Bordetella phage with good acid tolerance as well as composition, kit and application of Bordetella phage

Abstract

The invention discloses a bordetella bacteriophage with good acid tolerance as well as a composition, a kit and application of the bordetella bacteriophage, and belongs to the technical field of bacteriophages. The bacteriophage is bordetella bacteriophage BOP2ABONT11, the preservation number of the bacteriophage BOP2ABONT11 is GDMCC NO.67044-B1, and the bacteriophage BOP2ABONT11 has a nucleotide sequence as shown in SEQ ID No.1. The bacteriophage has a polyhedral stereosymmetric head and a short contracted tail, is strong in lysis capability, can identify 92% of bordetella, and has titer of 4.2 * 10 PFU / mL after being cultured for 8 hours under the condition that MOI is 0.001; the acid resistance and heat resistance are excellent, and the catalyst still has activity after being treated for 96 hours at pH 3 and is stable at 55-65 DEG C; the strain does not contain virulence genes, is high in safety and has no splitting action on non-host bacteria. The invention also provides a composition containing the bacteriophage, and a biological bactericide, a medicine, a feed additive and a kit prepared from the bacteriophage or the composition. The bacteriophage and related products can efficiently prevent and treat Bordetella infection, are applied to the fields of environment disinfection, food preservation, animal breeding, pathogenic bacterium detection and the like, and have wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of phage technology, specifically relating to a Bordetella phage with high lytic activity, excellent physicochemical stability, and especially good acid tolerance, as well as compositions containing the phage, kits, and their applications in fields such as biological sterilization, disease prevention and control, feed additives, and bacterial detection. Background Technology

[0002] In the livestock farming sector, infections caused by Bordetella bronchiseptica, a pathogenic bacterium, have become a key bottleneck restricting the healthy development of the industry. They not only cause huge economic losses, but also directly threaten human public health and safety through the food chain and other pathways.

[0003] For a long time, antibiotics have been the main means of preventing and controlling such bacterial infections. However, their irrational use and abuse have led to increasingly serious problems of antibiotic resistance in pathogens, creating a difficult dilemma to overcome. At the same time, other antibacterial agents currently used for the prevention and control of Bordetella infections not only have problems such as poor antibacterial efficacy and long treatment cycles, but also often involve hidden dangers such as interference with the normal intestinal microbiota balance of livestock and poultry, drug residues, and insufficient safety, failing to meet the technical requirements of modern farming for "green prevention and control".

[0004] As a type of virus that specifically lyses bacteria, bacteriophages are considered ideal candidates to replace antibiotics and other chemical agents for the prevention and treatment of bacterial infections due to their unique advantages such as high self-replication efficiency, strong host specificity (targeting only pathogenic bacteria), non-interference with the host's normal flora, and low likelihood of inducing drug resistance. They have shown great application potential in the field of animal husbandry.

[0005] Currently, some bacteriophages and related compositions targeting Bordetella have been published, but these existing bacteriophages generally have significant technical shortcomings: on the one hand, their lysis spectrum is narrow and their lysis efficiency is limited, making it difficult to cope with mixed infections in complex aquaculture environments; on the other hand, they lack tolerance to high temperatures above 50°C, failing to meet the temperature requirements of feed processing, transportation, and storage, and thus failing to meet the diverse application scenarios in actual production. Research on specific bacteriophages targeting Bordetella, a common pathogen in livestock farming, is still in its early stages, and related technological reserves are severely insufficient.

[0006] Therefore, developing a Bordetella phage with strong lysis ability, excellent environmental tolerance (especially acid and high temperature resistance), and high biosafety, as well as related products, while optimizing the performance of Bordetella phage, has become an urgent need for breakthroughs in pathogen control technology in the livestock breeding field. Summary of the Invention

[0007] In view of the shortcomings of existing technologies, such as limited phage lysis ability, insufficient acid and high temperature resistance, and lack of highly efficient and specific phages against Bordetella bronchiseptica, this invention provides a Bordetella bronchiseptica phage with good acid resistance, its composition, kit, and application. This phage has strong lysis ability, excellent acid and heat resistance, and high safety, which can effectively meet the application needs of Bordetella bronchiseptica infection prevention and control and related fields.

[0008] 1. Bordetella phage The Bordetella phage provided by this invention is Bordetella phage BOP2ABONT11, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 28, 2025, with the accession number GDMCC NO. 67044-B1, and has the nucleotide sequence shown in SEQ ID No. 1.

[0009] This bacteriophage possesses a polyhedral, three-dimensionally symmetrical head and a short, contracted tail. The head diameter is 62–72 nm, the tail length is 11–15 nm, and the tail diameter is 6–8 nm. When cultured for 8 hours at an MOI of 0.001, the titer can reach 4.2 × 10¹. 0 PFU / mL; good stability in the pH range of 4 to 10, and still active after treatment at pH 3 for 96 h; excellent stability at 55 to 65 °C, and good tolerance to ultraviolet light; can recognize 92% of Bordetella bronchiseptica, has a wide host range, and has no lytic effect on non-host pathogenic bacteria or non-pathogenic beneficial bacteria, and has high specificity.

[0010] 2. Phage composition The phage composition comprises at least the above-mentioned Bordetella phage BOP2ABONT11, and may further comprise one or more of the following: Klebsiella pneumoniae phage P6AKPJS20 (accession number CCTCC NO: M 20242965), Bordetella phage BOP3ABONT19 (accession number GDMCC NO. 67045-B1), or dodecyl dimethyl benzyl ammonium chloride at a final mass concentration of 0.1% (w / v).

[0011] 3. Biological bactericides The biocidal agent is suitable for lysing Bordetella lysate. It is prepared using the above-mentioned Bordetella phage or phage composition. The dosage form can be selected as solution, powder, gel, granule or lyophilized agent. It can be used in environmental disinfection, pollution prevention and control in food production workshops and other scenarios.

[0012] 4. Medications The drug is suitable for treating or preventing diseases caused by Bordetella infection. It is prepared using the aforementioned Bordetella phage, which can specifically kill Bordetella in animals without side effects.

