WO2019154032A1 - 一种宽谱沙门氏菌噬菌体及其应用 - Google Patents

一种宽谱沙门氏菌噬菌体及其应用 Download PDF

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WO2019154032A1
WO2019154032A1 PCT/CN2019/071903 CN2019071903W WO2019154032A1 WO 2019154032 A1 WO2019154032 A1 WO 2019154032A1 CN 2019071903 W CN2019071903 W CN 2019071903W WO 2019154032 A1 WO2019154032 A1 WO 2019154032A1
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salmonella
phage
pharmaceutical composition
bacteriophage
feed additive
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French (fr)
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潘强
任慧英
孙虎芝
刘广芹
王翠
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青岛诺安百特生物技术有限公司
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Priority to US16/967,882 priority Critical patent/US20210046131A1/en
Publication of WO2019154032A1 publication Critical patent/WO2019154032A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/40—Viruses, e.g. bacteriophages
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10332—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a long-tail wide host spectrum S. cerevisiae strong lytic phage and application thereof in a culture environment.
  • Chicken white pheasant is an acute systemic disease caused by Salmonella pullorum, which is one of the most serious bacterial infectious diseases in the chicken industry. It is mainly transmitted vertically through the egg, and can also pass through the digestive tract and respiratory tract infection. Eggs infected with chicken white sputum, mostly showing dead embryos or weak embryos, can not die or die after shelling, generally no special clinical symptoms. Chick infection is generally acute, with a peak incidence of 7 to 10 days of age. There are three common clinical symptoms: acute sepsis, arthritis and neurosis. Most of the clinical symptoms are thin white paste-like feces, and there is a phenomenon of paste anal. The sick chicks often die due to difficulty in breathing and heart failure.
  • the mortality rate of chicks aged 2 to 3 weeks is higher.
  • the chickens infected with chickens at 4 weeks of age are generally less likely to be infected with chickens.
  • the chickens are mainly local or chronic infections and recessive infections.
  • the hens show a decrease in egg production.
  • the mortality rate is low, but it can be sterilized for a long time.
  • the phage has a wide distribution in nature, a simple preparation process, a short development cycle, is not easy to produce drug resistance, and has low cost.
  • the phage has strong specificity, generally only infects the pathogen of a specific species, and does not destroy the normal flora; the phage is also highly proliferative, and can be used as a therapeutic preparation to continuously expand the therapeutic effect.
  • the current research has not found phage treatment. It can cause serious side effects and there is no report of oral allergic reactions of phage.
  • phage therapy is not limited by bacterial resistance, and phage has a bactericidal mechanism that is completely different from antibiotics and is not affected by the antibiotic resistance that bacteria have acquired. The phage only acts at the site of bacterial infection, and decreases with the death of the pathogen until it disappears, without causing secondary pollution.
  • Another object of the present invention is to provide an effective prevention and control product for the disease of Salmonella typhimurium in the breeding industry.
  • the present invention provides a long-tailed broad-spectrum host strain of Salmonella pullorum, a strong lytic phage, and a Salmonella typhimurium A phage preparation having strong lyticity, which can be used alone or in a cocktail. It provides a safe, non-toxic and non-residual phage product for the treatment of Salmonella pullorum infection, providing a phage source for industrial production of phage preparations.
  • Another object of the present invention is to provide a non-polluting and non-residue environmentally-friendly and effective prevention and treatment means, which prepares the phage SP4 into a liquid, lyophilized powder, alone or in combination with other phage, by oral or spraying, It is used to kill Salmonella in the living environment of animals and animals.
  • the object of the present invention is as follows: a long tail broad host spectrum S. cerevisiae strong lytic phage, characterized in that the deposit number is CGMCC No. 14332, and the deposit unit is the common microbiology center of the China Microbial Culture Collection Management Committee. The preservation date is July 27, 2017.
  • the phage is named SP4, and it has lytic activity against Salmonella hominis, Salmonella porcine, Salmonella typhimurium, Salmonella typhimurium and Salmonella typhimurium.
  • the phage has a polyhedral head structure and a non-shrinking tail, the head diameter is about 53 nm, and the tail is about 108 nm.
  • the phage can form a large translucent plaque on the double-layer agar medium plate, and there is no halo around the edge. Clear rules, about 2 ⁇ 3mm in diameter; the phage should belong to the long-tailed phage family.
  • the phage is placed at 40 to 50 ° C for 60 min, its activity is stable, placed at 80 ° C for 20 min, the titer is reduced by about 2 orders of magnitude, and placed in 70-80 ° C for 60 min is inactivated; When the pH is 6 to 9, the activity is stable.
