PREPARATION OF PHAGE COCKTAIL AS THERAPEUTIC AGENT FOR COW MASTITIS AND USE THEREOF

Abstract

Disclosed is preparation of a cocktail as a therapeutic agent for cow mastitis and use thereof. A compatibility of phages against the main pathogenic bacteria of cow mastitis is utilized in combination with a specific preparation method to prepare and obtain a therapeutic cocktail preparation. The therapeutic phage cocktail preparation for cow mastitis provided in the disclosure is easy to prepare, short in course of treatment and quick in effect. It has better therapeutic effect on both clinical cow mastitis and recessive mastitis than that of antibiotics and antimicrobial peptide and is expected to solve the problem of bacterial resistance in the treatment of cow mastitis, thereby reducing or getting rid of the troubles of diseases such as mastitis in cows, which is of potential development value in cow farming.

Claims

1. A therapeutic phage cocktail preparation for cow mastitis, wherein the therapeutic phage cocktail preparation for cow mastitis comprises a cow mastitis-derived Escherichia coli phage selected from the group consisting of Ecp1, Ecp3, Ecp5, Ecp16, Ecp17, and vB_EcoM_XJ2, a cow mastitis-derived Streptococcus phage selected from the group consisting of vB_StrM_L1 and vB_SagS_FSN1, and a cow mastitis-derived Staphylococcus aureus phage selected from the group consisting of P42 and vB_SauS_IMEP5.

2. The therapeutic phage cocktail preparation for cow mastitis of claim 1, wherein the cow mastitis-derived E. coli phage, the cow mastitis-derived Streptococcus phage, and the cow mastitis-derived S. aureus phage in the therapeutic phage cocktail preparation for cow mastitis are mixed in a volume ratio of 1:1:1.

3. The therapeutic phage cocktail preparation for cow mastitis of claim 1, wherein the cow mastitis-derived E. coli phages Ecp1, Ecp3, Ecp5, Ecp16, Ecp17, and vB_EcoM_XJ2 in the therapeutic phage cocktail preparation for cow mastitis are mixed in a volume ratio of 1:1:1:1:1:1.

4. The therapeutic phage cocktail preparation for cow mastitis of claim 1, wherein the cow mastitis-derived Streptococcus phages vB_StrM_L1 and vB_SagS_FSN1 in the therapeutic phage cocktail preparation for cow mastitis are mixed in a volume ratio of 1:1.

5. The therapeutic phage cocktail preparation for cow mastitis of claim 1, wherein the cow mastitis-derived S. aureus phages P42 and vB_Sau S_IMEP5 in the therapeutic phage cocktail preparation for cow mastitis are mixed in a volume ratio of 1:1.

6. A method for preparing the therapeutic phage cocktail preparation for cow mastitis of claim 1, wherein the method comprises following steps: (1) selecting a frozen cow mastitis-derived E. coli phage selected from the group consisting of Ecp1, Ecp3, Ecp5, Ecp16, Ecp17, and vB_EcoM_XJ2, a frozen cow mastitis-derived Streptococcus phage selected from the group consisting of vB_StrM_L1 and vB_SagS_FSN1, and a frozen cow mastitis-derived S. aureus phage selected from the group consisting of P42 and vB_SauS_IMEP5 for recovery; (2) taking 100 μL of a phage stock solution of each strain recovered in step (1), inoculating each solution into 5 mL of a host bacterial liquid in a logarithmic growth phase respectively, shaking and culturing at 37° C. at 180 r/min until turning clear, and centrifuging the solutions at 10,000 r/min for 10 min; taking each supernatant and obtaining phase dilutions with phosphate buffered solution at a concentration of 1×10.sup.9 pfu/mL; and (3) mixing the phage dilutions of each strain prepared in step (2) in proportion to prepare a therapeutic phage cocktail preparation for cow mastitis.