[0013] 5. Feed additives The feed additive was prepared using the aforementioned Bordetella phage, which can prevent Bordetella contamination during feed production, storage, and animal husbandry, inhibit Bordetella infection in animals, and enhance animal resistance.

[0014] 6. Reagent kit The kit contains the aforementioned Bordetella phage BOP2ABONT11, and is preferably a biosensor kit using Bordetella phage BOP2ABONT11 as a biorecognition element. It can be used for the rapid detection of Bordetella, including the detection of Bordetella in target samples and the screening of target pathogens in clinical samples. Beneficial effects

[0015] 1. The Bordetella phage of the present invention has strong lytic ability, recognizing and lysing 92% of Bordetella phages, and has high proliferation efficiency; under MOI=0.001 conditions, the titer can reach 4.2×10¹ after 8 hours of culture. 0 PFU / mL provides a high-quality source of strains for industrial production.

[0016] 2. This bacteriophage exhibits excellent environmental tolerance, being acid-resistant (still active after 96 hours of treatment at pH 3), heat-resistant (stable at 55–65°C), and UV-resistant (without order-of-magnitude decrease in titer after 1 hour of irradiation), making it suitable for a variety of complex application scenarios.

[0017] 3. Bacteriophages are safe and have no side effects. They do not contain virulence genes or harmful genes, have not undergone genetic modification, and toxicological experiments have shown that they have no adverse effects on animals such as mice, chicks, and piglets, and do not damage the normal microbial flora.

[0018] 4. Bacteriophages are highly specific, acting only against Bordetella bronchiseptica, and have no lytic effect on non-host pathogens or beneficial bacteria, thus not disrupting the balance of environmental microorganisms.

[0019] 5. It has diverse applications. It can be used alone or in combination with other bacteriophages and chemical bactericides to prepare compositions. It can also be made into various products such as biological bactericides, drugs, feed additives, and reagent kits. It has broad application prospects in the fields of Bordetella infection prevention and control, environmental disinfection, food preservation, and rapid detection. Attached Figure Description

[0020] Figure 1: Phage plaque morphology of Bordetella bronchiseptica phage BOP2ABONT11; Figure 2: Schematic diagram of the morphological structure of Bordetella phage BOP2ABONT11 under a transmission electron microscope; Figure 3: Distribution of unique endonuclease cleavage sites in the genome of Bordetella phage BOP2ABONT11; Figure 4: Phylogenetic tree analysis of the genome of Bordetella bronchiseptica phage BOP2ABONT11; Figure 5: Stability of Bordetella bronchiseptica phage BOP2ABONT11 at different temperatures; Figure 6: Stability of Bordetella phage BOP2ABONT11 under different pH conditions; Figure 7: Stability of Bordetella phage BOP2ABONT11 before and after preparation under different powder preparation conditions; Figure 8: The lytic ability of Bordetella bronchiseptica phage BOP2ABONT11 against Bordetella bronchiseptica; Figure 9: Lysis results of Bordetella bronchiseptica phage BOP2ABONT11 on non-host pathogenic bacteria; Figure 10: Lysis results of Bordetella bronchiseptica phage BOP2ABONT11 on non-pathogenic beneficial bacteria; Figure 11: Stability of Bordetella phage BOP2ABONT11 under UV conditions; Figure 12: Results of using Bordetella phage BOP2ABONT11 and its composition to prevent Bordetella contamination during pork sample preservation. Detailed Implementation

[0021] To make this application easier to understand, the following detailed description will be provided with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of this application. Unless otherwise specified, the raw materials or components used in this application can be obtained commercially or by conventional methods.

[0022] In the following examples, the strain codes used are all based on our company's naming convention.

[0023] The culture medium formulation used in the following examples is as follows: The TSB liquid culture medium formula is: 15g tryptone, 5g soybean peptone, 5g sodium chloride, 1000mL distilled water, autoclaved at 121℃ for 20min. The TSA solid culture medium formula is: 15g tryptone, 5g soybean peptone, 5g sodium chloride, 15g agar, 1000mL distilled water, autoclaved at 121℃ for 20min. The TSB semi-solid agar medium formula is: 15g tryptone, 5g soybean peptone, 5g sodium chloride, 7g agar, 1000mL distilled water, autoclaved at 121℃ for 20min. The SM solution formula is: 5.8g sodium chloride, 2g magnesium sulfate, 50mL 1mol / L Tris-HCl, 0.25g gelatin, 1000mL distilled water, autoclaved at 121℃ for 20min. Example 1: Isolation, preparation, purification, and culture of Bordetella bronchiseptica bacteriophage Phage isolation: An appropriate amount of wastewater collected from a livestock farm in Nantong City, Jiangsu Province, was centrifuged at low speed at room temperature and filtered through a 0.22 μm filter membrane. 10 mL of the filtered supernatant was added to an equal volume of TSB liquid medium (2 times the volume), along with 1 mL of the phage host bacterium – *Bordetella bronchiseptica* ABONT11 in logarithmic phase. The mixture was incubated at 37°C and 150 rpm for 8 h to enrich the phage and obtain a culture medium. The culture medium was centrifuged at 8000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane for sterilization. 100 μL of the filtrate was serially diluted 10-fold with SM buffer. 1 mL of each appropriate gradient dilution was mixed thoroughly with 200 μL of the host bacterium in logarithmic phase and allowed to stand for 15 min. This mixture was then added to 5 mL of 48°C semi-solid TSB medium and mixed well. The mixture was poured onto TSA plates to prepare a double-layer plate and incubated at 37°C for 8 h to obtain a double-layer plate with phage plaques.

[0024] Phage purification: A single clear phage plaque was picked from a double-layer plate and transferred to 1 mL of SM buffer. The plate was vortexed for 1 min, centrifuged at 6000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter for sterilization. The filtrate was serially diluted 10-fold. 1 mL of each appropriate serial dilution was added to 200 μL of logarithmic-phase host bacterial culture, mixed thoroughly, and allowed to stand for 15 min. Then, 5 mL of 48℃ semi-solid TSB medium was added, and the mixture was immediately poured onto TSA plates. After solidification, the plates were incubated at 37℃ for 8 h. This purification process was repeated at least 3-5 times to obtain uniformly shaped and sized single phage plaques on the double-layer plates. Figure 1 This is the purified phage, which is named Bordetella phage BOP2ABONT11.

