CN111549156A - Method for establishing PK-PD (PK-PD) synchronous model of tilmicosin on lawsonia intracellularis - Google Patents
Method for establishing PK-PD (PK-PD) synchronous model of tilmicosin on lawsonia intracellularis Download PDFInfo
- Publication number
- CN111549156A CN111549156A CN202010409968.5A CN202010409968A CN111549156A CN 111549156 A CN111549156 A CN 111549156A CN 202010409968 A CN202010409968 A CN 202010409968A CN 111549156 A CN111549156 A CN 111549156A
- Authority
- CN
- China
- Prior art keywords
- tilmicosin
- lawsonia intracellularis
- concentration
- establishing
- intracellularis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Toxicology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for establishing a PK-PD synchronous model of tilmicosin on lawsonia intracellularis, which comprises the following steps: drawing a growth curve of lawsonia intracellularis in IPEC-J2 cells; determining the minimum inhibitory concentration, the mutation-preventing concentration and the post-antibacterial effect of tilmicosin on the internal and external lawsonia intracellularis of the pig, and drawing in-vitro and half-body internal intracellular sterilization curves; according to the measured half-body intracellular sterilization curve of the lawsonia intracellularis, selecting a proper PK-PD parameter, fitting data, establishing a PK-PD synchronous model of tilmicosin to the lawsonia intracellularis, and formulating a reasonable administration scheme. The invention can formulate the drug delivery scheme of tilmicosin to lawsonia intracellularis, provide stable drug data support for scientific culture and scientifically guide clinical drug delivery; effectively relieves the drug resistance of the lawsonia intracellularis to tilmicosin, protects and maintains the effectiveness of the tilmicosin, and provides guidance for the research of the antibacterial drug on the PK-PD synchronous model of the lawsonia intracellularis.
Description
Technical Field
The invention relates to the technical field of PK-PD synchronous models, in particular to a method for establishing a PK-PD synchronous model of tilmicosin on lawsonia intracellularis of pigs.
Background
Lawsonia Intracellularis (Lawsonia Intracellularis, l.intracellularis) belongs to the genus devulcanium, gram-negative bacteria, are arcuately curved, comma-shaped or S-shaped, have a size of 1.25 to 1.75 μm × 0.25 to 0.43 μm, are obligately parasitic in animal intestinal cells, and can cause Porcine Proliferative Enteritis (PPE) characterized by adenomatous hyperplasia of immature intestinal cells in ileum and crypt colon. Although the death rate of PPE is not high, the PPE causes clinical symptoms such as diarrhea, slow growth and the like of sick pigs, and seriously reduces the feed utilization rate of animals, thereby causing huge economic loss.
With the increasing resistance of porcine proliferative enteritis to antibiotics, more and more drugs cannot be used for clinically treating porcine proliferative enteritis, so that the control and the slow-down of the generation and the spread of the drug resistance are far and heavily influenced by strict scientific means. Tilmicosin as a novel macrolide antibiotic has strong permeability to cell membranes, can quickly permeate the cell membranes to reach the intracellular state, and has good accumulation capacity in intestinal cells. In addition, the in vitro bacteriostasis test result of the lawsonia intracellularis shows that tilmicosin has stronger antibacterial activity to the lawsonia intracellularis than other antibacterial drugs. Therefore, tilmicosin can be used as the first choice medicine for the clinical lawsonia intracellularis infection at present. In order to scientifically and effectively use the tilmicosin in the treatment of the porcine proliferative enteritis, slow down the generation of drug resistance of the tilmicosin and help veterinarians in China to have rules in the clinical process, the formulation of a reasonable drug administration scheme has important significance for solving the using defects of the drugs in the prior art.