7. A method for treating cow mastitis, comprising administering the therapeutic phage cocktail preparation for cow mastitis of claim 1 to a cow in need thereof.

8. The method of claim 7, wherein the administering comprises directly injecting the phage cocktail into a gland cistern through a teat canal for perfusion.

9. The method of claim 7, wherein a dosage is 30 mL each for 2-3 times a day.

10. The method of claim 7, wherein the cow mastitis-derived E. coli phage, the cow mastitis-derived Streptococcus phage, and the cow mastitis-derived S. aureus phage in the therapeutic phage cocktail preparation for cow mastitis are mixed in a volume ratio of 1:1:1.

11. The method of claim 7, wherein the cow mastitis-derived E. coli phages Ecp1, Ecp3, Ecp5, Ecp16, Ecp17, and vB_EcoM_XJ2 in the therapeutic phage cocktail preparation for cow mastitis are mixed in a volume ratio of 1:1:1:1:1:1.

12. The method of claim 7, wherein the cow mastitis-derived Streptococcus phages vB_StrM_L1 and vB_SagS_FSN1 in the therapeutic phage cocktail preparation for cow mastitis are mixed in a volume ratio of 1:1.

13. The method of claim 7, wherein the cow mastitis-derived S. aureus phages P42 and vB_Sau S_IMEP5 in the therapeutic phage cocktail preparation for cow mastitis are mixed in a volume ratio of 1:1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows the clinical therapeutic effect of the therapeutic phage cocktail preparation for cow mastitis.

[0024] FIG. 2 shows the effect of phage cocktail as a therapeutic agent for cow recessive mastitis.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] Hereinafter, the present disclosure will be described with reference to examples, but the present disclosure is not limited to the following examples.

[0026] The reagents used in the present disclosure include: PEG8000 purchased from Tianjin Guangfu Fine Chemical Research Institute; SM buffer: NaCl 5.8 g, MgSO.sub.4.7H.sub.2O 2.0 g, 1 mol/L Tris-HCl (pH 7.5) 50 mL, 2% gelatin 5 mL, a balance of distilled water to 1 L, sterilized at 1×10Pa for 20 min, and stored at 4° C.; PBS buffer: NaCl 8.0 g, KCl 0.2 g, Na.sub.2HPO.sub.4 1.42 g, KH.sub.2PO.sub.4 0.27 g, a balance of distilled water to 1 L, pH adjusted to 7.4, sterilized at 1×10Pa for 20 min, and stored at 4° C.

[0027] The instruments and equipment used in the present disclosure are: DNP-9082 electro-thermostatic incubator from Shanghai Jinghong Experimental Equipment Co., Ltd.); MIKRO 120 high-speed desktop centrifuge from German HETTICH manufacturing company); Multifuge X1R desktop refrigerated centrifuge from Thermo Fisher Scientific (China) Co., Ltd.; HVE-50 autoclave from Japan HIRAYAMA company; ZHWY-2102 double-layer thermostatic shaker, Shanghai Zhicheng company; YHC-260 medicine storage cabinet from Shanghai Touching Technology Co., Ltd., and so on.

[0028] The reagents and materials are commercially available, and the equipment and instruments used in the process are common equipment in the art.

[0029] All materials, reagents and instruments used in the present disclosure are well known in the art, but do not limit the implementation of the present disclosure, and some other reagents and equipment well known in the art are suitable to the following embodiments of the present disclosure.

[0030] The following examples further illustrate the embodiments of the present disclosure but should not be construed as limiting the present disclosure. Modifications or substitutions made to the methods, steps or conditions of the present disclosure without departing from the spirit and essence of the present disclosure all fall within the scope of the present disclosure.