[0025] A single plaque of phage BOP2ABONT11 was picked and added to 1 mL of SM buffer, vortexed for 1 min, centrifuged at 6000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter for sterilization. 200 μL of the filtrate was added to 20 mL of TSB medium along with an equal volume of logarithmic-phase host bacteria and incubated at 37 °C for 8 h. The culture was then centrifuged at 8000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter to obtain the pure culture medium of *Bordetella phage* BOP2ABONT11.

[0026] The morphology of Bordetella phage under a transmission electron microscope is as follows: Figure 2 As shown, the bacteriophage has a polyhedral, three-dimensionally symmetrical head and a short, contracted tail. The head diameter is 62–72 nm, the tail length is 11–15 nm, and the tail diameter is 6–8 nm. The bacteriophage shown in the figure has a head diameter of 60 nm, a tail length of 13 nm, and a tail diameter of 7 nm.

[0027] The accession number for Bordetella phage BOP2ABONT11 is GDMCC NO. 67044-B1.

[0028] Example 2: Preparation of Bordetella phage particles, extraction, sequencing and whole genome analysis 1. Preparation of Bordetella phage particles and extraction and sequencing of genome. Phage nucleic acid was extracted and sequenced using a phage DNA extraction kit (Norgen Biotek). Nucleotide sequencing revealed that the *Bordetella phage* BOP2ABONT11 possessed the nucleotide sequence shown in SEQ ID No. 1.

[0029] 2. Genome sequence analysis of Bordetella phage Sequencing of the *Bordetella bronchiseptica* phage BOP2ABONT11 revealed that the phage genome is a linear double-stranded DNA with a total length of 44658 bp and a G+C content of 61.1%.

[0030] 3. Biological characteristics analysis of genomic restriction enzyme sites in Bordetella phage The unique endonuclease species and sites of the entire genome of *Bordetella bortezii* phage BOP2ABONT11 were analyzed using SnapGene software. The distribution of unique endonuclease cleavage sites in this genome is as follows: Figure 3 As shown.

[0031] 4. Genetic evolutionary analysis of Bordetella phage Phylogenetic tree analysis was performed using the genome of bacteriophage BOP2ABONT11 in NCBI. Figure 4 The results showed that the closest genetic distance to phage BOP2ABONT11 was Bordetella phage vB_BbrM_PBb002 (PV870137.1).

[0032] Based on the unique genomic information of the bacteriophage BOP2ABONT11 and its morphological characteristics under transmission electron microscopy, and according to the classification criteria established by the International Committee on Taxonomy of Viruses (ICTV), the bacteriophage BOP2ABONT11 is a strain of Bordetella phage, belonging to the class Caudoviricetes. Furthermore, this bacteriophage is highly likely to be a novel bacteriophage.

[0033] Example 3: Determination of the optimal multiple of infection (MOI) of Bordetella bronchiseptica phage against Bordetella bronchiseptica 1. Determination of Bordetella phage titer Using SM solution as a diluent, the pure culture medium of Bordetella phage BOP2ABONT11 (prepared from Example 1) was serially diluted 10-fold to 10-fold. 8 Times. Take l0 respectively. 5 l0 6 l0 7 and l0 8 1 mL of phage culture at each dilution was thoroughly mixed with 200 μL of host bacterial culture. After standing at room temperature for 15 min, the mixture was plated using the double-layer agar method. Once the agar solidified, the plates were incubated upside down at 37°C for 8 h. Three replicates were prepared for each dilution, and the average number of plaques from the three replicates at that dilution was used for counting. Phage titer (PFU / mL) = average number of plaques × dilution factor.

[0034] 2. Determination of the MOI of Bordetella phage A single colony of *Bordetella bronchiseptica* was picked and inoculated into a test tube containing 3 mL of TSB liquid medium. The culture was incubated at 37°C for 4 h to obtain a host bacterial suspension. The bacterial suspension was transferred to 10 mL of TSB medium at a ratio of 1:100 and incubated at 37°C until the pre-log phase. Pure culture medium of *Bordetella bronchiseptica* phage (prepared in Example 1) and host bacteria (MOI = phage count / bacterial count) were added according to the multiplicity of infection ratios in Table 1. TSB liquid medium was added to ensure the total volume of each tube was the same, and the tubes were incubated at 37°C for 8 h. After incubation, the tubes were centrifuged at 10000g for 10 min. The supernatant was filtered through a 0.22 μm filter and the titer of the filtrate was determined using the double-layer plate method. Double replicates were performed at each point, and the average value was taken. The MOI that produced the highest phage titer was considered the optimal multiplicity of infection. The experiment was repeated three times.

[0035] Table 1. Titer of Bordetella phage at different multiplicity of infection MOI Bordetella phage (PFU / mL) Host bacteria (cfu / mL) Bordetella phage titer (PFU / mL) 1000:1 <![CDATA[10 8 ]]> <![CDATA[10 5 ]]> <![CDATA[1.26x10 8 ]]> 100:1 <![CDATA[10 7 ]]> <![CDATA[10 5 ]]> <![CDATA[4.5x10 8 ]]> 10:1 <![CDATA[10 6 ]]> <![CDATA[10 5 ]]> <![CDATA[6.4x10 8 ]]> 1:1 <![CDATA[10 5 ]]> <![CDATA[10 5 ]]> <![CDATA[2x10 9 ]]> 1:10 <![CDATA[10 4 ]]> <![CDATA[10 5 ]]> <![CDATA[3.6x10 9 ]]> 1:100 <![CDATA[10 3 ]]> <![CDATA[10 5 ]]> <![CDATA[1x10 10 ]]> 1:1000 <![CDATA[10 2 ]]> <![CDATA[10 5 ]]> <![CDATA[4.2x10 10 ]]> Under the condition of a multiplicity of infection of 0.001, the titer of progeny phages produced by *Bordetella bronchiseptica* phage BOP2ABONT11 infecting the host bacteriology reached its highest value of 4.2 x 10⁻⁶. 10 PFU / mL.