  • the use of the above phage characterized in that the purified phage is capable of lysing Salmonella pullorum, and the 23 strains of 24 strains of Salmonella pullorum collected have a lysis rate of 95.83%.
  • Phage SP4 has a lysis effect on 64 strains of Salmonella in 28 strains (28 pigs, 11 ducks, 14 mink, 14 chickens, and 5 food sources). There are 28 pigs and 6 strains. The cleavage rate was 21.43%; 46 chickens were produced, 41 strains were lysed, and the lysis rate reached 89.13%; 11 ducks were produced, 9 strains were lysed, and the cleavage rate was 81.81%; 14 leeches were lysed, 7 strains were lysed, and the cleavage rate was 50%.
  • the phage In the use of the phage, it is characterized in that the phage is applied to chickens infected with Salmonella pullorum, and the mortality can be reduced by 60% within 2 weeks.
  • the phage In the application of the phage, it can also be used for the control of Salmonella swine, Salmonella typhimurium, Salmonella typhimurium and food source Salmonella infection.
  • the purified phage is prepared into a liquid, lyophilized powder form, or combined with other phage and antibiotics, orally or sprayed, for oral infection of different sources. control.
  • the invention has the advantages that the isolated Salmonella pullorum phage has lytic activity against Salmonella hominis, Salmonella typhimurium, Salmonella typhimurium, Salmonella sulphate and Salmonella sinensis, and is a broad host spectrum phage. It is a new type of environmentally friendly product and means for the prevention and treatment of avian salmonella disease.
  • the safety test of the chicks proved that the phage had no toxic side effects and high safety, and the use of the phage group in the incidence test significantly reduced the mortality of the chicks.
  • Figure 1 is an electron micrograph of SP4 phage.
  • Figure 2 is a picture of SP4 plaque.
  • Figure 3 is a picture of the cleavage map of SP4 phage.
  • M DL 2000 DNA Maker; 1SP4 nucleic acid + RNaseA; 2SP4 nucleic acid + DNaseI; 3SP4 nucleic acid; 4SP4 nucleic acid + BAL31.
  • Figure 4 is the thermal stability of SP4 phage.
  • Figure 5 shows the pH stability of SP4 phage.
  • Figure 6 is a one-step growth curve of SP4 phage.
  • the fecal sewage sample in the invention is collected from a chicken farm in Shandong province;
  • the host strain is Salmonella pullorum CVCC 533.
  • the bacterial solution of the host strain CVCC 533 was picked and streaked on a SS agar medium in three zones, and cultured in an incubator at 37 ° C for 16 to 24 hours to obtain a single colony. A single colony was picked, inoculated into a 5 mL LB broth tube, and cultured overnight at 37 ° C with shaking at 170 rpm to obtain a proliferating liquid.
  • phage stock solution Take 50 mL of chicken farm waste water, add 500 ⁇ L of host bacteria CVCC 533, add LB medium to 200 mL, and incubate overnight at 37 °C. On the next day, 5 mL of the liquid was taken out, centrifuged at 10,000 rpm for 10 min, and the supernatant was filtered through a 0.22 ⁇ m sterile microporous membrane to obtain a phage stock solution, which was stored at 4 ° C.
  • the phage was separated by double-layer plate method, and the phage stock solution was diluted 10-fold. 100 ⁇ L of each of 10 -2 and 10 -4 dilutions was mixed with 200 ⁇ L of host strain CVCC 533 proliferation solution, and incubated at 37 ° C for 5 min, then placed at about 50 ° C. The warmed upper agar (with agar concentration of 0.7%) was mixed and quickly poured onto the lower agar (1.5% agar concentration) plate, shaken evenly until the medium was solidified, and placed in an inverted culture at 37 ° C for 6-8 hours. A double layer plate forming a plaque.
  • Single plaques were picked up in 1 mL of LB broth on a double-layer medium in which plaques were formed, and placed in a 40 ° C water bath for 30 min to obtain a phage leaching solution.
  • a phage leaching solution Take 200 ⁇ L of phage leaching solution and 200 ⁇ L of host bacterial growth solution in 5 mL of liquid LB medium, shake culture at 37 ° C, 170 rpm until the liquid becomes clear, centrifuge the clear liquid at 10000 rpm for 10 min, take the supernatant, and use 0.22 ⁇ m sterile microfiltration. The membrane was filtered to obtain a phage proliferation solution.