Example 1: Therapeutic Phage Cocktail Preparation for Cow Mastitis

[0031] The present disclosure specifically provided a therapeutic phage cocktail preparation for cow mastitis, and the therapeutic phage cocktail preparation for cow mastitis included cow mastitis-derived E. coli phages Ecp1, Ecp3, Ecp5, Ecp16, Ecp17, and vB_EcoM_XJ2, cow mastitis-derived Streptococcus phages vB_StrM_L1 and vB_SagS_FSN1, and cow mastitis-derived S. aureus phages P42 and vB_SauS_IMEP5.

[0032] In the therapeutic phage cocktail preparation for cow mastitis, the cow mastitis-derived E. coli phages, the cow mastitis-derived Streptococcus phages, and the cow mastitis-derived S. aureus phages were mixed in a volume ratio of 1:1:1.

[0033] In the therapeutic phage cocktail preparation for cow mastitis, cow mastitis-derived E. coli phages Ecp1, Ecp3, Ecp5, Ecp16, Ecp17, and vB_EcoM_XJ2 were mixed in a volume ratio of 1:1:1:1:1:1.

[0034] In the therapeutic phage cocktail preparation for cow mastitis, the cow mastitis-derived Streptococcus phages vB_StrM_L1 and vB_SagS_FSN1 were mixed in a volume ratio of 1:1.

[0035] In the therapeutic phage cocktail preparation for cow mastitis, the cow mastitis-derived S. aureus phage P42 and vB_Sau S_IMEP5 were mixed in a volume ratio of 1:1.

Example 2: Method for Preparing Therapeutic Phage Cocktail Preparation for Cow Mastitis

[0036] The disclosure specifically provided a method for preparing the therapeutic phage cocktail preparation for cow mastitis, and the specific steps were as follows:

[0037] (1) frozen cow mastitis-derived E. coli phages Ecp1, Ecp3, Ecp5, Ecp16, Ecp17, and vB_EcoM_XJ2, cow mastitis-derived Streptococcus phages vB_StrM_L1 and vB_SagS_FSN1, and cow mastitis-derived S. aureus phages P42 and vB_Sau S_IMEP5 were selected for recovery;

[0038] (2) 100 μL of the phage stock solution of each strain recovered in step (1) was inoculated into 5 mL of a host bacterial liquid in a logarithmic growth phase respectively, then shaken and cultured at 37° C. at 180 r/min until each solution became clear, and centrifuged at 10,000 r/min for 10 min; a resulting supernatant was taken and diluted with phosphate buffered solution to a concentration of 1×10.sup.9 pfu/mL to obtain phase dilutions;

[0039] (3) the phage dilutions of each strain prepared in step (2) were mixed in the proportion according to Example 1 to prepare a therapeutic phage cocktail preparation for cow mastitis.

Example3: Therapeutic Phage Cocktail Preparation for Cow Mastitis

[0040] The disclosure specifically provided a method for preparing the therapeutic phage cocktail preparation for cow mastitis, and the specific steps were as follows:

[0041] (1) frozen cow mastitis-derived E. coli phages Ecp1, Ecp3, Ecp5, Ecp16, Ecp17, and vB_EcoM_XJ2, and cow mastitis-derived Streptococcus phages vB_StrM_L1 and vB_SagS_FSN1 were selected for recovery;

[0042] (2) 100 μL of a phage stock solution of each strain obtained by purification and screening in step (1) was inoculated into 5 mL of a host bacterial liquid in logarithmic growth phase respectively, shaken and cultured at 37° C. at 180 r/min until each solution became clear, and centrifuged at 10000 r/min for 10 min; a resulting supernatant was taken and diluted to a concentration of 1×10 .sup.9 pfu/mL to obtain phase dilutions;

[0043] (3) the phage dilutions of each strain prepared in step (2) were mixed in the proportion according to Example 1 to prepare a therapeutic phage cocktail preparation for cow mastitis.

