Bovine-derived coliphage and application thereof in prevention of cow foot rot

Abstract

The invention provides a bovine-derived Escherichia coli bacteriophage, which is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 2025205. The bovine-derived escherichia coli bacteriophage can survive in an environment with the pH value of 3.0-12.0, has good titer stability at the temperature of 4-50 DEG C, has good pH and temperature tolerance, and is suitable for preparing drugs and disinfectants for inhibiting bovine-derived escherichia coli.

Description

Field of the Invention

[0001] The present application belongs to the field of microbiology and veterinary medicine. Specifically, the present application provides a bovine Escherichia coli phage and its application in preventing cow foot rot. Background Art

[0002] Hoof disease is one of the most serious health problems affecting the economic profitability of large-scale dairy cattle farming. Hoof rot is one of these diseases. Hoof rot is typically caused by bacterial infection. Due to the harsh farm environment and damp ground, cow excrement is not cleaned promptly, leaving the cow's hooves permanently immersed in it, leading to hoof infection. Affected cows often limp, standing with the affected limb raised and afraid to touch the ground. The infected hoof is swollen, with deep rot in the hoof fork. After hoof trimming, pus will flow. Cows with hoof rot experience a decrease in milk production. Due to the long treatment cycle and high cost, affected cows are often culled, resulting in significant economic losses for the farm.

[0003] Escherichia coli belongs to the Enterobacteriaceae family, is a Gram-negative bacterium, and is a facultative anaerobic bacteria. Escherichia coli usually comes from feces. Due to the special environment of the farm, dairy cows cannot clean up their feces in time after defecation, and their feces will be spread into the environment, resulting in the presence of large numbers of Escherichia coli in the farm, causing serious pollution.

[0004] Farms commonly use antibiotics to treat foot rot, but overuse of antibiotics has become a serious public health issue. Long-term, high-volume use can lead to bacterial resistance and ultimately render the disease unusable. Furthermore, the combined use of multiple antibiotics can leave antibiotic residues in dairy products, potentially impacting human health. Therefore, this invention uses bacteriophages as an alternative to antibiotics for treatment.

[0005] Bacteriophages are viruses that can infect bacteria and other microorganisms. They are divided into virulent phages and temperate phages based on their lysis ability. Virulent phages can quickly and effectively lyse bacteria, only targeting the corresponding pathogens. They are highly specific, will not destroy normal bacterial flora, and are not prone to drug resistance. They are widely present in nature and can effectively replace antibiotics.

[0006] In recent years, bacteriophages have been increasingly used in the livestock industry. In 2011, Chinese researchers applied bacteriophages as a medicated bath to treat mastitis in dairy cows infected with Staphylococcus aureus. In 2010, American researchers used bacteriophages via gavage to treat diarrhea in piglets infected with Salmonella, both with significant therapeutic effects. This demonstrates the significant utility of bacteriophages in treating bacterial infections and holds great promise for treating foot rot in dairy cows. Summary of the Invention

[0007] On the one hand, the present application provides a bovine Escherichia coli phage, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025205.

[0008] On the other hand, the present application provides the use of the above-mentioned bovine Escherichia coli phage in the preparation of a drug for inhibiting bovine Escherichia coli.

[0009] On the other hand, the present application provides the use of the above-mentioned bovine Escherichia coli phage in the preparation of a drug for preventing or treating foot rot in dairy cows.

[0010] On the other hand, the present application provides a drug for inhibiting bovine Escherichia coli, wherein the drug comprises the above-mentioned bovine Escherichia coli phage.

[0011] On the other hand, the present application provides a drug for preventing or treating cow foot rot, wherein the drug comprises the above-mentioned bovine Escherichia coli phage.

[0012] Furthermore, the medicine is a bath liquid preparation.

[0013] In addition to liquid preparations for medicinal baths, those skilled in the art can also routinely select other dosage forms, including but not limited to injections, external powders, oral preparations, etc.

[0014] Furthermore, the medicine also contains pharmaceutically acceptable excipients.

[0015] Furthermore, the medicine also contains glycerol.

[0016] The pharmaceutically acceptable excipients include solvents, suspending agents, pH regulators, protective agents (such as glycerol), etc. The preparation may also contain cells, such as Escherichia coli cell components.

[0017] The titer of the phage in the drug can be determined according to factors such as the mode of administration, for example but not limited to 10 5 -10 8 PFU / mL.

[0018] On the other hand, the present application provides a disinfectant for dairy cattle breeding, which contains the above-mentioned bovine Escherichia coli phage.