  • phage proliferation solution Take 100 ⁇ L of phage proliferation solution and mix well with 200 ⁇ L of host strain CVCC 533 proliferation solution. After incubating at 37 ° C for 5 min, place it on the upper agar with a temperature of about 50 ° C (agar concentration of 0.7%). After mixing, quickly pour the lower agar (agar concentration is 1.5%) On the plate, shake it evenly until the medium is solidified. After incubating at 37 ° C for 6-8 hours, the plaque-forming double-layer plate is obtained again. Single plaques were picked up in 1 mL of LB broth on a double-layer medium in which plaques were formed, and placed in a 40 ° C water bath for 30 min to obtain a phage leaching solution.
  • Phage SP4 was observed under transmission electron microscopy (see Figure 1).
  • the phage head has a polyhedral structure with a slender tail.
  • the phage head has a diameter of about 55 nm, a transverse diameter of about 53 nm, and a tail of about 108 nm.
  • the phage morphology of this study is consistent with the characteristics of the long-tailed phage family and belongs to the long-tailed phage.
  • the phage SP4 can form a large translucent plaque on the double-layer agar medium plate, no halo around, clear edges and a diameter of about 2-3 mm (see Figure 2).
  • the phage nucleic acid was extracted using a viral genomic DNA/RNA extraction kit, and 5 ⁇ L of SP4 phage nucleic acid was mixed with 5 ⁇ L of DNase I, 5 ⁇ L of RNase A, and 5 ⁇ L of BAL31 nuclease and 25 ⁇ L of BAL31 buffer. The mixture was placed in a 37 ° C incubator for 1 h, and the product after the action was subjected to 1% agarose gel electrophoresis. According to the restriction enzyme map (see Fig. 3), the phage SP4 nucleic acid is a double-stranded DNA molecule (dsDNA).
  • dsDNA double-stranded DNA molecule
  • phage SP4 proliferation solution 100 ⁇ L of phage SP4 proliferation solution (potency: 3.8 ⁇ 10 10 PFU/mL) was dispensed into sterile EP tubes and treated in 40 ° C, 50 ° C, 60 ° C, 70 ° C, 80 ° C water bath for 20 min, 40 min. And 60min. Set 2 repetitions for each temperature. After the end of the action, the sample was taken, and the sample was immediately placed in an ice bath to be cooled, and the titer of the phage was measured by a double-layer plate method after dilution by 10 times. Taking the temperature as the abscissa and the logarithm of the phage titer as the ordinate, the phage SP4 thermostability curve was plotted.
  • phage titer was determined by a two-layer plate method for each tube sample, and two replicates were set for each pH.
  • the phage pH stability curve was plotted on the ordinate with the pH as the abscissa and the logarithm of the phage titer.
  • thermostability results showed that phage SP4 remained highly active after 60 min at 40 °C to 50 °C. After 1 h at 60 °C, the titer decreased by 5 titers, and at 70 °C to 80 °C for 60 min. Completely inactivated.
  • the phage of the present invention has good thermal stability and can be added to drinking water or feed.
  • the bacteriophage SP4 maintained its original effective price after 3 hours in the range of pH 6-9; it was active after 3 hours of pH 2-5, pH 10-13, and was more stable at pH 6-9.
  • the supernatant was diluted 10 times with physiological saline, and the phage titer was determined by double-layer plate method. Three parallels were performed, and the results were averaged.
  • the infection time was plotted on the abscissa, and the titer of the phage in the infection system was plotted on the ordinate.
  • a one-step growth curve yields an incubation period and an outbreak period of phage SP4.
  • the titer was basically unchanged within 15 min, and the titer was stable at 10 5 PFU/mL, indicating that the phage SP4 latency was about 15 min, and the phage infected the host strain.
  • the number of phage increased sharply.
  • the titer growth began to stabilize.
  • the titer reached 10 10 PFU/mL.
  • the phage SP4 burst period was about 50 min, and the burst amount was 70.
  • the bacterial solution of the host bacteria stored at -20 ° C was picked up with a sterile inoculating loop, streaked on a SS agar medium in three zones, and cultured in an incubator at 37 ° C for 16 to 24 hours to obtain a single colony.
  • Single colonies of resuscitation culture were picked with a sterilized white tip, inoculated into a test tube containing 5 mL of LB broth, and cultured at 37 ° C, shaking at 170 rpm for 16 h to obtain a single suspension of the host bacteria.
  • Adjust the concentration of host bacteria to 1 ⁇ 10 5 CFU/mL take 1 mL, mix with phage SP4 1 mL of 10 9 , 10 8 , 10 7 , 10 6 , 10 5 PFU/mL, and place at room temperature for 30 min.
  • 1 mL of SM solution was mixed for control treatment. After mixing gently, dilute the liquid by 10 -1 , 10 -2 , 10 -3 , 10 -4 , take 100 ⁇ L of each gradient to a common plate, spread evenly with a sterile coating bar, and incubate for 16 to 24 hours. Do 3 parallels. The number of plate colonies was counted.