Example 4: Therapeutic Phage Cocktail Preparation for Cow Mastitis

[0044] The disclosure specifically provided a method for preparing a therapeutic phage cocktail preparation for cow mastitis, and the specific steps were as follows:

[0045] (1) frozen cow mastitis-derived E. coli phages Ecp1, Ecp3, Ecp5, Ecp16, Ecp17, and vB_EcoM_XJ2, and cow mastitis-derived S. aureus phages P42 and vB_SauS_IMEP5 for recovery;

[0046] (2) 100 μL of the phage stock solution of each strain obtained by purification and screening in step (1) was inoculated into 5 mL of a host bacterial liquid in logarithmic growth phase respectively, shaken and cultured at 37° C. at 180 r/min until each solution became clear, and centrifuged at 10000 r/min for 10 min; a resulting supernatant was taken and diluted to a concentration of 1×10.sup.9 pfu/mL to obtain phase dilutions;

[0047] (3) the phage dilutions of each strain prepared in step (2) were mixed in the proportion according to Example 1 to prepare a therapeutic phage cocktail preparation for cow mastitis.

Example 5: Therapeutic Phage Cocktail Preparation for Cow Mastitis

[0048] The disclosure specifically provided a method for preparing a therapeutic phage cocktail preparation for cow mastitis, and the specific steps were as follows:

[0049] (1) frozen bovine mastitis-derived Streptococcus phages vB_StrM_L1, and vB_SagS_FSN1, and cow mastitis-derived S. aureus phages P42 and vB_SauS_IMEP5 were selected for recovery;

[0050] (2) 100 μL of the phage stock solution of each strain obtained by purification and screening in step (1) was inoculated into 5 mL of a host bacterial liquid in logarithmic growth phase respectively, shaken and cultured at 37° C. at 180 r/min until each solution became clear, and centrifuged at 10000 r/min for 10 min; a resulting supernatant was taken and diluted to a concentration of 1×10.sup.9 pfu/mL to obtain phase dilutions;

[0051] (3) the phage dilutions of each strain prepared in step (2) were mixed in the proportion according to Example 1 to prepare a therapeutic phage cocktail preparation for cow mastitis.

Example 6: Determination of Effect of Different Mixing Ratios of Phages

[0052] Phage cocktails as therapeutic agent for cow mastitis were prepared according to the Examples 2 to 5. After each group of phage preparations were diluted with PBS, they were added to a mixed suspension of main pathogenic bacteria of cow mastitis (E. coli, S. aureus, and Streptococcus). The growth of plaque was observed by double-layer agar method, and the experimental design of each group are shown in Table 1.

TABLE-US-00001 TABLE 1 Experimental Design Group Culture Control Suspension of main pathogens of cow mastitis Treatment therapeutic phage cocktail preparation for cow mastitis group 1 prepared in Example 3 Treatment therapeutic phage cocktail preparation for cow mastitis group 2 prepared in Example 4 Treatment therapeutic phage cocktail preparation for cow mastitis group 3 prepared in Example 5 Treatment therapeutic phage cocktail preparation for cow mastitis group 4 prepared in Example 2

[0053] According to the experimental design in the above table 1, the growth of plaques in each group was observed. No plaque appeared in the control group. The number of plaques in treatment group 4 was the largest. The numbers of plaques in treatment groups 1, 2 and 3 were lower than that of treatment group 4, which showed little difference among the groups. Therefore, it could be seen that in the preparation of the therapeutic phage cocktail preparation for cow mastitis, the combination of cow mastitis-derived E. coli phages, cow mastitis-derived Streptococcus phages and cow mastitis-derived S. aureus phages had the best lysis effect on the main pathogenic bacteria of cow mastitis.

Example 7: Determination of Therapeutic Effect of Therapeutic Phage

Cocktail Preparation for Cow Mastitis

[0054] Based on the screening results of Example 6, the therapeutic effect of the prepared therapeutic phage cocktail preparation for cow mastitis was determined.

