A high-temperature-resistant Salmonella pullorum phage, its phage composition and its application

Abstract

The present invention discloses a high-temperature resistant Salmonella pullorum phage, a phage composition thereof and its application. The high-temperature resistant Salmonella pullorum phage is named PC127 and was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on April 12, 2021, with a deposit number of CGMCC No. 22366. The phage composition includes phage PC127 and other matching phages. The phage has strong lysis and a wide lysis spectrum against Salmonella pullorum, can effectively prevent and treat pullorum, and has the effects of reducing the mortality rate of chickens, reducing the positive rate of pullorum antibodies, and increasing egg production. In addition, it can be used as an active ingredient and detection kit of an environmental disinfectant. While solving Salmonella infection, long-term application can purify pullorum in chicken farms.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a heat-resistant Salmonella pullorum phage, a phage composition thereof and applications thereof. Background Art

[0002] Salmonella pullorum, a host-specific pathogen, primarily infects chicks under 20 days of age, resulting in significant morbidity and mortality. This impacts the poultry industry globally, and is particularly severe in my country. S. pullorum primarily infects young chicks, primarily through horizontal and vertical transmission. At this age, the immune system of chicks is immature and unable to trigger a cellular immune response to prevent systemic infection. Pullorum has significant adverse effects on laying hens, breeder hens, and broiler chickens, leading to decreased egg production, increased mortality, decreased egg fertility, reduced hatchability, increased weak chicks, decreased brooding rate, increased mortality in broiler chickens, and decreased growth and uniformity. S. pullorum is highly resilient and can survive in chicken manure for up to 90 days. To mitigate the effects of pullorum, antibiotics are still used throughout my country. However, the use of antibiotics has proven to be a poor choice. Furthermore, due to the long-term and irrational use of antibiotics, bacterial resistance is becoming increasingly prevalent. Therefore, it is very necessary and urgent to find an effective method to prevent, control and treat Salmonella pullorum.

[0003] Bacteriophages are a type of virus that infects bacteria. They are often isolated from natural environmental samples such as sewage or feces. They have strict host specificity and only invade one or a few bacteria. Phages are not infectious to humans, animals, or plants, making them relatively safe. They also do not pollute the environment and are highly environmentally friendly. When phages come into contact with the corresponding host bacteria, they trigger the adsorption and intracellular proliferation process of phages, and release more phages through the lysis of the host bacteria. In this way, phages can achieve large-scale self-amplification by killing specific host bacteria in the environment. They have exponential growth characteristics, and the rate of drug resistance is much lower than that of antibiotics. Moreover, the bacteriophage's bacteriolytic effect is not limited by bacterial resistance. In addition, phage screening cycles are short, the preparation process is simple, and the cost is low, making it easy to produce on a commercial scale.

[0004] Pullorum disease is common in high-temperature and high-humidity areas in southern my country. The incubation period after pullorum disease infects chicks is very short, with symptoms usually appearing within 3 to 10 days. The peak mortality period is within 1 to 2 weeks. The temperature required for brooding chicks within 1 week is 30-35℃. Therefore, the bacteriophage used to purify pullorum disease in actual farms should have excellent high-temperature resistance.

[0005] Currently available Salmonella phages exhibit some heat resistance. For example, patent publication number CN108359644A, filed by the applicant in 2018, discloses a Salmonella phage strain, SP4, with a broad lytic spectrum and good efficacy against salmonellosis. Patent publication number CN111254121A, filed by the applicant in 2020, discloses another Salmonella phage strain, SPP11. However, these current Salmonella phages suffer from poor storage stability, making them unsuitable for long-term storage and exhibiting a short shelf life, significantly limiting their application.

[0006] Therefore, the existing technology needs to be further improved. Summary of the Invention

[0007] To address the above-mentioned problems, the present invention provides a new heat-resistant Salmonella pullorum phage and its application. The phage PC127 can be used to prepare drugs, environmental disinfectants, food preservatives, and detection kits for treating or preventing diseases caused by Salmonella pullorum infection. While resolving the problem of Salmonella pullorum infection, it avoids the problems of antibiotic residues and pathogen resistance caused by the use of antibiotics. Long-term use can also purify pullorum in farms and improve egg production rate and quality.

[0008] The technical solutions of the present invention are as follows:

[0009] In the first aspect, the present invention provides a strain of Salmonella pullorum phage with high temperature resistance isolated from chicken feather pollutants in a farm in Guangxi. The phage was named PC127 and was deposited in the General Microbiology Center of the China Culture Collection Administration on April 12, 2021. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 22366.

[0010] Bacteriophage PC127 has a polyhedral head structure and a contractile tail. The head is 80~88nm wide and 112~120nm long, and the tail is about 120~128nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the phage morphology of this application meets the characteristics of the Myotailed Phage family and belongs to the Myotailed Phage.