[0036] Example 4: Detection test for deletion of virulence genes or undesirable genes in Bordetella phage This invention selects 103 virulence genes (Table 2) that have been identified as originating from lysogenic bacteriophages in pathogenic bacteria. The whole genome of Bordetella phage BOP2ABONT11 is measured and subjected to bioinformatics analysis to determine whether it contains the above-mentioned virulence genes.

[0037] The results showed that the bacteriophage tested in this invention does not contain the following virulence genes or harmful genes, and therefore cannot encode proteins that may cause potential health risks. Therefore, the Bordetella phage BOP2ABONT11 will not affect the health of humans or animals.

[0038] Table 2. Major known virulence genes of lysogenic bacteriophages in pathogenic bacteria. Gene source Toxic genes Staphylococcus aureus Alpha toxin (alpha lysin), Beta-lysin (beta toxin); Gammalysin (Gamma toxin); Enterotoxins (SEA, SEB, SEC, SED, SEE).Pyrogenic exotoxins AB; beta-toxin; gamma-hemolysin component B; gamma-hemolysin chain IIprecursortoxic shock syndrome toxin-1 Staphylococcus aureus RF122 leukotoxin D subunit; leukotoxin E subunit Staphylococcus aureus subsp.Aureus MW2 alpha-hemolysin Clostridium botulism Neurotoxins (A to G); lysin; C2 toxin Escherichia coli O157:H7 Shiga-like toxin 1 and 2 (SLT-1 and 2 or Stx-1 and 2) Escherichia coli type III cytolethal distending toxin protein CdtA; cytolethal distending toxin type IV subunit A; hemolysin A Escherichia coli O111:H- str.11128 Shiga toxin 2 subunit A; Shiga toxin 2 subunit B Escherichia coli APEC O1 cytolethal distending toxin type IV subunit C Pseudomonas aeruginosa Exotoxin A; Proteases, Leukocidin;exotoxin A precursor;alkaline protease Bacillus pertussis Pertussis toxine (AB5-type Exotoxin);bifunctional hemolysin-adenylate cyclase precursor Bacillus Anthrax Toxin (Edema factor and Lethal factor) Helicobacter Pylori VacA toxin Cyanobacteria Hepatotoxin and neurotoxin Corynebacterium diphteriae Diphtheria toxin Clostridium tetani Tetanus toxin;Tetanus toxin tetX;Tetanolysin O Proteus mirabilis Neurotoxin Bacillus Cereus Cereolysin; Enterotoxin;Alveolysin Shigella dysenteriae Shigella toxin (Shiga toxin);Shiga toxin subunit A Clostridium difficile Toxin A;Toxin B;Toxin B – cytotoxin Legionella pneumophila Cytolysin Vibrio Cholera Cholera toxin Vibrio cholerae O395 zona occludens toxin Vibrio cholerae RC385 conserved hypothetical protein – toxin Streptococcus pyogens Streptolysin S; Erythrogenic toxin Corynephage omega diphtheria toxin Corynephage beta diphtheria toxin Corynebacterium ulcerans diphtheria toxin Pseudomonas phage phiCTX Cytotoxin Shigella dysenteriae Sd197 Shiga toxin subunit B precursor Clostridium botulinum C2 toxin, component I; C2 toxin, component II;BoNT / G; BoNT / F Clostridium botulinum B1 botulinum neurotoxin type B Clostridium phage c-st botulinum neurotoxin type C1 precursor Clostridium botulinum E3 botulinum neurotoxin type E3, BoNT / E3 Clostridium botulinum F botulinum neurotoxin type A1 Clostridium phage d-16 phi type D neurotoxin Clostridium botulinum D str.1873 tetanolysin O Proteus mirabilis HI4320 Toxin Proteus mirabilis ATCC 29906 Toxin; toxin Staphylococcus phage PVL LukF-PV Yersinia pestis CA88-4125 Toxin Brevibacillus laterosporus Isp2b protein weihenstephanensis Delta endotoxin central region subgroup 1 Bacillus thuringiensis Pesticidal crystal protein cry1Bc Clostridium novyi NT phospholipase C precursor (PLC) Clostridium novyi alpha-toxin Clostridium perfringens ATCC13124 perfringolysin O Clostridium perfringens SM101 phospholipase C Clostridium perfringens str. 13 Collagenase Clostridium perfringens Enterotoxin; delta toxin Clostridium perfringens B str.ATCC 3626 beta-toxinepsilon-toxin Clostridium septicum alpha-toxin Clostridium sordellii phospholipase C Listeria monocytogenesClip81459 listeriolysin O precursor Streptococcus pneumoniae TIGR4 Pneumolysin Bacillus anthracis hypothetical protein pxo1_122 edema factor Bacillus anthracis str. A2012 lethal factor Bordetella pertussis Tohama I Putative toxin; toxin subunit 2; toxin subunit 3 Prophage CP-933V shiga-like toxin 1 subunit A encoded within prophage CP-933V; shiga-like toxin 1 subunit B encoded within prophageCP-933V Vibrio mimicus VM573 heat-labile hemolysin Aeromonas hydrophila subsp.Hydrophila ATCC 7966 Hemolysin Streptococcus pyogenesMGAS10394 streptolysin O Streptococcuspyogenes mitogenic exotoxin Z precursor Streptococcus pyogenes phage5005.1 enterotoxin type A Streptococcus pyogenes MGAS6180 exotoxin type J precursor Streptococcus phage 370.3 streptococcal exotoxin H precursor Streptococcus pyogenes M1 GAS exotoxin G precursor Streptococcus equisimilis GGS_124 putative exfoliative toxin Yersinia enterocolitica heat-stable enterotoxin; heat-stable enterotoxin type B Example 5: Toxicological Experiment Twenty healthy, age- and weight-appropriate SPF (Specific Pathogen Free) mice (half male and half female) were randomly divided into two groups (phage group and control group) after three days of acclimatization, with 10 mice in each group (5 males and 5 females). The phage group received a tail vein injection of 100 μL (2.1 x 10⁻⁶) of physiological saline diluted with pure culture medium of Bordetella bronchiseptica phage BOP2ABONT11 (prepared in Example 3). 10The mice were administered sterile saline solution via tail vein injection (PFU / mL). The control group was also treated with this solution. Food intake, activity levels, body temperature, weight changes, and survival were observed. Ten days later, the mice were euthanized by neck dislocation, and their internal organs were examined.

