Bacteriophage of lytic salmonella pullorum and application of bacteriophage
By using phages of lysed Salmonella pullorum, the problems of drug resistance and drug residues in the control of Salmonella pullorum with antimicrobial drugs have been solved, achieving efficient and safe control of Salmonella pullorum and promoting the healthy development of the poultry industry.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGXI UNIV FOR NATITIES
- Filing Date
- 2026-03-19
- Publication Date
- 2026-05-12
Smart Images
Figure CN122012415A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, specifically to a phage of lytic Salmonella pullorum and its application. Background Technology
[0002] Salmonella pullorum is a host-specific pathogen that poses a serious threat to poultry farming, with widespread transmission routes and significant challenges in its control. This pathogen causes pullorum disease in chickens, exhibiting extremely high pathogenicity in chicks, leading to high morbidity and mortality rates and causing direct economic losses to farms. Adult and growing chickens infected with the pathogen often remain asymptomatic, becoming long-term carriers. This not only severely impacts their own reproductive performance but also allows the disease to spread through vertical transmission (affecting the reproductive system and infecting offspring) and horizontal transmission (spreading among flocks), creating a persistent infection cycle that severely restricts the healthy development of the poultry industry.
[0003] Currently, the control of Salmonella pullorum in my country's poultry industry still mainly relies on antibiotics. However, the long-term, repeated, and irrational use of antibiotics has led to a series of serious problems: on the one hand, bacterial resistance is increasing, causing a continuous decline in the effectiveness of existing antibiotics, and some strains have even developed into multidrug-resistant bacteria, leaving clinical control in a passive situation of "ineffective treatment"; on the other hand, antibiotic residues can enter the food chain through poultry products (meat and eggs), posing a potential threat to human health, while also polluting the farming environment, disrupting the ecological balance, and contradicting the industry's trend towards green farming. Therefore, developing a safe, efficient, environmentally friendly technology for the control and treatment of Salmonella pullorum that can avoid the problem of drug resistance has become a critical need that the poultry industry urgently needs to address.
[0004] Bacteriophages, as a class of viruses that specifically infect bacteria (including actinomycetes, fungi, and prokaryotes), offer an ideal solution to the aforementioned problems due to their unique characteristics. Widely found in natural environments such as sewage and feces, bacteriophages exhibit strict host specificity, attacking only one or a few specific bacteria. They are non-infectious to humans, animals, and plants, possessing extremely high safety and leaving no environmental pollutant residues, aligning with environmentally friendly farming principles. Their mechanism of action is as follows: upon contact with host bacteria, they rapidly adsorb and invade the bacterial cell, multiplying until they release progeny bacteriophages through lysis of the host bacteria. These progeny bacteriophages then continue to infect surrounding target bacteria, creating an exponential proliferation effect that efficiently kills target pathogens in a short time. Furthermore, the rate of antibiotic resistance development in bacteriophages is far lower than the rate of antibiotic resistance development in bacteria, and their lytic action is not limited by existing bacterial resistance. Additionally, bacteriophage screening cycles are short, preparation processes are simple, and production costs are low, facilitating large-scale commercial production and demonstrating significant potential to replace traditional antibiotics in controlling Salmonella pullorum disease in chickens. Summary of the Invention
[0005] The purpose of this invention is to provide a lytic phage of Salmonella pullorum to solve the problems of drug resistance and drug residues in the prior art of using antibacterial drugs to control Salmonella pullorum, as mentioned in the background section.
[0006] To achieve the above objectives, the present invention provides the following technical solution: a bacteriophage of lytic Salmonella pullorum, wherein the bacteriophage has a polyhedral head and a retractable tail, the average diameter of the head is about 71 nm and the tail is about 126 nm, and belongs to the Myocaudalidae family.
[0007] Preferably, it is capable of lysing 15 strains of Salmonella pullorum from 18 clinical isolates of Salmonella.
[0008] Preferably, the latency period is about 40 minutes, the lysis period is about 170 minutes, and the lysis volume reaches 142 PFU / cell.
[0009] Preferably, the optimal infection multiplicity is 0.0001.
[0010] Preferably, the cleavage activity is maintained in an environment of 4 ≤ pH ≤ 11.
[0011] Preferably, the activity is good under conditions of 30-50°C.
[0012] Preferably, the product includes feed additives, veterinary drugs, or disinfectants.