[0011] In this application, bacteriophage PC127 includes mutants that undergo point mutations, deletion mutations, or addition mutations with a homology exceeding 98% or 99% and that maintain substantially the same bactericidal activity. Because bacteriophages are highly susceptible to these mutations during replication, these mutants are also within the scope of protection claimed in this application. The sequence of bacteriophage PC127 can be sequenced using known methods based on the biological material deposited according to the present invention. Screening for mutants with extremely similar properties based on the bacteriophage provided by the present invention does not require creative effort for those skilled in the art.

[0012] In a second aspect, the present application further provides a phage composition comprising the aforementioned phage PC127. In practical applications, to further broaden the lysis spectrum of the phage preparation and fully utilize the differences in the lysis spectra of different phages, the aforementioned phage PC127 can be used in combination with other phages, such as SP4 (see patent publication number CN 108359644A) and SPP11 (see patent publication number CN 111254121A), both of which are Salmonella phages. This can broaden the bactericidal spectrum and eliminate as many Salmonella bacteria as possible in the environment, thereby preventing and treating salmonellosis.

[0013] Preferably, the phage composition comprises phage PC127, phage SP4, and phage SPP11.

[0014] In addition, the above-mentioned bacteriophage PC127 can also be combined with other different types of bacteriophages (inhibiting different pathogens that cause the same type of disease) for the prevention and treatment of the same type of disease.

[0015] In a third aspect, the present application also provides the use of the aforementioned bacteriophage PC127 or the aforementioned bacteriophage composition in the preparation of a drug, environmental disinfectant, and detection kit for preventing and treating Salmonella pullorum. The aforementioned prevention and treatment include both prophylaxis and treatment. The term "prevention" herein refers to all actions that inhibit or delay the disease by administering the composition. The term "treatment" herein refers to all actions that ameliorate or improve the disease by administering the composition.

[0016] In a fourth aspect, the present application further provides a phage pharmaceutical preparation, the active ingredient of which includes the aforementioned phage PC127 or phage composition; preferably, the phage pharmaceutical preparation also includes other antibacterial or bactericidal active ingredients; the pharmaceutical preparation is in the form of an oral dosage form, an external dosage form or a parenteral dosage form.

[0017] The application method of the phage drug preparation is: adding the phage or its composition as a therapeutic drug to the drinking water or feed of chickens, or administering it orally, subcutaneously, or intramuscularly to the chickens. The above methods can prevent and treat Salmonella pullorum disease, reduce the positive pullorum antibody, and improve egg production rate and survival rate.

[0018] Alternatively, the phage pharmaceutical preparation further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not cause significant irritation to an organism and does not eliminate the biological activity and characteristics of the active ingredient administered. In order to prepare the pharmaceutical composition as a liquid preparation, a pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, Ringer's solution, buffered physiological saline, albumin infusion, glucose solution, maltodextrin solution, glycerol, ethanol, various culture media, etc. They can be used alone or in any combination thereof. If necessary, other conventional additives such as antioxidants, buffers, and antibacterial agents can be added. When combined with a diluent, dispersant, surfactant, adhesive, and / or lubricant, the composition of the present invention can also be prepared into injection and oral dosage forms (e.g., aqueous solutions, suspensions, and emulsions, pills, capsules, granules), and other intermediate dosage forms such as lyophilized agents.

[0019] In a fifth aspect, the present application further provides a disinfectant, the active ingredient of which includes the bacteriophage PC127 or the aforementioned bacteriophage composition; preferably, the concentration of the bacteriophage is 10 8 PFU / ml or above.

[0020] Optionally, the disinfectant further comprises other active ingredients for inhibiting or eliminating bacteria in the chicken farm breeding environment; preferably, the environment to which the environmental disinfectant can be applied includes the breeding environment, which includes feed troughs, floors, walls, feces and bedding.

[0021] The disinfectant is used in a poultry farm environment to prevent contamination by pathogens in poultry slaughterhouses, poultry processing workshops and equipment, and poultry farming environments. The poultry farming environment includes sheds, feed troughs, floors, walls, feces, and bedding. The disinfectant can be used to disinfect and decontaminate water distribution systems, poultry facilities, feeding equipment, or other environmental surfaces, including but not limited to liquid immersion, spraying, or combined use with an aqueous carrier. This disinfectant can be used in place of antibiotics or traditional disinfectants, and is harmless to humans and poultry.

[0022] In a ninth aspect, the present application further provides a detection kit comprising the aforementioned bacteriophage PC127. Based on the lysis specificity of bacteriophage PC127 against host bacteria, the bacteriophage PC127 of the present invention can be used for the rapid detection of Salmonella pullorum, including but not limited to the detection of pathogens in clinical samples using test strips, test kits, etc., or the screening of target pathogens in clinical samples. The detection method is simple and highly sensitive.