Disclosure of Invention
The invention aims to provide a method for establishing a PK-PD synchronous model of tilmicosin on lawsonia intracellularis, a reasonable administration scheme of tilmicosin on lawsonia intracellularis is prepared by measuring pharmacodynamics of tilmicosin on lawsonia intracellularis and pharmacokinetic parameters in a pig body, so that stable administration data support is provided for scientific culture, clinical medication can be more scientifically guided, and porcine proliferative enteritis can be effectively treated and reference is provided for clinical medication; the drug resistance of lawsonia intracellularis to tilmicosin can be effectively relieved to a certain extent, and the effectiveness of tilmicosin is protected and maintained; can provide a guide for the research of a drug on a PK-PD synchronous model of intracellular bacteria.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for establishing a PK-PD synchronous model of tilmicosin on lawsonia intracellularis, which comprises the following steps:
Counting by adopting a peroxidase cell monolayer staining method, and determining the minimum inhibitory concentration MIC of tilmicosin on the internal and external cells of the lawsonia intracellularis, the specific process comprises the steps of paving the porcine jejunum epithelial cells in a 24-pore plate, and adding lawsonia intracellularis (the concentration is 5 × 10) when the confluency of the cells is about 60 percent5intercellularis/mL), culturing for 24h, removing the supernatant, and washing with PBS 3 times; thereafter, the cells were replaced with cell culture media containing tilmicosin at different concentrations (128. mu.g/mL, 64. mu.g/mL, 32. mu.g/mL, 16. mu.g/mL, 8. mu.g/mL, 4. mu.g/mL, 2. mu.g/mL, 1. mu.g/mL, 0.5. mu.g/mL, 0.25. mu.g/mL) every day for continuous culture for 5 days. Using a peroxidase single-layer staining method to stain, count and detect by microscopy to obtain the intracellular most abundant tilmicosin on the lawsonia intracellularis of the pigLow bacteriostatic concentration.
Lawsonia intracellularis (concentration 5 × 10)5intercellularis/mL), adding cell culture media containing tilmicosin at different concentrations ((128. mu.g/mL, 64. mu.g/mL, 32. mu.g/mL, 16. mu.g/mL, 8. mu.g/mL, 4. mu.g/mL, 2. mu.g/mL, 1. mu.g/mL, 0.5. mu.g/mL, 0.25. mu.g/mL), after 3 hours of action, adding the mixture to 24-well plates containing about 60% of cell confluency, changing the culture media to a medium containing no drug concentration every day, and continuously culturing for 5 days; and (3) performing staining counting by using a peroxidase single-layer staining method, and performing microscopic examination to obtain the extracellular minimum inhibitory concentration of tilmicosin on the lawsonia intracellularis of the pigs.
Counting by adopting a fluorescent quantitative PCR method, and drawing in-vitro and in-vivo intracellular sterilization curves of the tilmicosin on the lawsonia intracellularis in the porcine jejunum epithelial cells5intercellularis/mL), culturing for 3h, removing the supernatant, and washing with PBS 3 times; continuously culturing for 3 days to make the bacterial concentration reach 106And after L.intracellularlaris/mL, taking the measured minimum intracellular inhibitory concentration of tilmicosin to the lawsonia intracellularis of the pigs as a reference, adding cell culture media of tilmicosin with different concentrations (0MIC, 1/2MIC, 1MIC, 2MIC, 4MIC, 8MIC and 16MIC), sampling at 0h, 1h, 2h, 4h, 8h, 12h, 18h, 24h, 36h and 48h respectively, measuring the content of bacteria by adopting fluorescence quantitative PCR, and drawing an in-vitro intracellular sterilization curve of the lawsonia intracellularis.
Half-body intracellular sterilization curve because plasma and ileum contents of healthy and sick pigs collected in pharmacokinetic experiment are administrated, the sterilization characteristics of the drug in the healthy and sick pigs are measured in half-body, and the drawing of the half-body intracellular sterilization curve of lawsonia intracellularis in porcine ileum contents by tilmicosin is basically the same as the test method of the half-body intracellular sterilization curve of lawsonia intracellularis in porcine with tilmicosin, except that tilmicosin with different concentrations is added into a cell culture medium, so that the concentration of the drug in the intestinal contents measured at each time point is kept consistent, and then the lawsonia intracellularis (the concentration is 5 × 10)5L.intracellularis/mL). Sampling at 0h, 1h, 2h, 4h, 8h, 12h, 18h, 24h, 36h and 48h respectively, determining the content of bacteria by adopting fluorescent quantitative PCR, and drawing a semi-body intra-cell sterilization curve of the Lawsonia intracellularis.