  • Phage lysis efficiency (1 - number of colonies in the treatment group / number of colonies in the control group) ⁇ 100%
  • the phage cleavage profile was determined by the single spot method: 1 mL of fresh phage SP4 proliferation solution was taken, and the bacterial debris was sedimented by centrifugation at 10,000 rpm for 10 min. The phage stock solution was initially selected for testing. 104 strains of different origins of Salmonella in the laboratory were selected and streaked in SS plate to obtain single colonies. Single colonies were picked and inoculated into 5 mL nutrient broth, and cultured at 37 ° C, 170 rpm for 12 h to obtain bacterial strains of each strain. 100 ⁇ L of bacterial bacterial solution was uniformly coated on a common agar plate. After drying, 1 ⁇ L of SP4 phage proliferation droplets were taken on the plate, and cultured at 37 ° C for 8-12 hours after natural drying, and the results were observed.
  • phage SP4 has a broad spectrum of cleavage and can be used for the control of Salmonella infection from different sources.
  • Twenty one-day-old SPF chicks were purchased from a certain chicken farm in Qingdao and randomly divided into experimental group and blank control group.
  • the experimental group was administered with phage SP4 proliferation solution 1 ⁇ 10 10 PFU/mL/0.25 mL/only.
  • the rats were given an equal volume of sterile saline for 7 days. The behavior and growth of the chicks were observed. After 7 days, 5 chicks were dissected in each group to observe changes in visceral and digestive tract and mucosa.
  • 120 healthy 1 day old chicks were selected and divided into 3 groups, control group, infection group and treatment group, 40 in each group.
  • the infection was established as follows. Salmonella pullorum CVCC 533 monoclonal was picked in 5 mL LB medium and cultured for 24 h to adjust the concentration to 1 ⁇ 10 8 CFU/mL. The control group did not attack the bacteria.