[0023] The present invention has the following beneficial effects:

[0024] 1. The present invention provides a bacteriophage PC127 for the effective prevention and treatment of Salmonella pullorum, which has strong lytic activity and a broad lytic spectrum against Salmonella pullorum. This bacteriophage also exhibits excellent heat resistance, effectively preventing and treating pullorum, reducing chicken mortality and pullorum antibody positivity, and increasing egg production. This bacteriophage can be used as an active ingredient in environmental disinfectants and detection kits, effectively combating Salmonella infections and, with long-term application, purifying chicken farms from pullorum.

[0025] 2. The bacteriophages involved in the present invention are obtained from nature and are easy to industrialize. The drugs or disinfectants prepared from the bacteriophages can not only reduce costs but also have the advantages of being green and environmentally friendly.

[0026] 3. The phage has good long-term storage stability. Its potency is basically stable when stored at 37°C for half a year. Therefore, the phage has the advantage of a long-lasting effect. In addition, the phage has excellent reproduction performance and high burst volume, which has advantages in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a plaque image of bacteriophage PC127;

[0028] Figure 2 This is an electron microscope image of bacteriophage PC127;

[0029] Figure 3 The results of thermal stability test of bacteriophage PC127 are shown;

[0030] Figure 4 The pH stability test results of bacteriophage PC127;

[0031] Figure 5 This is the one-step growth curve of bacteriophage PC127. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in this field. The methods in the following embodiments, unless otherwise specified, are conventional methods in this field.

[0033] Example 1 Isolation and Screening of Salmonella Pullorum

[0034] 1. Experimental methods:

[0035] Isolation and Identification of Salmonella Pullorum: Forty liver tissue samples from chickens suspected of pullorum were collected in Guangxi, Guangdong, Jiangsu, and Anhui. The samples were aseptically treated and streaked onto SS agar to isolate the bacteria. The SS agar plates were incubated at 37°C for 16 hours, and the colonies were observed for growth. Representative single colonies were purified, examined by Gram staining, and identified by PCR using specific primers. Once confirmed as Salmonella pullorum isolates, they were stored for future use.

[0036] 2. Experimental results:

[0037] Fifteen strains of Salmonella pullorum were isolated and identified from 40 liver samples. Gram staining and PCR using specific primers showed that all the strains met the characteristics of Salmonella pullorum, proving that these 15 isolates were Salmonella pullorum with an isolation rate of 37.5%. These 15 isolates were numbered SPL1-SPL15.

[0038] Table 1 Isolation results of Salmonella pullorum

[0039]

[0040] Example 2 Isolation and Screening of Persistent Thermotolerant Salmonella Pullorum Phage

[0041] 1. Experimental methods

[0042] (1) Isolation of Salmonella pullorum phage

[0043] Appropriate amounts of chicken feathers and sewage samples were collected from farms in Guangxi, Guangdong, Anhui, and Jiangsu. The feathers and sewage samples were mixed, chopped, and immersed in a broth culture medium. The culture medium was then supplemented with the bacterial suspensions of the 15 clinically isolated Salmonella pullorum strains. The mixture was cultured in a constant temperature shaker at 37°C and 170 rpm for about 16 hours. The culture medium was filtered through a 0.22 μm sterile microporous filter membrane to obtain a phage proliferation liquid. The phage stock solution was diluted 10-fold, and appropriate gradient phage dilutions were mixed with 15 strains of Salmonella pullorum in a 1:1 ratio. After incubation at 37°C for 5 minutes, 200 μL of the mixture was pipetted and placed on the upper agar (agar concentration of 0.7%). After mixing, it was quickly poured onto the lower agar (agar concentration of 1.5%) plate, shaken and placed flat until the culture medium solidified. After incubation in a 37°C incubator for 4-6 hours, a double-layer plate with plaque formation was obtained.

[0044] Pick a single plaque from the double-layer agar medium where plaques have formed and place it in 1 mL of LB broth in a shaker at 37°C, 170 rpm, and incubate for approximately 30 minutes to obtain a phage extract. Mix the phage extract with the corresponding plaqued Salmonella pullorum (hereinafter referred to as the host strain) in a 1:1 ratio (incubate at 37°C for 5 minutes). Pipette 200 μL of the extract onto the top agar plate. Mix thoroughly and quickly pour the mixture onto the bottom agar plate. Shake the plate and place it flat until the medium solidifies. Incubate it upside down at 37°C for 4-6 hours. Once again, obtain a double-layer plate with plaques. Use sterile forceps to pick a single plaque from the double-layer medium where plaques have formed and place it in 1 mL of LB broth. Incubate it on a shaker at 37°C, 170 rpm, and incubate for approximately 30 minutes to obtain a phage extract. Repeat the above steps three times to obtain the purified phage extract. Take equal amounts of the purified phage extract and host bacterial proliferation liquid in 5 mL of liquid NB medium and culture at 37°C, 170 rpm, and shaking until the liquid becomes clear. Centrifuge the clear liquid at 11,000 rpm for 10 min, take the supernatant, and filter it using a 0.22 μm sterile microporous filter membrane to obtain the phage proliferation liquid, and measure the phage titer respectively.