[0039] Forty healthy, age- and weight-appropriate SPF (Specific Pathogen Free) mice (half male and half female) were randomly divided into four groups (phage group 1, phage group 2, phage group 3, and control group) after three days of acclimatization, with 10 mice in each group (5 males and 5 females). Phage group 1, phage group 2, and phage group 3 were administered 0.2 mL / mouse, 0.5 mL / mouse, and 1 mL / mouse, respectively, with a titer of 4.2 x 10⁻⁶. 9 The mice were given a pure culture medium of Bordetella bronchiseptica phage BOP2ABONT11 (prepared in Example 3) at a concentration of PFU / mL orally, while the control group was given an equal volume of physiological saline orally. The mice were observed for 15 consecutive days. The mice were then euthanized by cervical dislocation and their internal organs were examined.

[0040] Forty one-day-old chicks were randomly divided into four groups (phage group 1, phage group 2, phage group 3, and control group), with ten chicks in each group. Phage group 1, phage group 2, and phage group 3 were given 0.4 mL / chick, 1 mL / chick, and 2 mL / chick, respectively, with a titer of 4.2 x 10⁻⁶. 9 The experimental chicks were orally administered pure culture medium of Bordetella bronchiseptica phage BOP2ABONT11 (prepared in Example 3) at a concentration of PFU / mL, while the control group was given an equal volume of physiological saline orally. The administration was continued for 15 days. The experimental chicks were then euthanized and their internal organs were examined.

[0041] Thirty newborn piglets were randomly divided into 5 groups (phage group 1, phage group 2, phage group 3, phage group 4, and control group), with 6 piglets in each group. Phage group 1, phage group 2, phage group 3, and phage group 4 were given 2 mL / piglet, 6 mL / piglet, 10 mL / piglet, and 20 mL / piglet, respectively, with a titer of 4.2 x 10⁻⁶. 9 The pure culture medium of Bordetella bronchiseptica phage BOP2ABONT11 (prepared in Example 3) with a concentration of PFU / mL was administered orally, while the control group was given an equal volume of physiological saline orally. The administration was continued for 15 days.

[0042] The results showed that, in all the above treatment groups, intravenous injection of a large dose of Bordetella phage BOP2ABONT11 had no effect on the daily behavior of mice. During the experiment, the mice did not show any clinical symptoms such as lethargy, rough fur, loss of appetite, weight loss, or death. No abnormalities were found in the tissues and internal organs during autopsy. In other oral treatments, different doses of Bordetella phage BOP2ABONT11 did not affect the daily behavior of the tested animals. No symptoms such as depression, fever, vomiting, or diarrhea were observed. No abnormalities were found in the internal organs of the mice and chicks during autopsy.

[0043] The Bordetella phage BOP2ABONT11 of this invention is biosafe and can be applied in various fields, including but not limited to feed additives. Of course, Bordetella phage BOP2ABONT11 can also be used as a health product or pharmaceutical, and this toxicological experiment has also demonstrated its safety.

[0044] Example 6: Thermal stability test of Bordetella phage 5 mL of pure culture medium of *Bordetella bronchiseptica* phage BOP2ABONT11 (prepared in Example 3) was dispensed into sterile test tubes and incubated in water baths at 4℃ (CK), 55℃, 65℃, and 75℃ for 2 h, 24 h, and 48 h, respectively. After the incubation period, the sample tubes were removed and immediately placed in an ice bath to cool. After appropriate dilution, the phage titer was determined using the double-layer plate method. The experiment was repeated three times.

[0045] The results are as follows Figure 5 As shown, the activity of *Bordetella bronchiseptica* phage BOP2ABONT11 was relatively stable at 55℃. Compared with the control, its titer remained unchanged after a 2-hour water bath at 65℃, but decreased by four orders of magnitude after a 2-hour water bath at 75℃, indicating that *Bordetella bronchiseptica* phage BOP2ABONT11 has good heat resistance and can maintain a certain level of activity in high-temperature environments.

[0046] Example 7: pH stability test of Bordetella phage The valence is 4.2 x 10 9 PFU / mL of pure culture medium of Bordetella bronchiseptica phage BOP2ABONT11 (prepared in Example 3) was adjusted to pH 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, and 14.0, respectively. After treatment at room temperature for 1 h, 4 h, 8 h, 24 h, and 96 h, the titer was determined using the double-layer plate method. The experiment was repeated three times.

[0047] The results are as follows Figure 6 As shown, the phage BOP2ABONT11 of Bordetella bacteria exhibits relatively stable titers after treatment at pH 4 to pH 10 for 96 hours, indicating good stability under neutral, slightly acidic, and alkaline conditions. Phage BOP2ABONT11 remains active after treatment at pH 3 for 96 hours, demonstrating that this phage has good tolerance to acidic conditions and a wide range of adaptability.

[0048] Example 8: Bioactivity Test of Bordetella phage spray powder and lyophilized powder Bioactivity test of Bordetella phage spray powder Dissolve 2% starch (w / v) and 4% sorbitol (w / v) in a solution with a potency of 4.2 x 10⁻⁶. 9 Phage coating was performed by incubating PFU / mL *Bordetella bronchiseptica* phage BOP2ABONT11 pure culture medium (prepared in Example 3) with the phage at 37°C and 180 r / min for 2 h. The coated phage solution was then sprayed onto defatted rice bran carrier at a ratio of 1:4 (w / w), while stirring to ensure uniform mixing. The mixture was then dried at 50°C for 2 h with a humidity <10% and passed through a 40-mesh sieve. After preparation, samples were taken and the phage titer was determined using the double-layer plate method, with the experiment repeated three times. The phage spray powder was stored at room temperature.