And counting by adopting a fluorescent quantitative PCR method, and determining the mutation-preventing concentration of the lawsonia intracellularis in the porcine jejunal epithelial cells. The specific process is as follows: preparing cell culture media containing tilmicosin with different concentrations (0MIC, 1/2MIC, 1MIC, 2MIC, 4MIC, 8MIC and 16MIC) by taking the determined lowest intracellular inhibitory concentration of tilmicosin to Lawsonia as a reference; spreading the porcine jejunum epithelial cells in a 24-pore plate, adding the lawsonia intracellularis when the confluency of the cells is about 60%, culturing for 3h, removing supernatant, and washing for 3 times by PBS; continuously culturing for 5 days to make the bacterial concentration reach 107After l. intercellularis/mL, after centrifugation and resuspension, the concentration of bacteria was adjusted to achieve a concentration of about 109L. intracellularis/mL, adding cell culture media containing different drug concentrations, and placing in an incubator for 5 days; initial anti-mutation concentration (MPC) was set at the lowest drug concentration at which colonies still grew aseptically after 5 days of culturepr) Then using MPCprAnd linearly decreasing the concentration of the antibacterial drug by 20 percent to be a baseline, preparing the drug-containing cell culture medium with different concentrations (16 mu g/mL, 8 mu g/mL, 7 mu g/mL, 6.4 mu g/mL, 6 mu g/mL, 4 mu g/mL and 2 mu g/mL), repeating the steps, and obtaining the final MPC as the lowest drug concentration at which the colony growth does not occur after the MPC is put into an incubator for 5 days.
And counting by adopting a fluorescent quantitative PCR method, and determining the antibacterial after-effect of the lawsonia intracellularis in the porcine jejunal epithelial cells. The specific process is as follows: preparing a cell culture medium containing tilmicosin with different concentrations (1MIC, 2MIC and 4MIC) by taking the determined lowest intracellular bacteriostatic concentration of tilmicosin to lawsonia intracellularis as a reference, paving the porcine jejunum epithelial cells in a 24-hole plate, adding lawsonia intracellularis when the cell confluency is about 60%, culturing for 3 hours, removing a supernatant, cleaning for 3 times by PBS (phosphate buffer solution) and continuously culturing for 3 days to ensure that the bacterial concentration reaches 106Adding cell culture media containing different drug concentrations after L.intracellularis/mL, placing in an incubator for culturing for 1h and 2h, and inducing the generation of PAE; at the same time, is provided withControl groups, two groups A1, A2, contained cells and bacteria only (no drug). Removal and reconstitution of drug: after 1h and 2h for the experimental and control groups, respectively, the supernatant was removed, washed 3 times with PBS and immediately placed in the incubator, which is 0h after reconstitution. Establishing a bacterial growth kinetic curve: and (3) measuring the bacterial content of each time point by using fluorescent quantitative PCR (polymerase chain reaction) at 0, 1, 2, 4, 6, 8, 12 and 24 hours after reconstruction, establishing a kinetic curve of growth recovery of the control bacteria and the experimental bacteria after reconstruction, and calculating the PAE.
Step 2: selecting proper PK-PD parameters according to the in-vivo sterilization curve of tilmicosin to lawsonia intracellularis in the pig, which is measured in the step 1, and simulating Sigmoid E by using Winnolin softwaremaxAnd a PK-PD model equation, calculating pharmacodynamic target values of tilmicosin on the lawsonia intracellularis under different antibacterial effects, substituting the pharmacodynamic target values corresponding to different antibacterial effects of tilmicosin into a dose calculation equation, calculating to obtain the administration dose under the conditions of bacteriostasis, sterilization and eradication, making a reasonable administration scheme, and establishing a PK-PD synchronous model of the tilmicosin on the lawsonia intracellularis.