[0019] On the other hand, the present application provides a method for disinfecting a dairy cattle breeding site, which comprises using the above-mentioned disinfectant.

[0020] The phage XJA18 of the present application was deposited on February 13, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China. Its classification name is Escherichia coli phage XJA18 and the preservation number is CCTCC NO: M 2025205. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a picture of bacterial plaque.

[0022] Figure 2 Transmission electron microscopy images of bacteriophages (80,000 and 40,000 times).

[0023] Figure 3 This is the one-step growth curve of phage.

[0024] Figure 4 This is a diagram of bacteriophage thermal stability.

[0025] Figure 5 This is the pH stability diagram of phage.

[0026] Figure 6 The figure shows the results of in vitro antibacterial experiment. DETAILED DESCRIPTION

[0027] Example 1 Isolation and identification of bacteriophage

[0028] The host bacteria used in the experiment were Escherichia coli isolated from foot rot samples in a dairy farm in Changji, Xinjiang.

[0029] The liquid culture medium used in the experiment was LB liquid culture medium, the upper semi-solid culture medium was LB semi-solid culture medium with an agar content of 0.6%, and the lower solid culture medium was LB solid culture medium with an agar content of 1.5%.

[0030] Isolation, purification and enrichment of bacteriophages:

[0031] Wastewater was collected from farms and underground wells around Urumqi, Xinjiang, and the double-layer plate method was used to isolate and purify bacteriophages.

[0032] Separation: The collected sewage samples were mixed and centrifuged at 13400 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter membrane. 5 mL of the supernatant and 200 μL of the host bacteria cultured to the logarithmic phase were added to 5 mL of LB liquid medium and placed in a shaker at 37°C and 180 rpm for overnight culture. The phage stock solution after overnight culture was centrifuged at 13400 rpm for 10 minutes and filtered through a 0.22 μm filter membrane. The filtered stock solution was diluted to 10 by ten-fold dilution method. -8. Add 200 μL of phage stock solution of different concentration gradients and 200 μL of host bacteria to 5 mL of the upper layer (0.6% LB semi-solid medium), mix well, pour into the lower layer (1.5% LB solid medium), and culture in a constant temperature incubator at 37°C overnight. After culture, round, transparent single phage plaques can be seen on the plate ( Figure 1 ).

[0033] Purification: Pick a single plaque on the plate and add it to 1 mL of liquid culture medium. Place it in a shaker at 37°C and 180 rpm for 1 hour, then centrifuge it at 13400 rpm for 10 minutes, filter it with a 0.22 μm filter membrane, and dilute it tenfold to 10 -8 Pour the double-layer plate as above. Repeat this process 5 times until plaques of regular size and clear edges appear on the plate, indicating that the purification is successful.

[0034] Enrichment: Dilute the purified phage tenfold and pour it onto a double-layer plate. Select a plate covered with plaques and without bacterial moss, add 5mL of liquid culture medium, and let it stand at 4°C for 4-5h. Transfer the liquid in the plate to a centrifuge tube, centrifuge at 13400rpm for 10min, filter with a 0.22μm filter membrane, pour it onto a double-layer plate, repeat 3 times, and obtain phage enrichment liquid. Dilute the enrichment liquid tenfold, take 100μL of phage liquid and 100μL of bacterial liquid, mix them, pour them onto a double-layer plate, and culture them at 37°C overnight. Repeat three times for each dilution gradient. Select a plate with 30-300 plaques for counting. After counting, take the average value to calculate the titer. Phage titer (PFU / mL) = number of plaques × dilution factor × 100. Select a dilution factor of 10 -8 The plaque numbers on the plates were 40, 46, and 38, respectively. The titer of the phage after enrichment was 4.13×10 11 PFU / mL.

[0035] Phage morphology observation:

[0036] The phage was stained using the negative staining method: 20 μL of the enriched phage solution was dropped onto a copper grid, allowed to stand for 15 minutes, excess liquid was removed with filter paper, and then 20 μL of 2% phosphotungstic acid was added for staining for 5 minutes. Excess liquid was removed with filter paper, and after drying, the phage was observed using a Hitachi transmission electron microscope in high contrast mode at 80.0 kV with a magnification of 40,000-80,000 times. The phage was observed to have an icosahedral head with a diameter of approximately 67 nm and a tadpole-shaped tail with a diameter of approximately 171 nm, indicating a long-tailed phage ( Figure 2 Part A and Part B of the

[0037] Determination of host spectrum of phage:

[0038] The phage host spectrum was determined using the drop method: the strain to be tested was cultured to the logarithmic phase, 200 μL of the strain to be tested was added to 5 mL of the upper semi-solid medium, mixed well, and poured onto the lower solid medium. After the upper layer solidified, 10 μL of a 10 titer was added dropwise. 11 The phage was placed on the upper layer of semi-solid culture medium, dried, and then cultured overnight in a constant temperature incubator at 37°C. The phage's lysis effect was observed, and the appearance of plaques confirmed that the phage had a lytic effect on the test strain. The assay was performed on 87 strains of Escherichia coli isolated from cattle farms (5 batches of samples from different fixed-point locations—ABG, ABB, BN, XNB, and XBG) and found that 11 of them had a lytic effect (Table 1). Each fixed-point location had strains that could be lysed by phage XJA18, demonstrating the phage's universal effectiveness against foot rot pathogens.

[0039] Table 1 Host spectrum

[0040]

[0041]

[0042]

[0043]

[0044] Whole-genome sequencing analysis of bacteriophages

[0045] HiPure Lambda DNA Kits were used to extract the phage genome, and the concentration, purity, and total amount of the extracted genome were tested. If qualified, the genome was sequenced on the Illumina platform PE150, and the sequencing data was finally analyzed. The VAHTS Universal Plus DNA Library Prep Kit was used to construct a library with an initial input of 100 pg-1 μg. The input DNA was fragmented and the ends of the fragmented DNA were blunted. The 5' end was phosphorylated and the 3' end was tailed with a dA tail. Adapters were connected to the ends of the unrepaired products of the previous step. For the library with adapters, two rounds of VAHTS DNA Clean Beads (0.56×, 0.2×) were used for fragment size sorting. The original library was amplified using a high-fidelity polymerase. After the library was constructed, Qubit was used for preliminary quantification, and then the insert size of the library was detected using NGS3K / Caliper. After the insert size met the expectations, the effective concentration of the library (3 nM) was accurately quantified using qPCR, and the library was sequenced on the Illumina platform PE150. The sequencing data were filtered by fastp to obtain a total of 1,122,787,442 bp of effective sequencing data. The second-generation data were assembled using Unicycler, and the coding genes of the assembled genome were predicted using Prokka software. Finally, the predicted gene sequences were compared with the functional databases of Pfam, Gene, COG, KEGG, Uniprot, Refseq, NR, and TIGRFAMs by BLAST comparison to obtain gene function annotation results.

[0046] Whole-genome sequencing revealed that the phage is 50,572 base pairs long and belongs to the genus Caudoviricetes. It shares 95% identity with Escherichia phage vB_EcoS_SA12KD and 92% with Shigella phage Sfin-4. The GC content is 45.33%, and the genome is circular. Resistance genes were annotated using rgi based on the CARD database, and virulence genes were annotated using blastep based on the VFDB database. No virulence or resistance genes were found.

[0047] The phage was deposited in the China Type Culture Collection at Wuhan University, Wuhan, China on February 20, 2025. Its classification name is Escherichia coli phage and the preservation number is CCTCCNO: M 2025205.

[0048] Example 2 Biological characteristics of bacteriophage

[0049] Optimal multiplicity of infection:

[0050] The concentration of the bacterial solution cultured to the logarithmic phase was determined by the viable bacteria counting method, and the host bacteria were diluted to 10 -8 200 μL of diluted phage and 200 μL of bacterial suspension were added to 5 mL of liquid culture medium at a concentration ratio of phage: host bacteria = 100, 10, 1, 0.1, 0.01, and 0.001. The culture was placed on a shaker at 180 rpm and 37°C for 5 hours. The cultured phage solution was centrifuged at 13,400 rpm for 10 minutes, filtered through a 0.22 μm filter membrane, and poured onto double-layer plates for titer determination. Each multiplicity of infection was repeated three times. The phage titer reached the highest at a multiplicity of infection of 0.001, reaching 1.14×10 11 PFU / mL (Table 2).