[0049] Bioactivity test of Bordetella phage lyophilized powder Dissolve 2% starch (w / v) and 4% sorbitol (w / v) in a solution with a potency of 4.2 x 10⁻⁶. 9 Phage was coated by incubating PFU / mL *Bordetella bronchiseptica* phage BOP2ABONT11 pure culture medium (prepared in Example 3) at 37°C and 180 r / min for 2 h. The coated phage solution was then added to a material tray and freeze-dried under vacuum for 50 ± 2 h, followed by pulverization through a 40-mesh sieve. The moisture content of the freeze-dried powder was <10%. After preparation, samples were taken and the phage titer was determined using the double-layer plate method, with the experiment repeated three times. The freeze-dried phage powder was stored at room temperature.

[0050] The results are as follows Figure 7 As shown, the titer of Bordetella phage BOP2ABONT11 was not significantly different before and after preparation of spray powder and lyophilized powder, indicating that the phage has good tolerance and stability to the powder preparation conditions of spray powder and lyophilized powder.

[0051] Example 9: Lysis experiment of Bordetella bronchiseptica on Bordetella bacteriophage The lysis profile of bacteriophages was determined using a double-layer plate drop method. A total of 53 single colonies of *Bordetella bronchiseptica* from five different serotypes were selected and inoculated into test tubes containing 3 mL of TSB liquid medium. The cultures were incubated at 37°C for 8 h to obtain bacterial suspensions for each strain. 300 μL of the bacterial suspension was mixed with TSB semi-solid medium and plated onto ordinary agar plates. 5 μL of the suspension with a titer of 1 x 10⁻⁶ was then used. 6 PFU / mL of pure culture medium of Bordetella bronchiseptica phage BOP2ABONT11 (prepared in Example 3) was dropped onto each plate, allowed to air dry, and then incubated at 37°C for 8 hours. The results were then observed. Figure 8 As shown.

[0052] from Figure 8It is known that the Bordetella bacteriophage described in this application has a wide host range, can recognize various tested serotypes of Bordetella, and has a total lysis rate of up to 92%, and can be well applied in the preparation of biocides for lysing Bordetella.

[0053] Example 10: Lysis test of Bordetella bronchiseptica phage against non-host pathogenic bacteria The lysis profile of bacteriophages was determined using the double-layer plate drop method.

[0054] Single colonies of 120 non-host pathogenic bacteria were selected, including 15 strains each of *Clostridium perfringens*, *Acinetobacter baumannii*, *Klebsiella pneumoniae*, *Escherichia coli*, *Shigella*, *Salmonella*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa*. Each colony was inoculated into test tubes containing 3 mL of TSB and cultured to the logarithmic phase under optimal conditions for each bacterial strain. 300 μL of the bacterial suspension was mixed with semi-solid culture medium and plated onto ordinary agar plates. 5 μL of pure *Bordetella bronchiseptica* phage BOP2ABONT11 culture (prepared in Example 3) with a titer of 1 x 10⁶ PFU / mL was then added to each plate. After air-drying, the plates were incubated overnight under optimal conditions for each bacterial strain, and the results were observed.

[0055] Figure 9 Chinese: A1. Clostridium perfringens, A2. Acinetobacter baumannii, A3. Klebsiella pneumoniae, A4. Escherichia coli, A5. Shigella, A6. Salmonella, A7. Staphylococcus aureus, A8. Pseudomonas aeruginosa Depend on Figure 9 It is evident that the Bordetella phage described in this application cannot recognize each of the tested bacteria. This indicates that the tested phage has strong intrageneric specificity and does not interfere with other microbial communities in the environment.

[0056] Example 11: Lysis test of Bordetella phage on non-pathogenic beneficial bacteria Sixty single colonies of non-pathogenic beneficial bacteria were selected, including 10 strains each of non-pathogenic rhizobia, non-pathogenic Bacillus licheniformis, non-pathogenic Bacillus subtilis, non-pathogenic Bacillus megaterium, Bacillus coagulans, and Clostridium butyricum. Each colony was inoculated into test tubes containing 3 mL of TSB and cultured to the logarithmic phase under optimal conditions to obtain bacterial suspensions. 300 μL of the bacterial suspension was mixed with semi-solid culture medium and plated onto ordinary agar plates. 5 μL of the suspension with a titer of 1 x 10⁻⁶ was then used. 6 PFU / mL of pure culture medium of Bordetella bronchiseptica phage BOP2ABONT11 (prepared in Example 3) was dropped onto each plate. The plates were incubated overnight under the optimal culture conditions for each tested bacterium, and the results were observed.

[0057] Figure 10Chinese: B1. Non-pathogenic rhizobium, B2. Non-pathogenic Bacillus licheniformis, B3. Non-pathogenic Bacillus subtilis, B4. Non-pathogenic Bacillus megaterium, B5. Bacillus coagulans, B6. Clostridium butyricum Depend on Figure 10 It is evident that none of the Bordetella phages described in this application could recognize any of the 60 tested non-pathogenic beneficial bacteria strains. This indicates that the tested phages did not damage the beneficial bacterial microbial community.

[0058] Example 12: UV tolerance test of Bordetella phage Take 10 mL, the potency is 6.4 × 10. 8 PFU / mL of pure culture medium of Bordetella bronchiseptica phage BOP2ABONT11 (prepared in Example 3) was spread evenly in a 90 mm sterile culture dish and placed under a UV lamp (20 W, 30 cm, 50 μW / cm) in a sterilized laminar flow hood. 2 Irradiation was performed under [a specific irradiation method]. Samples were taken at 0 min, 20 min, 40 min, 60 min, 2 h, 3 h, 4 h, and 5 h, and placed in the dark at room temperature for 30 min before phage titer was determined using the double-layer plate method. The experiment was repeated three times. Titer was determined using the double-layer plate method.

[0059] The results are as follows Figure 11 As shown: Bordetella phage BOP2ABONT11 exhibits good resistance to ultraviolet radiation; its titer did not decrease by orders of magnitude after 1 hour of ultraviolet irradiation; and after 5 hours of irradiation, BOP2ABONT11 still possessed a titer of 10. 4 PFU / mL potency.

[0060] Example 13: Preparation of a composition of Bordetella phage With a valence of 1x10 9 A pure culture medium of Bordetella phage BOP2ABONT11 (prepared in Example 3) and a pure culture medium of Bordetella phage BOP3ABONT19 were prepared by uniformly mixing equal volumes of the two phages to form a 1:1 composition of Bordetella phage BOP2ABONT11 and Bordetella phage BOP3ABONT19 (Composition 1).