  • each chicken was orally administered with 100 ⁇ L.
  • the treatment group was orally administered with bacteriophage SP4 at the same time as the oral bacteria, and the infected group was orally administered with an equal volume of PBS for 5 days, followed by normal feeding for 14 days, and the chicks of each group were observed. Death status, calculate mortality.

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Abstract

提供一种沙门氏菌噬菌体，特别涉及一种长尾宽宿主谱鸡白痢沙门氏菌强裂解性噬菌体。上述鸡白痢沙门氏菌噬菌体命名为SP4，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏日期为2017年7月27日，保藏编号为CGMCC No.14332。该噬菌体对沙门氏菌具有较强的裂解作用，该噬菌体还可以降低感染鸡白痢的雏鸡的死亡率。该制剂可以单独或鸡尾酒使用，为鸡源沙门氏菌、鸭源沙门氏菌、水貂源沙门氏菌、食品源沙门氏菌以及猪源沙门氏菌感染的治疗提供一种安全、无毒副及无残留作用的噬菌体产品来源。

Description

一种宽谱沙门氏菌噬菌体及其应用 技术领域
本发明属于生物技术领域，尤其涉及一种长尾宽宿主谱鸡白痢沙门氏菌强裂解性噬菌体及其在养殖环境中的应用。
背景技术
鸡白痢是由鸡白痢沙门氏菌引起的急性系统性疾病，危害养鸡业最严重的细菌性传染病之一，主要经卵垂直传播，亦可经消化道，呼吸道感染。蛋内感染鸡白痢者，多表现死胚或弱胚，不能出壳或出壳后死亡，一般无特殊的临床症状。雏鸡感染一般呈急性经过，发病高峰在7～10日龄，常见的临床症状有三型：急性败血型、关节炎型和神经型。临床症状多为排稀薄白色浆糊状粪便，出现糊肛现象，病雏多因呼吸困难及心力衰竭而死。2～3周龄的雏鸡感染死亡率较高，4周龄以上鸡感染鸡白痢一般较少死亡，成鸡以局部或慢性感染、隐性感染为主，母鸡表现产蛋量下降，感染后死亡率较低，但可长期带菌排菌。
目前临床防治鸡白痢的主要方法为定期检疫，净化种鸡群，全进全出和自繁自养的管理措施及生产模式。由于沙门氏菌病可呈水平传播和垂直传播，上述预防作用并不理想，目前多数养殖场以药物预防为主，大量抗生素预防导致细菌耐药性问题严重，继控制沙门氏菌污染成为世界性难题后，沙门菌耐药性问题也已经是一个全球性的问题。因此，研究开发无污染、无残留、安全、可替代抗生素的新型产品解决沙门氏菌污染问题，引起各界广泛关注。
噬菌体自然界分布广泛，制备工艺简单，研发周期短，不易产生耐药性，成本低廉。噬菌体具有很强的特异性，一般只感染特定种属的病原菌，不会破坏正常菌群；噬菌体的增殖能力也非常强，作为治疗制剂使用能不断扩大作用疗效，目前的研究还没有发现噬菌体治疗会引起严重副反应，也没有噬菌体口服过敏现象的报道。最重要的是噬菌体治疗不受细菌耐药性的限制，噬菌体具有完全不同于抗生素的杀菌机制，不受到细菌已经获得的抗生素耐药性的影响。噬菌体只在细菌感染的部位发生作用，随着病原菌的死亡而减少，直至消失，不会造成二次污染。
发明内容
本发明的目的在于：提供一种长尾宽宿主谱的鸡白痢沙门氏菌噬菌体。
本发明的另一目的是：针对目前养殖业中鸡白痢沙门氏菌疾病提供一种有效防治的产品，本发明提供一种长尾宽宿主谱鸡白痢沙门氏菌强裂解性噬菌体，开发一种对鸡白痢沙门氏菌具有强裂解性的噬菌体制剂，该制剂可以单独或鸡尾酒使用。为鸡白痢沙门氏菌感染的治疗提供一种安全、无毒副及无残留作用的噬菌体产品，为工业化生产噬菌体制剂提供噬菌体来源。
本发明的又一目的是：提供一种无污染、无残留的环保有效的防治手段，将所述的噬菌体SP4制备成液体、冻干粉，单独或配合其他噬菌体，经口服、喷雾等方式，用于杀灭动物体内外、养殖空间环境中的沙门氏菌。
本发明的目的是这样实现的：一种长尾宽宿主谱鸡白痢沙门氏菌强裂解性噬菌体，其特征在于：保藏号为CGMCC No.14332，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心，保藏日期为2017年7月27日，该噬菌体命名为SP4，其对鸡源沙门氏菌、猪源沙门氏菌、鸭源沙门氏菌、貂源沙门氏菌以及食品源沙门氏菌均有裂解活性。