[0045] (2) Screening of long-lasting Salmonella pullorum phage

[0046] The isolated Salmonella pullorum phages were adjusted to a uniform initial concentration (5.00×10 9 PFU / mL) and stored at 37°C for 6 months. Phage titers were then re-evaluated. A total of 30 Salmonella strains, including 15 clinically isolated Salmonella pullorum strains SPL1-SPL15 and 15 previously isolated, identified, and stored Salmonella strains SPL16-SPL30, were selected for lysis spectrum analysis. Phages with the highest titers and the best lysis spectrum were selected as the final screening phage strains. The results are shown in Table 2 below.

[0047] (3) The phages screened in the previous step were tested for storage stability at 37°C with other phages (e.g., Clostridium perfringens phage PMQ06 (see patent publication number CN111690620A), Escherichia coli phage BP7 (see patent publication number CN103289963B), and Staphylococcus phage SA1 (see patent publication number CN107779440B). The phages were adjusted to a uniform initial concentration (5.00×10 9 The phage titers were determined after 1 week, 1 month, 3 months, and 6 months of storage at 37°C. The results are shown in Table 3 below.

[0048] 2. Experimental results:

[0049] (1) Thirteen Salmonella pullorum phages were screened using the 15 aforementioned Salmonella pullorum isolates, numbered PC118 to PC130. These 13 Salmonella pullorum phages all formed clear plaques on double-layer agar plates, with no halo around them and clearly visible edges, with diameters of approximately 0.5 mm to 1.2 mm. The titers and lysis spectra of these 13 phages after 6 months of storage were measured and compared, and finally, the Salmonella pullorum phage PC127 with the best long-term storage stability at 37°C, high lysis rate, and the best overall performance was screened (see Table 2).

[0050] (2) As shown in Table 3, the Salmonella pullorum phage PC127 provided by the present invention has excellent long-term storage stability compared to existing phages. After being stored at 37°C for 6 months, its potency remains essentially unchanged, indicating that its shelf life is longer than 6 months. It can be inferred that the shelf life of this phage is even longer at 4°C. The above results indicate that the shelf life of this phage is much longer than that of other phages at room temperature. In practical applications, its long shelf life can greatly reduce its application cost.

[0051] Table 2 Titer and lysis rate of 13 Salmonella pullorum phages stored at 37℃ for 6 months

[0052]

[0053] Table 3 Titers of different phages stored at 37°C for different times (PFU / mL)

[0054]

[0055] Example 3 Morphological Observation and Identification of Salmonella Pullorum Phage PC127

[0056] 1. Experimental methods:

[0057] Take 20 μL of phage sample and drop it on a copper grid with a carbon coating. Allow it to precipitate naturally for 15 minutes. After blotting it with filter paper, stain it with 2% (W / V) phosphotungstic acid (PTA) for 1-2 minutes. After blotting it with filter paper, observe and photograph it under a transmission electron microscope.

[0058] 2. Experimental results:

[0059] like Figure 2 As shown, phage PC127 has a polyhedral head structure and a contractile tail. The head is 80~88nm wide and 112~120nm long, and the tail is about 120~128nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the phage morphology of the present application conforms to the characteristics of the Myotailed Phage family and belongs to the Myotailed Phage.

[0060] Example 4 Whole genome analysis of Salmonella pullorum phage PC127

[0061] The genome of bacteriophage PC127 was extracted and whole genome sequencing and sequence analysis were performed. The genome sequence obtained by sequencing is in NCBI GenBank No. ON754979.1. The sequence was analyzed:

[0062] (1) The genome of bacteriophage PC127 is 39169 bp in length, with a G+C content of 48.28% and an A+T content of 51.72%. The base contents of C, G, A, and T are 25.69%, 22.59%, 24.87%, and 26.85%, respectively. The results of online annotation of the whole genome by RAST showed that the genome contains 47 open reading frames (ORFs). Among these 47 ORFs, 26 structural proteins were found, mainly including phage structural and packaging proteins (capsid and scaffold proteins, tail tube proteins, tail fiber proteins, head-to-tail linker proteins, terminase large subunit and terminase small subunit, etc.), phage lysis-related proteins (endolysin, perforin), proteins related to DNA replication and modification (exonucleases, DNA polymerases, primases / helicases, endonucleases, DNA binding proteins, RNA polymerases, DNA linker proteins, endopeptidases, etc.), and other functional proteins (internal viral proteins, etc.). Among the 47 ORFs, 45 had ATG start codons, 1 had GTG start codons, and 1 had ATC start codons. Analysis using the tRNAscan-SE software revealed that the genome contained no tRNA genes. Analysis using the online CGE server tool revealed that the genome contained no drug resistance or virulence genes. Analysis using PHASTER revealed that the genome contained no lysogeny-related genes.