Preferably, the lawsonia intracellularis is a strain separated from a suspected porcine proliferative enteritis ileal disease material, and the concentration of the strain is qualitatively detected by PCR and quantitatively detected by fluorescent quantitative PCR.
Preferably, the acquisition of pharmacokinetic parameters of lawsonia intracellularis by tilmicosin in step 1 comprises the following steps: establishing a piglet fistula model, setting an experimental group and a control group, using the separated lawsonia intracellularis strain to detoxify the experimental group, establishing a fistula model for artificially infecting lawsonia intracellularis, intragastrically administering two groups of animals according to body weight, taking plasma and ileum contents at different time points after administration, and detecting tilmicosin drug concentration in the plasma and the ileum contents by using a high performance liquid chromatography.
Preferably, the determination of the mutation-proof concentration is carried out by taking the determined minimum intracellular inhibitory concentration of tilmicosin to Lawsonia as a reference, preparing cell culture media containing tilmicosin with different concentrations, and taking the minimum drug concentration without bacterial colony after observation for 5 days as an initial mutation-proof concentration MPCprThen using MPCprFor baseline, linearly decreasing the concentration of 20% antibacterial agent, preparing tilmicosin cell culture media with different concentrations, and taking the lowest concentration of the antibacterial agent at which colonies do not appear after 5 days as the final MPC.
Preferably, the antibacterial after-effect is that a cell culture medium containing tilmicosin with different concentrations is prepared by taking the measured minimum intracellular inhibitory concentration of tilmicosin to lawsonia, the cell culture medium induces the production of PAE, two control groups only containing IPEC-J2 and porcine lawsonia intracellularis are arranged at the same time, the experimental group and the control groups are subjected to drug removal and reconstruction, the porcine lawsonia intracellularis content at different time points is measured by fluorescence quantitative PCR, a growth recovery kinetic curve after the reconstruction of the control group and the experimental group is established, and the PAE is calculated.
Preferably, the appropriate PK-PD parameter, i.e. (AUC/MIC), is selected based on the measured in vivo in-vivo sterilization profile of tilmicosin for Lawsonia intracellularis suisexSimulation of Sigmoid E using Winnolin softwaremaxA PK-PD model equation, calculating pharmacodynamic target values of tilmicosin under different antibacterial effects on lawsonia intracellularis of pigs, and corresponding (AUC/MIC) to the tilmicosin with different antibacterial effectsexSubstituting the values into a dose calculation equation I to calculate the required dose of tilmicosin for achieving different antibacterial effects:
wherein, in formula I, Dose represents the administered Dose; (AUC/MIC)exThe expression refers to the PK-PD parameter values for different therapeutic effects (bactericidal, bacteriostatic and eradication); CL refers to the clearance rate of tilmicosin in the pig digestive tract; f is bioavailability; MIC is the intracellular MIC value of lawsonia intracellularis; fu represents the ratio of the free drug concentration.
The invention also provides a method for establishing the PK-PD synchronous model of the tilmicosin to the lawsonia intracellularis, and a dosing scheme of the tilmicosin to the lawsonia intracellularis is established.