该噬菌体具有呈多面体的头部结构和无收缩性的尾部，头部直径约53nm，尾部长约108nm；噬菌体在双层琼脂培养基平板上可以形成较大透亮空斑，周围无晕环，边缘清晰规则，直径约2～3mm；该噬菌体应属于长尾噬菌体科。
在本发明中：所述的噬菌体在40～50℃中放置60min，其活性稳定，在80℃中放置20min，效价降低约2个数量级，在70～80℃中放置60min则失活；在pH为6～9时，活性稳定。
一种上述噬菌体的应用，其特征纯化后的噬菌体能够裂解鸡白痢沙门氏菌，对所收集的24株鸡白痢沙门氏菌中的23株有裂解作用，裂解率为95.83％。
一种上述噬菌体的应用，其特征纯化后的噬菌体能够裂解不同宿主来源的沙门氏菌。噬菌体SP4对104株(猪源28株、鸭源11株、水貂源14株、鸡源46株、食品源5株)沙门氏菌中的64株菌具有裂解作用，猪源28株，裂解6株，裂解率为21.43％；鸡源46株，裂解41株，裂解率达到89.13％；鸭源11株，裂解9株，裂解率为81.81％；水貂源14株，裂解7株，裂解率为50％；食品源5株，裂解1株，裂解率为20％；对46株鸡源沙门氏菌中24株鸡白痢 沙门氏菌可裂解23株，裂解率高达95.83％。。
在所述的噬菌体的应用中，其特征在于：在感染鸡白痢沙门氏菌雏鸡中应用此噬菌体，2周内可以减少60％的死亡率。
在所述噬菌体的应用中，还可以用于猪沙门氏菌、鸭沙门氏菌、水貂沙门氏菌以及食品源沙门氏菌感染的控制。
在所述的噬菌体的应用中，其特征在于：将纯化后的噬菌体制备成液体、冻干粉形式，单独或与其他噬菌体、抗生素复配后通过口服或喷雾等途径用于不同来源沙门氏菌感染的控制。
本发明的优点在于：分离到的鸡白痢沙门氏菌噬菌体对鸡源沙门氏菌、猪源沙门氏菌、鸭源沙门氏菌、水貂源沙门氏菌以及食品源沙门氏菌均有裂解活性，是一种宽宿主谱的噬菌体。是一种新型环保的防治禽沙门氏菌疾病的产品和手段。经雏鸡安全性试验证明，该噬菌体无毒副作用，安全性高，在发病试验中服用本噬菌体组明显降低了雏鸡死亡率。
附图说明
图1为SP4噬菌体的电镜图片。
图2为SP4噬菌斑图片。
图3为SP4噬菌体酶切图谱图片。其中，M：DL 2000DNA Maker；1SP4核酸+RNaseA；2SP4核酸+DNaseⅠ；3SP4核酸；4SP4核酸+BAL31。
图4为SP4噬菌体的热稳定性。
图5为SP4噬菌体的pH值稳定性。
图6为SP4噬菌体的一步生长曲线。
具体实施方式
下面结合具体的实施来进一步阐述本发明。应理解，这些实施仅用于说明本发明，而不是用来限制本发明的范围。下述实施中所使用的实验方法如无特殊说明，均为常规方法。
实施例1噬菌体SP4的分离制备
本发明中的粪液污水样品采自山东省某鸡场；
宿主菌为鸡白痢沙门氏菌CVCC 533。
1、宿主菌CVCC 533的复苏培养与增值液制备
挑取宿主菌CVCC 533的菌液，在SS琼脂培养基上三区划线，37℃的恒温箱中培养16～24h，得到单菌落。挑取单菌落，接种于5mL的LB肉汤试管中，37℃，170rpm振荡培养过夜，得到增殖液。
2、粪液污水样品处理
取50mL鸡场的粪液污水，加入500μL宿主菌CVCC 533，再加入LB培养基至200mL，37℃浸泡孵育过夜。次日，取出5mL的液体，10000rpm离心10min，上清液经0.22μm的无菌微孔滤膜滤过，得到噬菌体原液，置于4℃保存。
3、噬菌体的分离
利用双层平板法分离噬菌体，将噬菌体原液10倍稀释，取10 -2和10 -4稀释液各取100μL与200μL宿主菌CVCC 533增殖液混合均匀，37℃孵育5min后，置于约50℃保温的上层琼脂(琼脂浓度为0.7％)，混匀后迅速倾倒下层琼脂(琼脂浓度为1.5％)平皿上，摇匀平置至培养基凝固，置于37℃倒置培养6～8h后，获得形成噬菌斑的双层平板。
实施例2噬菌体的增殖和纯化
1、噬菌体的增殖
在形成噬菌斑的双层培养基上用灭菌镊子挑取单个噬菌斑置于1mL的LB肉汤中，置于40℃水浴锅中孵育30min，得到噬菌体浸出液。取200μL噬菌体浸出液与200μL宿主菌增殖液于5mL液体LB培养基中，37℃，170rpm振荡培养，直至液体变清亮，将清亮液体10000rpm离心10min，取上清，使用0.22μm的无菌微孔滤膜滤过，得到噬菌体增殖液。
2、噬菌体的纯化