[0063] (2) In the genome of bacteriophage PC127: the gene sequence of the tail fiber protein related to phage host recognition is shown in sequence 1 in the sequence listing; the sequence of the highly conserved terminase large subunit protein gene is shown in sequence 2 in the sequence listing; the sequence of the DNA polymerase gene is shown in sequence 3 in the sequence listing; the sequence of the endolysin gene related to lytic ability is shown in sequence 4 in the sequence listing. For detailed information, please see Table 4 below.

[0064] Table 4 Gene sequence information

[0065]

[0066] Example 5 Determination of the Optimal Multiplicity of Infection (MOI) of Salmonella Phage PC127 Against Salmonella

[0067] 1. Experimental methods:

[0068] Pick a single Salmonella colony and inoculate it into 5 ml of NB broth medium. Incubate at 37°C and 170 rpm for 12-16 hours to obtain a bacterial solution. Determine the bacterial concentration by pouring the solution into the broth and adjust the concentration to 1×10 9 cfu / mL, 1×10 8 cfu / mL…1×10 5 cfu / mL. The concentration of phage isolated in Example 2 was adjusted to 1×10 5 ~1×10 7 PFU / mL. Add phage and bacterial solution to NB medium according to the phage to bacterial count ratio in Table 4. Proliferate at 37°C, 170 rpm, and shake until the liquid becomes clear. Record the proliferation time. Take an appropriate amount of the clear liquid and centrifuge at 11,000 rpm for 10 minutes. Remove the supernatant and filter it through a 0.22 μm sterile microporous filter. Determine the phage titer in the filtrate using the double-layer plate method. The MOI (number of phages / number of bacteria) with the highest phage titer is the optimal multiplicity of infection for that phage.

[0069] 2. Results

[0070] The results in Table 5 below show that when the MOI is 1:1000, the titer of PC127 reaches the highest, which is 1.02×10 11 PFU / mL, at this time the initial input of bacteriophage PC127 is small, and the highest reproduction yield can be achieved, which shows that the above properties of this phage are conducive to large-scale industrial production.

[0071] Table 5 The titer of Salmonella phage PC127 at different multiplicity of infection

[0072]

[0073] Example 6 Temperature Stability of Salmonella Pullorum Phage PC127

[0074] 1. Experimental methods:

[0075] The same volume of 1×10 10 Containers of PC127 phage proliferation fluid (100 pfu / mL) were placed at 40°C, 50°C, 60°C, 70°C, 80°C, and 90°C, with two replicates set up at each temperature. After incubation for 20, 40, and 60 minutes, samples were taken after the incubation period and immediately cooled in an ice bath. Each sample was then diluted 10-fold, and the titer was determined using an appropriate dilution gradient. A phage thermostability curve was plotted with temperature as the horizontal axis and the logarithm of the phage titer as the vertical axis.

[0076] 2. Experimental results and analysis:

[0077] The results are visible Figure 3 The activity of phage PC127 was still high after being placed at 40-60℃ for 60min, with little change from the initial titer. The titer of phage PC127 decreased by 4 orders of magnitude after being placed at 80℃ for 20min, and only decreased by 2 orders of magnitude after being placed at 70℃ for 60min. After being placed at 90℃ for 20min, the titer of phage PC127 was still 3.00×10 3 PFU / mL, so PC127 has strong heat resistance.

[0078] Example 7 pH Stability of Salmonella Pullorum Phage PC127

[0079] 1. Experimental methods:

[0080] Take three sterile test tubes and add 4.5 mL of NB broth with different pH values ​​(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Then place the test tubes in a 37°C water bath. After the temperature stabilizes, add 500 μL of 3×10 10 Pfu / mL of phage growth medium was mixed and incubated in a 37°C water bath for 1, 2, or 3 hours. Immediately after incubation, an appropriate amount of 1 mol / L HCl or NaOH was added to the mixture to adjust its pH to approximately 7. The mixture was diluted 10-fold and titered using an appropriate dilution gradient. Two replicates were performed for each pH value. A phage pH stability curve was plotted with pH as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0081] 2. Experimental results and analysis

[0082] from Figure 4 The results showed that the titer of phage PC127 was maintained at 10 in the range of pH 5.0-11.0. 9 pfu / mL, stable activity; at pH 3.0 for 1h, the titer is 10 7 pfu / mL, can resist certain strong acid environment; at pH 12, the titer is 10 for 3h. 7 pfu / mL, at pH 13 for 3h, the titer was 10 3 pfu / mL, therefore, bacteriophage PC127 has strong stability in a wide pH range and can resist a certain range of strong acid and strong alkaline environments.