[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: The phage of the lytic Salmonella pullorum isolated by the present invention has the advantages of broad host spectrum, high lysis efficiency, low optimal multiple of infection, and low production cost. Clinical trials have shown that it can significantly reduce the mortality rate of infected chicks, reduce the detection rate of Salmonella in organs, and alleviate pathological damage. Long-term use can also purify chicken flocks and improve egg production rate and quality, providing a safe, efficient, economical and environmentally friendly new solution for the prevention and control of Salmonella pullorum in poultry farming. Attached Figure Description
[0014] Figure 1 : A diagram showing the morphology of phage plaques formed by bacteriophages on a double-layer plate;
[0015] Figure 2 Transmission electron micrograph of bacteriophage morphology;
[0016] Figure 3 : One-step growth curve of bacteriophage;
[0017] Figure 4 : Activity change curves of bacteriophages under different pH conditions;
[0018] Figure 5 : Activity change curves of bacteriophages under different temperature conditions;
[0019] Figure 6 Comparison of liver pathological sections of chicks in the phage treatment group and the control group. Detailed Implementation
[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.
[0021] Please see Figure 1-6 The present invention provides a technical solution: a phage of lytic Salmonella pullorum, the phage having a polyhedral head and a retractable tail, the average diameter of the head being about 71 nm and the tail being about 126 nm, belonging to the Myocaudidae family.
[0022] The example described herein describes the ability to lyse 15 out of 18 clinical isolates of Salmonella pullorum.
[0023] Its latency period is approximately 40 minutes, its lysis period is approximately 170 minutes, and its lysis rate reaches 142 PFU / cell.
[0024] The optimal multiplicity of infection is 0.0001.
[0025] It can maintain good cleavage activity in an environment of 4 ≤ pH ≤ 11.
[0026] The substance exhibits good activity at temperatures of 30-50℃.
[0027] The application of the bacteriophage of the lytic Salmonella pullorum, wherein the product includes feed additives, veterinary drugs or disinfectants.
[0028] Phage isolation: Wastewater from poultry farms in Beihai City was collected and allowed to stand at 4℃ for 24 h. The supernatant was collected, and CaCl2 was added to a final concentration of 1 mol / L. The mixture was centrifuged at 8000 r / min for 10 min, and 20 mL of the supernatant was collected and filtered through a 0.22 μm filter. 10 mL of the sterilized filtrate was added to 20 mL of TSB liquid medium and mixed well. 10 mL of fresh Salmonella S38 bacterial culture (OD600nm approximately 0.6) was added to the mixture, and the mixture was incubated overnight at 37℃ and 180 r / min. Subsequently, the culture was centrifuged at 8000 r / min for 5 min, and 100 μL of the supernatant was mixed with 100 μL of fresh Salmonella S38 bacterial culture (OD600nm approximately 0.6). The mixture was then mixed with 0.6% LB agar at approximately 45℃ and poured onto LB agar plates. After solidification, the plates were incubated at 37℃ for 12 h, and phage plaques were observed. A single phage plaque was selected and dissociated in SM buffer at room temperature for 4 hours. 100 μL each of the dissociation solution and the host bacterial culture were taken, mixed thoroughly, and poured into a double-layer plate. The plate was then incubated at 37°C for 12 hours. This process was repeated 4-5 times to obtain purified phage P429.
[0029] Phage concentration and ultracentrifugation: Cesium chloride gradient solutions were prepared with densities of cesium chloride (p = 1.45, 1.50, 1.70 g / mL). Cesium chloride was added to the phage solution to achieve a final concentration of 1.15 g / mL. 3 mL of each of the different density gradient cesium chloride solutions was added to an ultracentrifuge tube, followed by 6 mL of phage solution. The tubes were centrifuged at 4°C and 12000 rpm for 4 h. The phage layer was collected and stored.
[0030] Morphological observation: 3 μL of liquid was taken by pipette with a titer of 1×10⁻⁶. 8 Phage solution of PFU / mL was added to a carbon-coated 400-mesh copper grid, allowed to stand for 10 min, stained with 1% phosphotungstic acid for 2 min, and then the morphology of the phage was observed by transmission electron microscopy. The results showed that phage P429 has a polyhedral head structure and a retractable tail. The average diameter of the head is about 71 nm and the tail is about 126 nm, belonging to the Myocaudae family.
[0031] Determination of bacteriophage biological characteristics:
[0032] Host spectrum: The host spectrum of P429 against 18 strains of Salmonella pullorum was determined using the double-layer plate method. The results showed that the bacteriophage had a lytic effect on 15 clinical isolates of Salmonella, with a relatively broad lytic spectrum.
[0033] Optimal Multiple of Infection (MOI): 100 μL of diluted phage solution was mixed with 100 μL of host bacterial culture at different MOIs (0.0001, 0.001, 0.01, 0.1, 1, 10, and 100), and added to LB broth. The mixture was incubated at 37°C and 180 r / min for 6 h using a shaker. After incubation, the mixture was centrifuged at 10000 × g for 10 min, and the supernatant was collected and filtered through a 0.22 μm anhydrous membrane. The titer was determined using the double-layer plate method. The results showed that the highest number of progeny phages were produced when the MOI was 0.0001, with an average titer of 4.26 × 10⁻⁶. 8 PFU / mL, therefore the optimal multiplicity of infection is 0.0001.