取噬菌体增殖液100μL与200μL宿主菌CVCC 533增殖液混合均匀，37℃孵育5min后，置于约50℃保温的上层琼脂(琼脂浓度为0.7％)，混匀后迅速倾倒下层琼脂(琼脂浓度为1.5％)平皿上，摇匀平置至培养基凝固，置于37℃倒置培养6～8h后，再次获得形成噬菌斑的双层平板。在形成噬菌斑的双层培养基上用灭菌镊子挑取单个噬菌斑置于1mL的LB肉汤中，置于40℃水浴锅中孵育30min，得到噬菌体浸出液。取200μL噬菌体浸出液与200μL宿主菌增殖液于5mL液体LB培养基中，37℃，170rpm振荡培养，直至液体变清亮，将清亮液体10000rpm离心10min，取上清，使用0.22μm的无菌微孔滤膜滤过，得到 噬菌体增殖液。如此循环3次，得到纯化后的噬菌体悬液。
实施例3噬菌体的生物学特性
1、噬菌体SP4的形态学特性
噬菌体SP4在透射电镜下观察(见图1)。该噬菌体头部呈多面体结构，有细长的尾部，噬菌体头部长径约55nm，横径约53nm，尾部约108nm。根据噬菌体分类，本研究噬菌体形态符合长尾噬菌体科的特征，属于长尾噬菌体。
2、噬菌体SP4培养特性
噬菌体SP4在双层琼脂培养基平板上可以形成较大透亮空斑，周围无晕环，边缘清晰规则，直径约2-3mm(见图2)。
3、噬菌体SP4基因组核酸类型鉴定
将大规模增殖的噬菌体SP4用PEG-NaCl法浓缩后，使用病毒基因组DNA/RNA提取试剂盒对噬菌体核酸进行提取，取5μL SP4噬菌体核酸分别与5μL DNaseⅠ、5μLRNaseA以及5μL BAL31核酸酶和25μL BAL31buffer混合,将混合物置于37℃温箱中作用1h，取作用之后的产物进行1％的琼脂糖凝胶电泳。根据酶切图谱(见图3)分析得出，该噬菌体SP4核酸是双链DNA分子(dsDNA)。
实施例4温度及pH对噬菌体SP4的影响
各取100μL噬菌体SP4增殖液(效价为3.8×10 10PFU/mL)分装于无菌EP管中，分别于40℃、50℃、60℃、70℃、80℃水浴中作用20min、40min和60min。每一温度设2个重复。待作用结束后取样，并立即将样品置于冰浴中冷却，通过10倍倍比稀释后用双层平板法测定噬菌体的效价。以温度为横坐标，以噬菌体效价的对数值为纵坐标，绘制噬菌体SP4热稳定性曲线。
以实施例4的噬菌体SP4为基础，于无菌试管加入不同pH值(2、3、4、5、6、7、8、9、10、11、12、13)的LB肉汤4.5mL，然后将上述试管置于37℃的水浴锅中，待温度平衡后各加入500μL噬菌体增殖液混匀后，37℃水浴作用1h、2h和3h。每管样品采用双层平板法测定噬菌体效价，并且每个pH值设定2个重复。以pH值为横坐标，噬菌体效价的对数值为纵坐标绘制噬菌体pH值稳定性曲线。
热稳定性结果发现(图4)，噬菌体SP4在40℃～50℃作用60min后依然保持较高活性，在60℃作用1h后效价下降5个滴度，在70℃～80℃作用60min 时完全失活。说明本发明噬菌体的热稳定性较好，能够在饮水或饲料中添加使用。
如图5所示，噬菌体SP4在pH 6～9范围内3h后保持原有效价；在pH 2～5、pH 10～13范围内作用3h后均有活性，在pH 6～9噬菌体更稳定。
实施例5噬菌体SP4的一步生长曲线
以实施例4的噬菌体SP4为基础，将感染复数为10的噬菌体SP4增殖液和宿主菌新鲜增殖液各1mL，充分混匀(此时开始计时)，37℃孵育5min，13000g离心30s，用微量移液器尽量吸去上清，再用5mL LB肉汤洗涤1次(13000g离心30s)，弃上清。用预热的LB肉汤混悬沉淀(总体积为5mL)并充分混匀，迅速置于37℃摇床中170rpm振荡培养，在0时刻和每隔10min取出150μL，10000rpm离心1min，吸取100μL的上清用生理盐水10倍倍比稀释后用双层平板法测定噬菌体效价，做3个平行，结果取平均值，以感染时间为横坐标，感染体系中噬菌体的滴度为纵坐标，绘制一步生长曲线，得到噬菌体SP4的潜伏期、爆发期。
通过图6噬菌体SP4一步生长曲线发现，噬菌体感染宿主菌后，在15min内效价基本不变，效价稳定在10 5PFU/mL，表明噬菌体SP4潜伏期约为15min，噬菌体侵染宿主菌后的10～60min内，噬菌体数量急剧增加，在60min时效价增长开始趋于平稳，此时效价可达10 10PFU/mL，可见噬菌体SP4的爆发期约为50min，爆发量为70。
实施例6噬菌体SP4的体外裂解效率
用无菌接种环挑取于-20℃保存的宿主菌的菌液，在SS琼脂培养基上三区划线，37℃的恒温箱中培养16～24h，得到单菌落。用灭菌的白色枪头挑取复苏培养的单菌落，接种盛有5mL的LB肉汤的试管中，37℃，170rpm震荡培养16h，得到单一的宿主菌悬液。调整宿主菌浓度为1×10 5CFU/mL，取1mL，分别用10 9，10 8，10 7，10 6，10 5PFU/mL的噬菌体SP4 1mL与之混合，室温放置30min，同时设与1mL的SM液混合的对照处理。轻微混匀后，将液体稀释10 -1，10 -2，10 -3，10 -4，每个梯度取100μL至普通平板，用无菌涂布棒涂布均匀，倒置培养16～24h，每个做3个平行。进行平板菌落数计数。