[0083] Example 8 Determination of the One-Step Growth Curve of Salmonella Pullorum Phage PC127

[0084] 1. Experimental methods:

[0085] 1 mL of phage PC127 growth medium and 1 mL of fresh host bacterial growth medium at a multiplicity of infection of 10 were mixed thoroughly (timer started at this point), incubated at 37°C for 5 minutes, centrifuged at 13,000 rpm for 30 seconds, and the supernatant removed as much as possible with a micropipette. The pellet was then washed once with 5 mL of NB broth (centrifuged at 13,000 rpm for 30 seconds), and the supernatant discarded. The pellet was resuspended in preheated NB broth (total volume 5 mL) and mixed thoroughly. The pellet was immediately incubated in a shaker at 37°C at 170 rpm. At time 0 and every 5 minutes, 150 μL aliquots were removed and centrifuged at 10,000 rpm for 1 minute. The phage titer was determined by double-layer plating in triplicate, and the results were averaged. A one-step growth curve was constructed with infection time as the abscissa and phage titer in the infection system as the ordinate to determine the latent and burst periods of phage PD328 and to calculate the burst titer.

[0086] Burst volume = total number of phages at the end of the outbreak / total number of bacteria at the beginning of the outbreak

[0087] 2. Experimental results and analysis

[0088] from Figure 5 The results show that after phage PC127 infects the host bacteria, the phage lysis cycle is about 50 minutes, the incubation period is about 5 minutes, and the phage outbreak period is about 40 minutes; after 60 minutes, the phage number remains basically unchanged and enters a stable period, at which time the titer can reach 10 10 pfu / mL, and the burst size of bacteriophage PC127 was 126.

[0089] Example 9 Determination of the lysis spectrum of Salmonella pullorum phage PC127

[0090] 1. Experimental methods:

[0091] Fifteen clinical isolates of Salmonella pullorum (SPL1-15) from Example 1, 20 experimentally preserved animal-derived Salmonella strains of different serotypes (including Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum, Salmonella Typhisuis, and Salmonella Duck), and 10 strains of human-derived Salmonella (SF1-SF10) were selected. The lysis rates of PC127 against these 45 Salmonella strains were determined by a double-layer plate assay. At the same time, the lysis spectra of the above-mentioned Salmonella were determined using SP4 (chicken source) in the company's authorized patent CN108359644A and the bacteriophage SPP11 (chicken source) disclosed in another authorized patent CN111254121A of the company. The differences in the lysis of the above-mentioned 45 Salmonella strains by the three different Salmonella phages were compared.

[0092] 2. Experimental results and analysis

[0093] (1) From the lysis spectrum experimental results in Table 6 below, it can be seen that the phage PC127 of Salmonella pullorum can lyse 37 of the 45 Salmonella strains, with a lysis rate of up to 82.22%. Among them, its lysis rate for 15 of the 45 Salmonella strains is 100%, while its lysis rate for human Salmonella is 60%. This shows that phage PC127 has a specific lysis advantage against Salmonella pullorum.

[0094] Bacteriophage SP4 was only able to lyse 31 of the 45 Salmonella strains, with a lysis rate of 68.89%; Salmonella SPP11 was able to lyse 29 of the 45 Salmonella strains, with a lysis rate of 64.44%; obviously, the lysis rate of bacteriophage PC127 on the following 45 Salmonella strains was much higher than that of bacteriophage SP4 and SPP11.

[0095] (2) The results in Table 6 below also show that the lysis spectra of phages PC127, SP4, and SPP11 are complementary. The lysis rate of the three phages combined against the above 45 Salmonella strains can reach 100%. This shows that the phage combination composed of these three phages has a great advantage in the width of the lysis spectrum, which has guiding significance for the application of phage products.

[0096] Table 6 Lysis spectrum of Salmonella pullorum phage PC127 against 45 strains of Salmonella

[0097]

[0098]

[0099] Example 10 Lysis test of Salmonella pullorum phage PC127 on non-host bacteria

[0100] 1. Experimental methods

[0101] A total of 25 different types of non-host bacteria, including 10 strains of Escherichia coli, 5 strains of Staphylococcus, 5 strains of Proteus and 5 strains of Clostridium perfringens, were selected, and the lysis spectrum of Salmonella pullorum phage PC127 was determined according to the above-mentioned lysis spectrum determination method.