[0061] With a valence of 1x10 9A pure culture medium of Bordetella phage BOP2ABONT11 (prepared in Example 3) and a pure culture medium of Klebsiella pneumoniae phage P6AKPJS20 were prepared by uniformly mixing equal volumes of the two phages to form a 1:1 composition of Bordetella phage BOP2ABONT11 and Klebsiella pneumoniae phage P6AKPJS20 (Composition 2).

[0062] With a valence of 1x10 9 Pure culture medium of Bordetella phage BOP2ABONT11 (prepared in Example 3), pure culture medium of Bordetella phage BOP3ABONT19 and Klebsiella pneumoniae phage P6AKPJS20 were prepared by uniformly mixing equal volumes of the three phages to prepare a 1:1:1 composition of Bordetella phage BOP2ABONT11, Bordetella phage BOP3ABONT19 and Klebsiella pneumoniae phage P6AKPJS20 (Composition 3).

[0063] With a valence of 1x10 9 A composition was prepared by uniformly mixing an equal volume of PFU / mL Bordetella phage BOP2ABONT11 pure culture medium (prepared in Example 3) with dodecyl dimethyl benzyl ammonium chloride, and the final application mass concentration of dodecyl dimethyl benzyl ammonium chloride in the composition was 0.1% (W / V) (Composition 4).

[0064] Example 14: Prevention and control of Bordetella bronchiseptica contamination during pork sample preservation using Bordetella bronchiseptica bacteriophage and its composition Preparation methods of compositions 1 to 4: same as those described in Example 13.

[0065] Pure culture medium of Bordetella phage BOP2ABONT11: prepared in Example 3.

[0066] 3. Cut the autoclaved pork into 105 1cm cubes and divide them into 7 groups (single phage BOP2ABONT11 group, composition 1, composition 2, composition 3, composition 4, control group, and blank group), with 15 cubes in each group, and place them in sterile Petri dishes. In the experimental groups, each cube was inoculated with 2 x 10- phages. 5 cfu / mL Bordetella bronchiseptica and a dose of 10 6 The test phage was inoculated at PFU / kg; the control group was inoculated with 2 x 10 PFU / kg per patch. 5 The treatment group received CFU / mL *Bordetella bronchiseptica* and an equal volume of sterile water; the control group received an equal volume of sterile physiological saline. All treatments were incubated at 37°C. Every 8 hours, a piece of pork was placed in 10 mL of sterile water, shaken thoroughly, and the *Bordetella bronchiseptica* content in the liquid was determined by the spread plate method. The experiment was repeated three times. Results are shown below. Figure 12 .

[0067] Figure 12 The results showed that after 32 hours, a large number of *Bordetella bronchiseptica* bacteria grew on the surface of pork in the control group; while in the experimental groups, due to the addition of *Bordetella bronchiseptica* phage BOP2ABONT11 and its combinations 1-4, the level of *Bordetella bronchiseptica* on the surface of pork was consistently kept low. *Bordetella bronchiseptica* phage BOP2ABONT11 and its combinations 1-4 can be used as a biocidal agent to effectively kill *Bordetella bronchiseptica* on the surface of pork and prevent contamination of pork during storage. Of course, *Bordetella bronchiseptica* phage BOP2ABONT11 can also be prepared into health products or pharmaceuticals for application.

[0068] Example 15: The preventive and therapeutic effects of Bordetella phage and its composition on animals infected with Bordetella phage. The effect of Bordetella phage and its combination on preventing Bordetella infection in mice Three hundred healthy SPF-grade laboratory mice, aged 6 weeks and of similar weight (half male and half female), were selected and, after three days of acclimatization, randomly divided into 6 groups (phage BOP2ABONT11 group, composition 1, composition 2, composition 3, positive control group, and negative control group), with 50 mice in each group (25 males and 25 females). The pure culture medium of Bordetella phage BOP2ABONT11 was prepared in Example 3.

[0069] Before the experiment, the mouse house, feed troughs, waterers, etc., were thoroughly cleaned and disinfected, and the entire mouse house was fumigated for disinfection. The experimental mice had free access to food and water, and the house was cleaned and disinfected regularly.

[0070] Mice in the phage BOP2ABONT11 group were intraperitoneally injected with 100 μL / mouse at a concentration of 1x10⁻⁶. 7Two hours after injection of *Bordetella bronchiseptica* bacterial suspension at cfu / mL, 100 μL / animal with a titer of 1x10 was injected intraperitoneally. 8 Pure culture medium of the test phage BOP2ABONT11 (prepared from Example 3) with a concentration of PFU / mL.

[0071] Mice of compositions 1-3 were administered 100 μL / mouse via intraperitoneal injection, representing a concentration of 1x10⁻⁶. 7 Two hours after injection of *Bordetella bronchiseptica* bacterial suspension at cfu / mL, 100 μL / animal with a titer of 1x10 was injected intraperitoneally. 8 The corresponding PFU / mL phage pure culture medium (prepared from Example 13).

[0072] The positive control mice were intraperitoneally injected with 100 μL / mouse at a concentration of 1x10⁻⁶. 7 Two hours after injecting a CFU / mL solution of Bordetella bacteria, an equal volume of sterile saline was injected into the peritoneal cavity of each individual.

[0073] The negative control mice were injected intraperitoneally with 100 μL of sterile saline, and 2 hours later, they were injected intraperitoneally again with the same amount of sterile saline.

[0074] During the experiment, all groups were fed a basal diet. Seven days after intraperitoneal injection, the mortality rate of mice in each group was recorded, and all mice were necropsyed to determine the rate of visceral lesions. The results are shown in Table 3.

[0075] Mortality rate = (number of mice that died / total number of mice) × 100%.

[0076] Lung lesion rate = (number of mice with lung lesions / total number of mice) × 100%.

[0077] Protection rate = 1 - mortality rate.