The invention discloses the following technical effects:
the method obtains the lawsonia intracellularis through clinically separating the ileum disease material suspected to be the porcine proliferative enteritis, draws a growth curve of the lawsonia intracellularis, determines the minimum inhibitory concentration, the mutation prevention concentration and the post-antibacterial effect of tilmicosin on the lawsonia intracellularis, draws in-vitro and half-body intracellular sterilization curves, and carries out the pharmacokinetic research of the tilmicosin on the porcine body; according to the measured in-vivo sterilization curve of tilmicosin to the half-body of the lawsonia intracellularis, selecting a proper PK-PD parameter (the selection standard is that according to the measured in-vivo sterilization curve of tilmicosin to the half-body of the lawsonia intracellularis, if the sterilization effect is enhanced along with the increase of the drug concentration and shows obvious concentration dependence, AUC/MIC is selected, and if the sterilization effect is enhanced along with the increase of the drug action time and shows obvious time dependence, T > MIC is selected), fitting data, formulating a reasonable administration scheme of the tilmicosin to the lawsonia intracellularis, and establishing a PK-PD synchronous model of the tilmicosin to the lawsonia intracellularis. By adopting the method, a reasonable administration scheme of tilmicosin to lawsonia intracellularis can be worked out, stable administration data support is provided for scientific culture, and clinical administration can be guided more scientifically; the method can effectively relieve the drug resistance of the lawsonia intracellularis to tilmicosin to a certain extent, protect and maintain the effectiveness of the tilmicosin, and provide a guide for the research of the antibacterial drug on the PK-PD synchronous model of the lawsonia intracellularis.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the results of PCR qualitative detection of isolated strains according to the present invention;
FIG. 2 shows the results of quantitative detection of isolated strains by fluorescent quantitative PCR in accordance with the present invention;
FIG. 3 is a graph showing the growth curve of L.intracellularis in porcine jejunal epithelial cells in accordance with the invention;
FIG. 4 is a schematic diagram showing the intracellular bactericidal curve of tilmicosin on lawsonia intracellularis in porcine jejunal epithelial cells in accordance with the present invention;
FIG. 5 is a schematic diagram showing the extracellular bactericidal curve of tilmicosin on lawsonia intracellularis in porcine jejunal epithelial cells in accordance with the present invention;
FIG. 6 shows the Mlxplore software of the present invention simulating bacterial growth under different dosing regimens; wherein adm 1: the preventive dose is as follows: 3.93mg/kg, 2 times/d; and adm 2: the treatment dose is as follows: 14.20mg/kg, 2 times/d; adm 3: eradication dose: 21.50mg/kg, 2 times/d; adm 4: prophylactic dose: 3.93mg/kg, 1 time/d; adm 5: the treatment dose is as follows: 14.20mg/kg, 1 time/day; adm 6: eradication dose: 21.50mg/kg, 1 time/d.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
The method for establishing the PK-PD synchronous model of the tilmicosin to the lawsonia intracellularis comprises the following steps:
step 1: firstly, separating and detecting lawsonia intracellularis: taking out ileal disease material suspected to be porcine proliferative enteritis, opening the intestinal tract by scissors, and washing residues by PBS; taking intestinal mucosa with slide, homogenizing, adding equal volume of PBS containing trypsin, and incubating at 37 deg.C for 35 min; passing the supernatant through two layers of gauze to obtain filtrate, respectively passing through 5.0 μm, 1.2 μm and 0.65 μm porous membrane filters, centrifuging (10000r/min, 3min) to obtain precipitate, resuspending bacteria with cell frozen stock solution, and freezing at-80 deg.C.
And (3) taking the separated lawsonia intracellularis as a template, and qualitatively detecting the separated strain by using PCR (polymerase chain reaction) and quantitatively detecting the concentration of the separated strain by using fluorescent quantitative PCR.
The PCR qualitative detection result shows that: the amplification result is consistent with the expected result, and the target band (319bp) is amplified, and the result is shown in FIG. 1. The fluorescent quantitative PCR qualitative detection result shows that: measured CtThe value was 16.01, and the number of the isolated strains was converted to a concentration of 5 × 10 by substituting-3.275 x +29.597 for the standard curve y (3.275 x +29.597) previously established before the method disclosed in the present invention was established5intracellularis/mL, results are shown in figure 2.
Step 2: drawing a growth curve of lawsonia intracellularis in porcine jejunum epithelial cells, and the specific process comprises the following steps: spreading the porcine jejunum epithelial cells in a 24-well plate to wait for the cellsWhen the confluence degree is about 60%, Lawsonia intracellularis (concentration 5 × 10) is added5l.intercellularis/mL), after 3h of culture, removing the supernatant, washing 3 times with PBS, which is recorded as 0h at this time, replacing the fresh DMEM cell culture medium, sampling at 0h, 6h, 12h, 18h, 24h, 2d, 3d, 4d, 5d, 6d, 7d, and 8d, respectively, measuring the content of bacteria by fluorescent quantitative PCR, and drawing the growth curve of lawsonia intracellularis.