[0102] 2. Experimental results and analysis

[0103] The results showed that no clear plaques were found in the double-layer plates of Salmonella pullorum phage PC127 and 25 strains of non-host bacteria, indicating that phage PC127 was unable to recognize the above-mentioned 10 strains of Escherichia coli, 5 strains of Staphylococcus aureus, 5 strains of Proteus and 5 strains of Clostridium perfringens. This shows that the test phage has extremely strong host specificity and has no destructive effect on the microbial community, and can be used to prepare detection kits.

[0104] Example 11 Environmental Disinfection Test of Salmonella Pullorum Phage PC127

[0105] 1. Experimental methods

[0106] A large number of Salmonella were detected in a chicken hatchery, and chemical disinfectants were ineffective in killing bacteria. This experiment was designed to use bacteriophage PC127 to spray disinfect the hatchery.

[0107] The preparation method of the bacteriophage disinfectant is as follows: take the bacteriophage liquid and host bacteria in NB broth medium in a ratio of 1:1, incubate at 37°C, 170 rpm for 4-6 hours, until the liquid is clear and bacterial fragments are visible. The clear liquid is filtered to remove bacterial fragments and the phage titer is determined to be 1×10 10 pfu / ml, the phage liquid was diluted 100 times to prepare a titer of 1×10 8 pfu / mL disinfectant, seal and store at 2~8℃ for future use.

[0108] (1) Air disinfection method:

[0109] The air in the hatchery was sampled using the natural sedimentation method. Five test points were selected, including the center and four corners. The sampling points were 0.5 m from the ground, and the four corners were 1 m from the wall. A 9 cm diameter XLD agar plate was placed at each point. Before disinfection, two XLD agar plates were used at each sampling point. The petri dish lid was opened and the sample was collected for 10 minutes. The phage preparation was disinfected using the farm's own mist line (10 mL / m 3 , phage titer 1×10 8The samples were disinfected with 100 μg / mL of XLD agar (0.1 μg / mL) and benzalkonium bromide (1:25 dilution of benzalkonium bromide). After 30 minutes of disinfection, two XLD agar plates were placed at each of the five sampling points. The Petri dishes were opened and sampled for 10 minutes. The pre- and post-disinfection sampling plates were placed in a 37°C incubator for 12–24 hours, and the cultured bacteria were counted.

[0110] According to the Ostwald formula, the total number of colonies is C = 50000N / AT, where C is the total number of colonies per cubic meter (CFU / m 3 ); N: number of colonies per dish; A: area of ​​culture dish (cm 2 ); T: sampling time (min).

[0111] (2) Eggshell surface disinfection method

[0112] Sampling before disinfection: Pipette 0.5 mL of sterile PBS into a sterile centrifuge tube, dip a sterile cotton ball in PBS and smear the sampling surface on the eggshell. After repeated smearing, place it in a sterile centrifuge tube and store at 4°C. Randomly collect 10 eggs.

[0113] After the egg fumigation room was disinfected with bacteriophage spray for 30 minutes, post-disinfection sampling was performed at 10 randomly selected locations using the pre-disinfection sampling method. Salmonella counts were determined according to GB 4789.2-2016, National Food Safety Standard for Food Microbiology, and the elimination rate was calculated.

[0114] Elimination rate = (average bacterial count in samples before disinfection - average bacterial count in samples after disinfection) / average bacterial count in samples before disinfection × 100%

[0115] 2. Experimental results and analysis

[0116] As shown in Table 7, after disinfection with bacteriophage PC127, the Salmonella elimination rate in the hatchery air and on the eggshell surface was between 68% and 80%. The disinfection effect on the hatchery air was particularly good, with a Salmonella elimination rate of 80.77%. This indicates that bacteriophage PC127 has a significant effect in disinfecting Salmonella and can be promoted and applied as a new biological environmental disinfectant.

[0117] Table 7 Salmonella colony counts before and after bacteriophage PC127 disinfection

[0118]

[0119] Example 12 Experiment on the Elimination of Salmonella by Salmonella Pullorum Phage PC127

[0120] 1. Experimental method: 40 3-day-old SPF chickens were divided into two groups, the phage group and the control group. The chickens in the phage group were orally administered with 1×10 PC127 phage. 9PFU / feather, and the control group was orally administered with the same amount of sterile saline. Both the phage group and the control group were orally administered with 1×10 7 Seven days after infection, feces were collected for the determination of Salmonella by fluorescence quantitative method, and liver lesions were observed and Salmonella in the liver was determined by autopsy.