噬菌体裂解效率＝(1-处理组菌落数/对照组菌落数)×100％
通过噬菌体SP4体外裂解效率测定发现，噬菌体浓度≥10 8PFU/mL，对宿主菌完全裂解，噬菌体浓度≤10 7PFU/mL时裂解率逐渐下降。
实施例7噬菌体裂解谱的分析
以实施例4的噬菌体SP4为基础，采用单斑法测定噬菌体的裂解谱：取1mL新鲜噬菌体SP4增殖液，10000rpm离心10min沉降细菌碎片。初步选择噬菌体原液进行试验。分别挑取实验室保存的104株不同来源沙门氏菌在SS板划线培养得到单菌落，挑取单菌落分别接种于5mL营养肉汤中，37℃，170rpm培养12h，得到各株细菌菌液，取100μL细菌菌液分别均匀涂布在普通琼脂平板上，待干燥后，取1μL SP4的噬菌体增殖液滴于平板上，待自然干燥后37℃培养8～12h，观察结果。
通过裂解谱测定实验发现，试验选用的细菌菌液在平板上生长良好，噬菌体SP4对104株(猪源28株、鸭源11株、水貂源14株、鸡源46株、食品源5株)沙门氏菌中的64株菌具有裂解作用，猪源28株，裂解6株，裂解率为21.43％；鸡源46株，裂解41株，裂解率达到89.13％；鸭源11株，裂解9株，裂解率为81.81％；水貂源14株，裂解7株，裂解率为50％；食品源5株，裂解1株，裂解率为20％。其中对46株鸡源沙门氏菌中24株鸡白痢沙门氏菌可裂解23株，裂解率高达95.83％。说明噬菌体SP4有较宽的裂解谱，可用于不同来源的沙门氏菌感染的控制。
实施例8噬菌体的安全性试验
选择20只1日龄SPF雏鸡，购自青岛某种鸡场，随机分成实验组和空白对照组2组，试验组灌服噬菌体SP4增殖液1×10 10PFU/mL/0.25mL/只，试验组灌服等体积无菌生理盐水，连续服7d，观察小鸡的行为表现及其生长情况，7d后每组剖检5只小鸡，观察内脏和消化道及黏膜变化情况。
结果发现试验组和对照组小鸡生长情况一致，无不良反应，两组剖检的鸡内脏、消化道黏膜等未见异常，从而确定噬菌体SP4安全无毒副作用。
实施例9噬菌体SP4的雏鸡治疗试验
选择120只健康的1日龄的雏鸡，分为3组，对照组、感染组、治疗组，每组40只。按以下方式建立感染，挑取鸡白痢沙门氏菌CVCC 533单克隆于5mL LB培养基中，培养24h，调整其浓度为1×10 8CFU/mL。对照组不攻菌，感染组每只小鸡口服100μL，治疗组在口服细菌的同时口服噬菌体SP4，感染组口服等体积PBS，连续灌服5天，后期正常饲养14天，观察各组小鸡死亡情况，计算死亡率。
结果显示，对照组雏鸡全部成活，感染组雏鸡死亡率100％，治疗组雏鸡死亡率40％，可知感染鸡白痢沙门氏菌的雏鸡服用噬菌体SP4可以减少60％的死亡率。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种沙门氏菌噬菌体，其特征在于，其保藏号为：CGMCC No.14332。
  2. 权利要求1所述的沙门氏菌噬菌体在制备治疗或预防沙门氏菌感染的疾病的药物中的用途。
  3. 根据权利要求2所述的用途，其中，所述沙门氏菌选自鸡源沙门氏菌、猪源沙门氏菌、鸭源沙门氏菌、水貂源沙门氏菌、食品源沙门氏菌；所述鸡源沙门氏菌选自鸡白痢沙门氏菌、鸡伤寒沙门氏菌和鸡肠炎沙门氏菌。
  4. 根据权利要求2所述的用途，其中，所述沙门氏菌感染的疾病选自鸡白痢、禽伤寒、禽副伤寒、仔猪副伤寒、水貂沙门氏菌病、鼠伤寒沙门氏菌肠炎。
  5. 一种药物组合物或饲料添加剂，其包含权利要求1所述的沙门氏菌噬菌体。
  6. 根据权利要求5所述的药物组合物或饲料添加剂，所述药物组合物中还包含药学上可接受的载体。
  7. 根据权利要求5或6所述的药物组合物或饲料添加剂，所述的药物组合物的药物制剂形式为口服给药剂型或喷雾剂型或肠外给药剂型；
    优选地，所述药物制剂形式为口服给药剂型。
  8. 根据权利要求5-7任一项所述的药物组合物或饲料添加剂，所述的药物组合物或饲料添加剂中还包含至少一种治疗沙门氏菌感染疾病的活性成分；所述治疗沙门氏菌感染疾病的活性成分选自其他种类沙门氏菌噬菌体。
  9. 根据权利要求5-8任一项所述的药物组合物或饲料添加剂，所述的药物组合物或饲料添加剂中权利要求1所述的沙门氏菌噬菌体的效价≥10 5PFU/mL，优选为≥10 8PFU/mL。
  10. 一种环境消毒剂，其包含权利要求1所述的沙门氏菌噬菌体。
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