The results are shown in FIG. 3: lawsonia intracellularis is in a delayed phase for 0-1 day, in a log phase for 2-5 days, in a stationary phase for 6 days, and in a decline phase for 7-8 days.
The method for determining the minimum extracellular inhibitory concentration of tilmicosin on lawsonia intracellularis comprises determining the minimum extracellular inhibitory concentration of lawsonia intracellularis (concentration 5 × 10)5intercellularis/mL), adding culture media containing tilmicosin with different concentrations (128 mug/mL, 64 mug/mL, 32 mug/mL, 16 mug/mL, 8 mug/mL, 4 mug/mL, 2 mug/mL, 1 mug/mL, 0.5 mug/mL and 0.25 mug/mL), adding the mixture into a 24-well plate with about 60% of cell confluency after 3 hours of action, changing the mixture into DMEM cell culture media without drug concentration every day, and continuously culturing for 5 days; and (3) performing staining counting by using a peroxidase single-layer staining method, and performing microscopic examination to obtain the extracellular minimum inhibitory concentration of tilmicosin on the lawsonia intracellularis of the pigs.
The staining count by the peroxidase monolayer staining method shows that: the minimum intracellular inhibitory concentration of tilmicosin to lawsonia intracellularis of 2 mug/mL and the minimum extracellular inhibitory concentration of 4 mug/mL.
Drawing a semiintracellular sterilization curve of the tilmicosin to the lawsonia intracellularis in the porcine jejunum epithelial cells, and the specific process is that the porcine jejunum epithelial cells are paved in a 24-pore plate, and the lawsonia intracellularis (with the concentration of 5 × 10) is added when the cell confluency is about 60 percent5intercellularis/mL), after 3h of culture, the supernatant was removed, washed 3 times with PBS and cultured continuously for 3 days to reach a bacterial concentration of 106And L.intracellularis/mL, adding culture media with different concentrations of tilmicosin (the tilmicosin medicament concentration in ileum contents measured at each time point), sampling at 0, 1, 2, 4, 8, 12, 18, 24, 36 and 48 hours respectively, measuring the content of bacteria by adopting fluorescent quantitative PCR (polymerase chain reaction), and drawing an intra-cell sterilization curve of the lawsonia intracellularis.
As shown in fig. 4 and 5: tilmicosin has stronger bactericidal capacity on lawsonia intracellularis of pigs, and the bactericidal effect on lawsonia intracellularis of pigs is increased along with the increase of the concentration of tilmicosin. Intracellular bactericidal effects have been shown at tilmicosin concentrations of 2. mu.g/mL. With the increase of the concentration of the drug, the drug shows a remarkable bactericidal ability in a short time. According to the results, the bactericidal capacity of the tilmicosin on the lawsonia intracellularis in the porcine jejunal epithelial cells shows concentration dependence, namely the higher the tilmicosin concentration is, the stronger the bactericidal effect on the lawsonia intracellularis is.
And 5:the method for measuring the anti-mutation concentration of lawsonia intracellularis in the porcine jejunum epithelial cells comprises the following specific steps: taking the determined minimum intracellular inhibitory concentration of tilmicosin to Lawsonia as a reference, preparing tilmicosin cell culture media with different concentrations (0MIC, 1/2MIC, 1MIC, 2MIC, 4MIC, 8MIC and 16MIC, namely 0 mu g/mL, 1 mu g/mL, 2 mu g/mL, 4 mu g/mL, 8 mu g/mL, 16 mu g/mL and 32 mu g/mL). Spreading the porcine jejunum epithelial cells in a 24-well plate, adding Lawsonia inermis when the confluency of the cells is about 60%, culturing for 3h, removing supernatant, and washing with PBS for 3 times. Continuously culturing for 5 days to make the bacterial concentration reach 107After l. intercellularis/mL, after centrifugation and resuspension, the concentration of bacteria was adjusted to achieve a concentration of about 109Intracellularis/mL, DMEM cell culture media containing different drug concentrations (16. mu.g/mL, 8. mu.g/mL, 7. mu.g/mL, 6.4. mu.g/mL, 6. mu.g/mL, 4. mu.g/mL, 2. mu.g/mL) were added and placed in an incubator for 5 days. Initial anti-mutation concentration (MPC) was set at the lowest drug concentration at which colonies still grew aseptically after 5 days of culturepr). Then using MPCprAnd linearly decreasing (20%) the concentration of the antibacterial drug to obtain cell culture media containing drugs with different concentrations as a baseline, repeating the steps, and culturing in an incubator for 5 days until the lowest drug concentration at which bacterial colony growth does not appear is the final MPC.