[0121] 2. Experimental results and analysis

[0122] Seven days after challenge with Salmonella pullorum using bacteriophage PC127, the mortality rate in the control group was 45%, while there were no deaths in the phage-treated group. The detection rate of Salmonella in feces was 80% lower in the phage-treated group than in the control group. The rate of liver lesions was 75% lower in the phage-treated group than in the control group. The detection rate of Salmonella in the liver was 80% lower in the phage-treated group than in the control group. These data demonstrate that the use of bacteriophage PC127 against pullorum infection significantly reduces mortality and effectively eliminates Salmonella.

[0123] Table 8 Results of the bacteriophage PC127 clearance experiment on Salmonella

[0124]

[0125] Example 13: Experimental Study on Purification of Pullorum in Chicken Farms by Bacteriophage PC127

[0126] 1. Experimental method: A laying hen farm in the south with a high positive rate of pullorum antibody was selected, and two 7,000-bird houses were selected as the experimental and control groups. Treatment of the experimental group: bacteriophage PC127 was made into a spray-dried powder, 1×10 9 PFU / g. Bacteriophage PC127 powder was added to the feed at a rate of 500g / ton from the brooding period until 250 days of age. A control group was treated without phage supplementation. Other immunization and medication procedures were performed according to farm procedures. Layers at 70 and 200 days of age were screened for pullorum antibody positivity using the whole blood plate agglutination test specified in the Chinese agricultural industry standard "Diagnostic Techniques for Chicken Typhoid and Pullorum (NY / T 536-2002)."

[0127] 2. Experimental results and analysis

[0128] The long-term addition of bacteriophage PC127 to the feed significantly reduced the positive rate of Salmonella pullorum antibodies in the control and experimental groups at 70 and 200 days of age. The phage-treated group showed a decrease in the positive rate from 2% at 70 days to 0.05% at 200 days, while the control group showed a positive rate of 8.05% to 10%. Long-term addition of bacteriophage PC127 to the feed significantly reduced the positive rate of Salmonella pullorum antibodies, demonstrating the effectiveness of long-term pullorum purification.

[0129] Table 9 Results of positive test for Salmonella pullorum antibodies

[0130]

[0131] Example 14 Safety Test of Bacteriophage

[0132] 1. Experimental methods:

[0133] Forty one-day-old SPF chickens were selected and divided into a phage group and a control group. The phage group was orally administered with 1×10 PC127. 10 The mice were fed 1 mL of PFU of phage and observed for 7 days. A control group received an equivalent dose of sterile saline orally. Necropsies were performed to observe lesions in the heart, liver, spleen, lungs, kidneys, brain, and intestines. During the feeding period, their mental state and feeding habits were monitored.

[0134] 2. Experimental results and analysis

[0135] Throughout the dosing period, no symptoms of illness or toxicity were observed in either the phage or control groups, and their mental state and food intake remained normal. Detailed clinical autopsies revealed normal function of the animals' major organs and intestines in both the phage and control groups. This demonstrates that phage PC127 is highly safe and has no adverse effects on the animals.

[0136] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solutions and concepts of the present invention, and all these changes or substitutions should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A thermostable Salmonella pullorum phage, characterized in that: It was named PC127 and its deposit number is CGMCC No.22366.

2. A phage composition comprising the heat-resistant Salmonella pullorum phage according to claim 1, characterized in that: Also included are one or both of Salmonella phage SP4 and Salmonella phage SPP11.

3. Use of the heat-resistant Salmonella pullorum phage according to claim 1 or the phage composition according to claim 2 in the preparation of drugs, environmental disinfectants and detection kits for preventing and treating Salmonella pullorum disease.

4. A phage pharmaceutical preparation, the active ingredient of which comprises the heat-resistant Salmonella pullorum phage according to claim 1 or the phage composition according to claim 2.

5. The bacteriophage pharmaceutical preparation according to claim 4, characterized in that The bacteriophage pharmaceutical preparation also includes other bacteriostatic or bactericidal active ingredients.

6. The bacteriophage pharmaceutical preparation according to claim 4, characterized in that The pharmaceutical preparation is in the form of an oral dosage form, an external dosage form or a parenteral dosage form.

7. An environmental disinfectant, characterized in that The active ingredient comprises the heat-resistant Salmonella pullorum phage according to claim 1 or the phage composition according to claim 2.

8. The environmental disinfectant according to claim 7, characterized in that It also contains other active ingredients designed to inhibit or eliminate bacteria in the environment.

9. Use of the environmental disinfectant according to claim 7 in the disinfection of a chicken farm environment, characterized in that: The environmental disinfectant can be used to disinfect the breeding environment or breeding equipment from salmonella by spraying or soaking, and the breeding environment includes feed troughs, floors, walls, feces and bedding.

10. A detection kit, characterized in that The method comprises the heat-resistant Salmonella pullorum phage according to claim 1 or the phage composition according to claim 2.

Images