[0051] Table 2 Optimal multiplicity of infection of Escherichia coli phage

[0052]

[0053] One-step growth curve

[0054] First, determine the initial titer of the phage and bacterial suspension. Add 500 μL of each titer of phage and bacterial suspension to a 1 mL centrifuge tube at the optimal multiplicity of infection (MOI) of 1:1000, mix thoroughly, and incubate at 37°C for 15 minutes. After incubation, centrifuge at 13,400 rpm for 5 minutes, discard the supernatant, and resuspend in 1 mL of liquid culture medium. After resuspension, centrifuge at 13,400 rpm for 5 minutes, discard the supernatant, and repeat the wash twice to remove free phage. After the final wash, add 15 mL of liquid culture medium to the phage, mix thoroughly, and incubate on a shaker at 180 rpm at 37°C for 120 minutes. At 0, 5, 10, 20, 30, 40, 50, 60, 80, 100, and 120 minutes, aspirate 1 mL, centrifuge at 13,400 rpm for 5 minutes, filter through a 0.22 μm filter, and pour onto double-layer plates. Determine the titer at each time point, with three replicates per time point. Observe the incubation period and outbreak period of the phage and calculate the phage lysis amount. Lysis amount = phage titer at the end of lysis / host bacterial concentration at the beginning of infection.

[0055] The incubation period of the phage was 10 min, the outbreak period was 30 min, and the lysis capacity was 2220 pfu / cell ( Figure 3 ).

[0056] Temperature stability:

[0057] The initial titer is 10 8The phage was divided into 200uL in 16 centrifuge tubes, and each two tubes of phage were placed in a water bath at 4℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 1 hour. One tube was taken out every 30 minutes and the titer of the phage was determined by the double-layer plate method. Each temperature and time were repeated three times. The titer of the phage remained basically stable when it was warmed at 4℃ to 60℃ for 30 minutes, the titer was reduced by half at 70℃, and the titer was greatly reduced at 80℃; the titer was basically stable when it was warmed at 4℃ to 50℃ for 1 hour, the titer was slightly reduced at 60℃, the titer was reduced by half at 70℃, and it was basically inactivated at 80℃ ( Figure 4 ). The phage has good temperature tolerance and can survive stably at 4°C to 50°C.

[0058] pH stability:

[0059] HCl and NaOH were used to adjust the pH of PBS buffer to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively. 100 uL of 10 titer was added to 900 uL of PBS buffer at different pH values. 8 After mixing, the phage was incubated in a shaker (37°C, 180 rpm) for 1 hour. The titer was determined by double-layer plate method with three replicates for each pH value. The titer of the phage remained stable from pH 4 to pH 12, decreased slightly at pH 3, and was essentially inactivated at pH 2 ( Figure 5 ). The phage has good acid and alkali resistance and can survive at pH = 3 to pH = 12.

[0060] In vitro antibacterial experiment of phage:

[0061] In vitro antibacterial experiments were performed using Escherichia coli isolated from foot rot samples from a dairy farm in Changji, Xinjiang. The bacterial solution was shaken to the logarithmic phase, and the number of viable bacteria was determined by colony counting. After diluting the bacterial solution and phage, they were mixed at a ratio of MOI = 100, 10, 1, 0.1, 0.01, and 0.001 and added to a 96-well plate. Three replicates were set for each gradient. The positive control (PC) used liquid culture medium instead of phage, and the blank control (NC) added liquid culture medium. The OD600 value was measured with a microplate reader at 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, and 10h. The OD600 of each group showed an upward trend within 4h, and gradually leveled off after 4h. Compared with PC, the OD600 under each MOI was reduced, and the OD600 was lowest when MOI = 0.001 ( Figure 6 ). It was proved that the phage had an antibacterial effect on Escherichia coli, especially when MOI = 0.001, the antibacterial ability was the strongest, which fully confirmed that the optimal MOI of the phage was 0.001.

Claims

1. A bovine Escherichia coli phage, characterized in that: The bovine Escherichia coli phage is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025205.

2. Use of the bovine Escherichia coli phage according to claim 1 in the preparation of a drug for inhibiting bovine Escherichia coli.

3. Use of the bovine Escherichia coli phage according to claim 1 in the preparation of a medicament for preventing or treating foot rot in dairy cows.

4. A drug for inhibiting bovine Escherichia coli, comprising the bovine Escherichia coli phage according to claim 1.

5. A drug for preventing or treating foot rot in dairy cows, comprising the bovine Escherichia coli phage according to claim 1.

6. according to the medicine described in claim 4 or 5, described medicine is medicated bath preparation.

7. The drug according to claim 6, further comprising a pharmaceutically acceptable excipient. The medicine according to claim 7 , further comprising glycerol.

9. A disinfectant for dairy cattle breeding, characterized in that: The disinfectant contains the above-mentioned bovine Escherichia coli phage.

10. A method for disinfecting a dairy cattle breeding site, characterized in that: The disinfection method comprises applying the disinfectant according to claim 9 to a breeding site.

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