(1) Experimental Grouping

[0055] 1. The therapeutic phage cocktail preparation for cow mastitis prepared according to Example 2 with the best in vitro lysis effect was used to perfuse the udders of diseased cows with mastitis as a phage treatment group. A concentration of 1×10.sup.9-1×10.sup.8 pfu/mL phage was directly injected into the gland cistern through the teat canal, with a dosage of 30 mL each for 2-3 times a day for 1-2 weeks.

[0056] 2. Commercially available antimicrobial peptide products were used to perfuse the udders of cows with mastitis as the antimicrobial peptide treatment group, which was directly injected into the gland cistern through the teat canal. The dosage was 30 mL each for 2-3 times a day for 1-2 weeks.

[0057] 3. Routine treatment with antibiotics (penicillin sodium and streptomycin sulfate) was set as the antibiotic treatment group for control.

(2) Determination of Therapeutic Effect

[0058] The typical clinical mastitis and recessive mastitis cases were selected, and the lytic phage or lyase gene expression product was injected into the udder by an udder perfusion method, twice a day, for 1-2 weeks; at the same time, the clinical routine drug treatment group was selected as control. The California Mastitis Test (CMT) was used to measure the incidence of recessive mastitis and the level of SCC in milk samples of the tested cows. Changes in the disease incidence and in the detected negative number of head and mammary region with mastitis across time periods were the main indexes for judgment. The measurement results are shown in Table 2.

TABLE-US-00002 TABLE 2 Determination of the effect of different regimens on cow mastitis. Clinical mastitis Recessive mastitis treatment treatment Cure rate Cure rate Cure rate Cure rate of diseased of mammary of diseased of mammary Treatment cow region cow region Phage 66.67% 75.00% 83.33% 87.50% treatment Antimicrobial Not Not 75.00% 80.00% peptide conducted conducted treatment Antibiotic 58.33% 71.43% 83.33% 85.71% treatment

[0059] The cocktail preparation prepared according to the embodiments of the present disclosure was applied to the treatment of clinical mastitis in cows. After treatment, the SCC in the milk sample of the diseased cow was significantly reduced. The specific determination results are shown in FIG. 1 and FIG. 2. As shown by the measurement results in the above Table 2, the therapeutic effect of the therapeutic phage cocktail preparation for cow mastitis provided in the present disclosure was better than that of the antimicrobial peptide treatment group and the antibiotic treatment group for control. The cocktail preparation prepared from 6 strains of cow mastitis-derived E. coli phage, 2 strains of cow mastitis-derived S. aureus phage, and 2 strains of cow mastitis-derived Streptococcus phage, and the teat perfusion method were used to treat cow mastitis caused by E. coli, S. aureus, and Streptococcus. The cure rate of clinical mastitis of diseased cows by phages was 66.67% (8/12), and the cure rate of clinical mastitis of mammary regions was 75.00% (15/20); the cure rate of recessive mastitis of diseased cows was 83.33% (10/12), and the cure rate in mammary regions was 87.50% (14/16); of the 7 mammary regions that were not cured, 5 were mammary regions from cows with clinical mastitis, and 2 were mammary regions from cows with recessive mastitis. The control group was treated with antibiotics (penicillin sodium and streptomycin sulfate). For clinical cow mastitis, the cure rate was 58.33% (7/12) in the diseased cows, and 71.43% (15/21) in the mammary regions. For recessive cow mastitis, the cure rate was 83.33% (10/12) in diseased cows and 85.71% (18/21) in the mammary regions. Statistical analysis showed that there was a significant difference in the cure rate of the two treatments for cows with clinical mastitis (P=0.033, P<0.05); the cure rate for the recessive mastitis was not significantly different (P=0.087, P>0.05).

[0060] The present disclosure will be well implemented according to the embodiments above. The above-mentioned embodiments are only to describe the preferred embodiments of the present disclosure, and do not limit the scope of the present disclosure. Various changes and improvements to the technical solutions of the present disclosure made by those skilled shall fall within the protection scope determined by the present disclosure.