The results show that the initial determination of the anti-mutation concentration MPC of tilmicosin on lawsonia intracellularis pr8. mu.g/mL, and a final antimutagenic concentration MPC of 6.4. mu.g/mL, measured after a linear decrease in 20% concentration. According to the mutation selection window theory, the mutation selection window of the lawsonia intracellularis is 2-6.4 mug/mL, and the generation of drug-resistant mutant strains can be reduced by adjusting the drug concentration.
Step 6: drawing the antibacterial after-effect of lawsonia intracellularis in the porcine jejunum epithelial cells, and the specific process is as follows: taking the determined minimum intracellular inhibitory concentration of tilmicosin to lawsonia, preparing a tilmicosin cell culture medium containing different concentrations (1MIC, 2MIC and 4MIC), paving the porcine jejunum epithelial cells in a 24-hole plate, adding lawsonia intracellularis when the cell confluency is about 60%, culturing for 3h, removing the supernatant, and washing for 3 times by PBS. Continuously culturing for 3 days to reach bacterial concentration106Adding DMEM cell culture media containing different drug concentrations after L.intracellularis/mL, placing the DMEM cell culture media in an incubator for 1h and 2h, and inducing the generation of PAE. The control group, A1 and A2, contained cells and bacteria only (no drug). Removal and reconstitution of drug: after 1h and 2h for the experimental and control groups, respectively, the supernatant was removed, washed 3 times with PBS and immediately placed in the incubator, which is 0h after reconstitution. Establishing a bacterial growth kinetic curve: and (3) measuring the bacterial content of each time point by using fluorescent quantitative PCR (polymerase chain reaction) 0, 1, 2, 4, 6, 8 and 12 hours after reconstruction, establishing a kinetic curve of growth recovery of the control bacteria and the experimental bacteria after reconstruction, and calculating the PAE.
The results of 1h and 2h PAE exposure of Lawsonia intracellularis to tilmicosin at different concentrations of 1MIC, 2MIC, 4MIC, respectively, are shown in Table 1: when the incubation time is 2h, the PAE is slightly longer than that of the incubation time of 1h, namely, the PAE has a tendency of slowly increasing with the increase of the drug concentration; at 1h and 2h of incubation, the time to generate PAE was also significantly longer with increasing concentration, exhibiting a significant concentration dependence.
TABLE 1 antibacterial Effect of tilmicosin on Lawsonia intracellularis in pigs
And 7: the pharmacokinetics research of tilmicosin on the pig body comprises the following specific processes: selecting 12 weaned piglets with weight of about 20Kg, installing a T-shaped fistula at the ileum section, establishing a piglet fistula model, then randomly dividing into 2 groups, 6 groups of experimental groups and control groups respectively, using separated lawsonia strains to detoxify the experimental groups, establishing a fistula model of artificially infected lawsonia intracellularis, intragastrically administering two groups of animals according to 10mg/Kg weight, taking 5mL of blood plasma and 3-4mL of ileum contents at different time points (0, 1, 2, 4, 8, 12, 18, 24, 36 and 48h) after administration, and detecting tilmicosin drug concentrations in the blood plasma and the ileum contents by high performance liquid chromatography.
