage used for Nocardia spp. in fish is 48.05 kd. The phage used for Nocardia spp. in fish contains genes such as phage terminal enzymes and capsid proteins that may be related to lysis function.

[0031] This invention also provides the application of bacteriophages for Nocardia fish in the preparation of Nocardia fish agents. For example, in the production of bacteriophage pharmaceutical formulations, feed additives, disinfectants, etc.

[0032] The bacteriophages used for Nocardia in fish exhibit significant lytic activity against different species of Nocardia, with the best lytic effect against Nocardia in fish. This suggests that they have great potential for application in the prevention or treatment of Nocardiasis in fish.

[0033] Bacteriophages for Nocardia spp. or compositions containing bacteriophages for Nocardia spp. can be used as feed additives for aquatic animals, with a bacteriophage titer of 1×10⁻⁶. 6 PFU / mL or higher. Under these conditions, feed additives are more effective in preventing or treating nocardiosis in fish.

[0034] In addition, the present invention provides a disinfectant whose active ingredient includes a bacteriophage for Nocardia flammatories or a composition containing a bacteriophage for Nocardia flammatories. The disinfectant also contains other active ingredients or adjuvants that inhibit or kill bacteria; wherein the adjuvants include reagents that can prolong the shelf life of the bacteriophage. This water disinfectant can be used for disinfection of ponds or cages in aquaculture or for aquaculture supplies, and can be used by spraying, soaking, etc. This water disinfectant can be used as a substitute for antibiotics or traditional disinfectant products.

[0035] This invention also provides a detection kit comprising the Nocardia spp. spp. phage or a combination of the aforementioned phages, as described above. Experimental results show that the phage for Nocardia spp. ...

[0036] This invention provides a method for screening and purifying phages of Nocardia flavonoids from fish, as follows:

[0037] Multiple sampling points were collected from fishponds in Zhongshan City, Guangdong Province, where an outbreak of nocardiosis in fish had occurred. Water and mud samples were collected. The mud samples were treated with sterile water and then preliminarily filtered. The filtrate and water samples were centrifuged at 5000g for 10 min using a high-speed centrifuge, and then filtered through a 0.22μm filter membrane (the mud sample filtrate required multiple centrifugations). The supernatant obtained was mixed with BHI liquid medium, and then Nocardia fish bacteria culture was added. After standing for 1 h, the mixture was incubated at 28℃ and 130rpm for 72 h on a shaker.

[0038] After filtration and centrifugation, a sterile stock solution was obtained. 2 mL of the sterile stock solution was mixed with 2 mL of Nocardia fish bacterial suspension, and then added to 8 mL of semi-solid BHI medium to prepare a double-layer plate. Plaques were observed. Figure 1 As shown.

[0039] The phage fluids obtained from the initial screening were mixed with SM buffer at a 1:1 ratio and stored overnight at 4°C. The next day, 50 mL of BHI liquid medium was mixed with 1 mL of Nocardia fish bacterial suspension, serving as a control compared to adding only 100 mL of BHI liquid medium and 1 mL of Nocardia fish bacterial suspension. Growth changes in the bacterial suspensions were observed. Figure 2 As shown.

[0040] The present invention provides a method for identifying bacteriophages used in Nocardia fish as follows:

[0041] I. Electron Microscopic Detection of Bacteriophages

[0042] 1. Experimental Method:

[0043] After centrifugation using an ultra-high-speed centrifuge, the bacteriophages were collected, negatively stained, and observed using a transmission electron microscope.

[0044] 2. Experimental Results and Analysis

[0045] Transmission electron microscopy revealed that the fish Nocardia bacteriophage is a tailed bacteriophage with a tail length of 165±2 nm, a width of 8±2 nm, and a head diameter of 66.1±4 nm. The head is circular and has a distinct tail structure. Figure 3 As shown, it is named YS-1.

[0046] II. Analysis of bacteriophage-specific genes

[0047] Whole-genome sequencing analysis of phage YS-1, using COG / KEGG / Uniprot annotation, revealed that the genome contains highly conserved phage terminal enzymes and capsid proteins, such as... Figure 4 As shown.

[0048] This invention also conducted experiments on the bacteriophage used for Nocardia in fish on snakehead fish. The experimental methods and results are shown below:

[0049] (I) Experimental Methods

[0050] 1. After temporarily holding the purchased snakehead fish for one week, they were divided into 8 groups. Groups A, B, C, and D were prevention groups: they were fed treated feed first, then challenged with the virus. Groups E, F, G, and H were treatment groups: they were challenged with the virus first, then treated with treated feed. Control groups A and E were fed normal feed, while groups B and F were fed feed containing *Bacillus amyloliquefaciens*, an antagonist to *Nocardia spp.* (80 mL of 1×10⁻⁶ solution per 1000 g of feed). 8 Antagonistic bacteria (PFU / mL), groups C and G were fed diets containing Nocardia fish phage YS-1 (100mL of 1×10⁻⁶ PFU / mL added per 1000g of feed). 8 The fish were fed a compound diet (90 mL of phage and antagonistic bacteria compound solution per 1000 g of feed) with PFU / mL lysate. The feed amount was 3% of the average body weight of the experimental fish, and the fish were fed twice a day (9:00 and 15:00). The water was changed twice a week, with 2 / 3 of the water replaced.