[0078] Table 3. Efficacy of Bordetella phage in preventing Bordetella infection in mice. bacteriophage BOP2ABONT11 group Composition 1 Composition 2 Composition 3 Positive control group negative control group mortality rate 0 0 0 0 96% 0 Lung lesion rate 10% 0 10% 0 94% 0 As shown in Table 3, compared with the positive control group, the mortality rate of mice treated with Bordetella phage or its combination was 0, and the lung lesion rate was no higher than 10%. The protection rate of Bordetella phage and its combination against the tested mice reached 100%. This indicates that Bordetella phage can effectively prevent mice from being infected with Bordetella.

[0079] Therefore, Bordetella phages can be well used in the preparation of drugs for the treatment or prevention of diseases caused by Bordetella infection.

[0080] Example 16: Preparation and use of a kit for Bordetella phage and its composition The kit contains 5–10 mL of a potency of 1×10⁻⁶. 9 The contents included: pure culture medium of Bordetella phage BOP2ABONT11, 1 L of TSB liquid medium, 1 L of TSB semi-solid medium, and 1 L of LTSA solid medium; wherein, Bordetella phage BOP2ABONT11 was the pure culture medium of phage BOP2ABONT11 prepared in Example 1, or the pure culture medium of phage BOP2ABONT11 prepared in Example 3.

[0081] The method of using the Bordetella phage BOP2ABONT11 kit is as follows: Take a titer of 1×10⁻⁶. 9 PFU / mL Bordetella phage BOP2ABONT11 liquid was used to determine the lysis profile of the test phage using a double-layer plate drop method. A single colony to be tested was picked and inoculated into the target liquid medium. The culture was then shaken and incubated at the target temperature according to the growth characteristics of the test strain to prepare the bacterial suspension. 300 μL of the test strain suspension was mixed with 5 mL of TSB semi-solid medium and plated onto a TSA solid plate. 10 μL of Bordetella phage BOP2ABONT11 liquid was then dropped onto the plate. After air drying, the plates were incubated at the target temperature according to the growth characteristics of the test strain. The results were then observed to determine whether the target strain was present.

[0082] The biosensor kit contains 20–50 mL of a titer of 1×10⁻⁶. 8 Bordetella phage BOP2ABONT11.

[0083] The method for using the Bordetella bronchiseptica phage BOP2ABONT11 biosensor kit is as follows: Take the sample to be tested, add sterile physiological saline to just cover the sample, and prepare a homogenate. The titer is 1×10⁻⁶. 8 PFU / mL of Bordetella phage BOP2ABONT11 liquid was used to immobilize the phage on the surface of a magnetoelastic sensor and incubated in a humid environment at 30°C for 30 min. The magnetoelastic sensor was then placed in the sample to be tested and left to stand at the target temperature for bacterial growth for 30 min. The resonant frequency shift before and after sensor detection was measured by an electromagnetic coil, thus obtaining the number or concentration of target bacteria in the sample.

[0084] The Bordetella phage BOP2ABONT11 of the present invention can be used to prepare compositions, reagents, kits or biosensor kits for the rapid detection of Bordetella, including but not limited to detecting Bordetella in target samples in the form of reagents, kits or biosensor kits, or screening target pathogens in clinical samples, effectively ensuring the sensitivity of the detection.

[0085] It should be noted that the embodiments described above are only for explaining this application and do not constitute any limitation on this application. This application has been described with reference to typical embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to this application within the scope of the claims, and revisions can be made to the invention without departing from the scope and spirit of this application. Although the application described herein relates to specific methods, materials, and embodiments, it does not mean that this application is limited to the specific examples disclosed herein; on the contrary, this application can be extended to all other methods and applications with the same function.

Claims

1. A Bordetella phage with good acid tolerance, characterized in that, The bacteriophage is Bordetellaphage BOP2ABONT11, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on September 28, 2025, with the accession number GDMCC NO. 67044-B1, and has the nucleotide sequence shown in SEQ ID No.

1.

2. The Bordetella phage according to claim 1, characterized in that, The bacteriophage has a head with polyhedral three-dimensional symmetry and a short, contracted tail. The head diameter is 62–72 nm, the tail length is 11–15 nm, and the tail diameter is 6–8 nm.

3. The Bordetella phage according to claim 1, characterized in that, The bacteriophage was cultured for 8 hours at an MOI of 0.001, and its titer was 4.2 × 10⁻⁶. 10 PFU / mL.

4. The Bordetella phage according to claim 1, characterized in that, The bacteriophage remained active after treatment at pH 3 for 96 hours and exhibited good stability at 55–65°C.

5. The Bordetella phage according to claim 1, characterized in that, The phage has a recognition rate of 90%-96% against Bordetella bronchiseptica.

6. A bacteriophage composition, characterized in that, It includes the Bordetella phage BOP2ABONT11 as described in any one of claims 1-5.

7. The phage composition according to claim 6, characterized in that, It also contains Klebsiella pneumoniae phage P6AKPJS20 and / or Bordetella phage BOP3ABONT19, wherein the Klebsiella pneumoniae phage P6AKPJS20 has the accession number CCTCC NO: M 20242965 and the Bordetella phage BOP3ABONT19 has the accession number GDMCC NO. 67045-B1.

8. The phage composition according to claim 6, characterized in that, It also includes Bordetella phage BOP2ABONT11, with accession number GDMCC NO. 67044-B1.

9. The phage composition according to claim 6, characterized in that, It also includes dodecyl dimethyl benzyl ammonium chloride with a final mass concentration of 0.1% (w / v).

10. A biological bactericide, characterized in that, Suitable for lysing Bordetella lysate, the active ingredient of which includes the Bordetella phage as described in any one of claims 1-5 or the phage composition as described in any one of claims 6-9; the dosage form of the biocide is a solution, powder, gel, granule or lyophilized agent.

11. A medicine for treating or preventing Bordetella bronchiseptica infection, characterized in that, The raw materials for its preparation include Bordetella phage as described in any one of claims 1-5.

12. A kit containing Bordetella phage, characterized in that, The kit includes the Bordetella phage BOP2ABONT11 as described in any one of claims 1-4, and the kit is a biosensor kit using Bordetella phage BOP2ABONT11 as a biorecognition element.

13. A feed additive, characterized in that, The raw materials for its preparation include Bordetella phage as described in any one of claims 1-4.

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