[0034] One-step growth curve: Phage fluid was added to the culture medium of Salmonella Pullorum S38 (OD600nm approximately 0.6) at an optimal multiplicity of infection of 0.0001, and the cells were incubated on a shaker at 37°C and 180 rpm. Samples were taken at different time points, and phage titers were determined to plot a one-step growth curve. The results showed that the latency period of P429 was approximately 40 minutes, the lysis period was approximately 170 minutes, and the lysis yield was approximately 142 PFU / cell.
[0035] pH tolerance: SM buffer solutions with different pH values (pH range 3-13) were prepared. 1 mL of phage fluid was added to 9 mL of SM buffer solution at different pH values, and the solutions were incubated at 37°C for 1 h. The phage titer was then determined. The results showed that in an environment of 4 ≤ pH ≤ 11, the titer of phage P429 did not change significantly, maintaining good lytic activity, with the highest activity at pH 9.
[0036] Temperature tolerance: 500 μL of phage solution was incubated in water baths at different temperatures (10–80 °C) for 1 h, and the phage titer was determined. The results showed that the titer of phage P429 remained at a high level and exhibited good activity under conditions of 30–50 °C. At temperatures ≥50 °C, the titer gradually decreased, and it was completely inactivated after incubation at 80 °C for 1 h.
[0037] Application of bacteriophages in chicks:
[0038] Determination of the median infectious dose (LC50) of Salmonella pullorum in chickens: Twenty-five 1-day-old SPF chicks were randomly divided into 5 groups of 5 chicks each. The control group was administered physiological saline by gavage, while the experimental groups were administered different concentrations of S38 bacterial suspension by gavage. The results showed that when the challenge concentration was 1×10⁻⁶, the median infectious dose of Salmonella pullorum was significantly higher than that of S38. 7 When CFU / mL is above a certain level, at least 80% of chicks are infected, and the determined challenge dose is 1×10⁻⁶. 7 CFU / mL.
[0039] Safety test of bacteriophage: Ten 1-day-old SPF chicks were selected and divided into two groups. The control group was administered physiological saline by gavage, and the experimental group was administered 1×10⁻⁶ phage by gavage. 8 The phage fluid was at a concentration of PFU / mL. During the experiment, the chicks in both groups showed normal mental state and feed intake, and necropsy revealed normal major organs and intestines, indicating that phage P429 has high safety.
[0040] Phage therapy for Salmonella infection: Forty 1-day-old SPF chicks were selected and divided into two groups. All chicks were orally administered Salmonella pullorum S38 (1×10⁻⁶). 7 CFU / feather), phage treatment group orally administered phage P429 (1×10⁻⁶) 8 The control group received an equal volume of sterile saline orally. Results showed that the mortality rate in the control group was 15%, while there were no deaths in the phage group. The detection rates of Salmonella in the liver, ileum, and cloaca were reduced by 75%, 70%, and 45% respectively in the phage group compared to the control group, and it significantly reduced organ pathological damage. This completes the entire study. Any content not described in detail in this specification is prior art known to those skilled in the art.
[0041] The terms “center,” “longitudinal,” “lateral,” “front,” “rear,” “left,” “right,” “vertical,” “horizontal,” “top,” “bottom,” “inner,” and “outer,” etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are merely simplified descriptions for the convenience of describing the present invention and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting the scope of protection of the present invention.
[0042] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. 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 of a lytic Salmonella pullorum, characterized in that, The bacteriophage has a polyhedral head and a retractable tail. The average diameter of the head is about 71 nm and the tail is about 126 nm. It belongs to the Myocaudalidae family.
2. The phage of lytic Salmonella pullorum according to claim 1, characterized in that: It is said to be able to lyse 15 of the 18 clinical isolates of Salmonella pullorum.
3. The phage of lytic Salmonella pullorum according to claim 1, characterized in that: Its latency period is approximately 40 minutes, its lysis period is approximately 170 minutes, and its lysis rate reaches 142 PFU / cell.
4. The phage of lytic Salmonella pullorum according to claim 1, characterized in that: The optimal multiplicity of infection is 0.0001.
5. The phage of Salmonella pullorum lysing according to claim 1, characterized in that: It can maintain good cleavage activity in an environment of 4 ≤ pH ≤ 11.
6. The phage of Salmonella pullorum lysing according to claim 1, characterized in that: The substance exhibits good activity at temperatures of 30-50℃.
7. The application of the bacteriophage of lytic Salmonella pullorum according to any one of claims 1-6, characterized in that: The products include feed additives, veterinary drugs, or disinfectants.