[0051] 2. Feed treatment containing Nocardia antagonistic bacteria for fish: ① Activate the bacterial solution in advance; ② Expand the culture by inoculating the activated bacterial solution at a rate of 1%; ③ Dilute the bacterial solution and count the bacteria using the spread plate method; ④ Collect the bacteria by centrifugation at 10000g for 5 minutes, and adjust the concentration to 1×10⁻⁶ with PBS. 8 cfu / mL; ⑤ Place the adjusted concentration of antagonistic bacteria and feed together in a clean sterilization box, shake well, and place in an air-conditioned room at 16℃ to air dry before feeding the next day (ensure fresh feeding).

[0052] 3. Feed treatment containing Nocardia fowl for fish: ① Pre-concentrate multiple 100mL bottles of Nocardia fowl lysis buffer for fish (cultured for 36h at 28℃ and 120rpm); ② Adjust the PBS concentration to 1×10⁻⁶. 6 cfu / mL; ⑤ The night before, place the adjusted concentration of phage and feed together in a clean sterilization box, shake well, and place in an air-conditioned room at 16℃ to air dry. Feed the feed the next day, and store any excess feed in a refrigerator at 4℃. (Ensure freshness for feeding).

[0053] 4. Treatment of feed containing phage and Bacillus amyloliquefaciens: ① Mix the above-treated antagonistic bacteria and phage at a volume ratio of 1:1; ② Place the antagonistic bacteria and phage combined feed at the adjusted concentration in a clean sterilized box, shake well, place in an air-conditioned room at 16℃ and air dry in the shade for feeding the next day, and store any excess in a refrigerator at 4℃ (to ensure fresh feeding).

[0054] 5. The experimental fish used for virus challenge were intraperitoneally injected with sterile PBS diluted to a concentration of 1×10⁻⁶. 7 100 μL of PFU / mL Nocardia fish bacteria were injected, and the control group was injected with the same amount of sterile PBS. Three fish were randomly collected from the infected group and the control group at 0 days and 14 days after infection for dissection. The spleens were fixed with 4% paraformaldehyde for pathological section analysis.

[0055] 6. Blood was drawn from 6 fish in each group and stored at 4℃. After 12 hours, the fish were centrifuged at 3500 rpm for 10 minutes at 4℃. The supernatant was collected and placed in a 200 μL centrifuge tube and stored at -80℃ for the determination of catalase (CAT), lysozyme (LZM), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), and enzyme activity analysis of lipase (LPS).

[0056] 7. Pathological tissues from different groups were analyzed using HE staining and scanning electron microscopy.

[0057] (II) Experimental Results and Analysis

[0058] 1. After being challenged with Nocardia spp. in fish, farmed snakehead fish showed mortality within about a week, exhibiting symptoms such as swelling of the genital pore, ascites, and enteritis. After one week, the livers and tissues of fish that died showed significant congestion and a small number of visible nodules. Antagonistic bacteria and bacteriophages specifically formulated for Nocardia spp. in fish effectively reduced mortality caused by Nocardia spp. infection. Furthermore, the combined preparation of bacteriophages and antagonistic bacteria showed the most significant effect in treating diseases caused by Nocardia spp. in fish, as shown in Table 1.

[0059] 2. In the evaluation experiment of prevention and treatment effects, compared with the control group, the SOD enzyme activity was significantly enhanced in the experimental fish fed with compound bacteriophage and antagonistic bacteria diet, such as... Figure 6 As shown.

[0060] 3. In the spleen tissue of the control group of snakehead fish, obvious nodules were visible, with necrotic tissue fragments in the center and congested blood vessels within the spleen; while the experimental fish fed with compound bacteriophage and antagonistic bacteria diet showed no obvious nodule formation, only a small amount of erythrocyte proliferation, such as... Figure 7 As shown.

[0061] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A bacteriophage for Nocardia fish, characterized in that, The bacteriophage is used to lyse Nocardia fish, which is derived from the water of a fish farm pond; the bacteriophage was deposited on June 17, 2025 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 46495.

2. The bacteriophage for Nocardia fish as described in claim 1, characterized in that, The genome length of the bacteriophage is 48.05 kd.

3. The bacteriophage for Nocardia fish as described in claim 2, characterized in that, The titer of the bacteriophage is greater than or equal to 1 × 10⁻⁶. 6 PFU / mL.

4. The use of the bacteriophage for Nocardia fish as described in claim 1 in the preparation of Nocardia fish preparations.

5. A bacteriophage composition, characterized in that, Includes the bacteriophage for Nocardia fish as described in claim 1.

6. A bacteriophage drug formulation, characterized in that, The active ingredient of the phage drug formulation includes the phage for Nocardia fish as described in claim 1 or the phage composition as described in claim 5.

7. A phage drug formulation as described in claim 6, characterized in that, The phage drug formulation further includes a pharmaceutically acceptable carrier; the dosage form of the phage drug formulation includes one or more of the following: solution, powder, gel, granule, emulsion, suspension, and lyophilized agent.

8. A feed additive, characterized in that, The feed additive includes the bacteriophage for Nocardia fish as described in claim 1 or the bacteriophage composition as described in claim 5.

9. A disinfectant, characterized in that, The disinfectant includes the bacteriophage for Nocardia fish as described in claim 1 or the bacteriophage composition as described in claim 5.

10. The disinfectant as described in claim 9, characterized in that, The disinfectant also includes auxiliary agents.

Images