And 8: the method for formulating the tilmicosin administration scheme by lawsonia intracellularis comprises the following specific processes: intracellular response to tilmicosinIntracellular sterilization profile in Lawsonia in vivo, selection of appropriate PK-PD parameters, i.e. (AUC/MIC)ex. Simulation of Sigmoid E using Winnolin softwaremaxA PK-PD model equation, calculating pharmacodynamic target values of tilmicosin under different antibacterial effects on lawsonia intracellularis of pigs, and corresponding (AUC/MIC) to the tilmicosin with different antibacterial effectsexSubstituting the values into a dose calculation equation I to calculate the required dose of tilmicosin for achieving different antibacterial effects:
wherein, in formula I, Dose represents the administered Dose; (AUC/MIC)exThe expression refers to the PK-PD parameter values for different therapeutic effects (bactericidal, bacteriostatic and eradication); CL refers to the clearance rate of tilmicosin in the pig digestive tract; f is bioavailability; MIC is the intracellular MIC value of lawsonia intracellularis; fu represents the ratio of the free drug concentration.
The pharmacokinetic parameters of the tilmicosin premix in the ileum contents of the pigs are obviously superior to those of plasma (such as drug concentration, area under a curve of time of administration and the like), lawsonia intracellularis colonizes in the digestive tract of the pigs, and the infected target is located in the ileum, so that the pharmacokinetic data and the pharmacodynamic data of the tilmicosin premix in the ileum of the sick animals are finally selected to establish the dosing scheme of the tilmicosin on the lawsonia intracellularis of the pigs in the experiment.
After the data are substituted, the administration dosage under different medication purposes is obtained, and the results show that: the prophylactic, therapeutic and eradication doses were 4.14mg/kg, 15.45mg/kg and 25.03mg/kg, respectively.
The growth of the bacteria at three doses (prevention, treatment and eradication) and at different dosing intervals was simulated and predicted by the Mlxpore software to obtain the optimal dosing regimen and dosing intervals. As can be seen from fig. 6, in the case of the administration interval of 12h, the 4.14mg/kg b.w administration dose failed to achieve the bacteriostatic effect, the bacterial count began to rise after a short drop and was higher than the initial bacterial count, the 15.45mg/kg b.w administration dose began to drop after the administration, but the bactericidal effect was not ideal, and the 25.03mg/kg b.w administration dose achieved a better bactericidal effect. Under the condition of 24h dosing interval, the 4.14mg/kg b.w dosing dose can not achieve the bacteriostatic action and the bacterial amount is increased sharply, the 15.45mg/kg b.w dosing dose can obtain the ideal bactericidal effect, the number of bacteria can be predicted to be inhibited or killed to a great extent after 3d dosing, the very ideal bactericidal effect can be achieved under the 25.03mg/kg b.w dosing dose, but in the aspect of dosage, too much medicine can cause waste and drug resistance, and the medicine concentration in tissues of once-a-day dosing dose is higher than that of twice-a-day dosing from the aspect of a simulated and predicted dosing interval chart, so the dosing scheme of 15.45mg/kg b.w dosing interval of 3d is optimal.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (7)
1. The method for establishing the PK-PD synchronous model of the tilmicosin to the lawsonia intracellularis is characterized by comprising the following steps:
step 1: determining pharmacodynamic and pharmacokinetic parameters of tilmicosin on lawsonia intracellularis in pigs: counting by adopting a fluorescent quantitative PCR method, and drawing a growth curve of the lawsonia intracellularis in the porcine jejunum epithelial cells IPEC-J2;
counting by adopting a peroxidase cell monolayer staining method, and determining the minimum inhibitory concentration MIC of tilmicosin to the lawsonia intracellularis and the extracellular lawsonia intracellularis of the pig;
counting by adopting a fluorescent quantitative PCR method, and drawing in-vitro and in-vivo intracellular sterilization curves of tilmicosin to the lawsonia intracellularis;
counting by adopting a fluorescent quantitative PCR method, and determining the mutation-preventing concentration of tilmicosin on lawsonia intracellularis; counting by adopting a fluorescent quantitative PCR method, and determining the antibacterial effect of tilmicosin on lawsonia intracellularis;
step 2: selecting PK-PD parameters according to the in-vivo sterilization curve of tilmicosin to lawsonia intracellularis in the pig, which is measured in the step 1, and simulating Sigmoid E by using Winnolin softwaremaxAnd a PK-PD model equation, calculating pharmacodynamic target values of tilmicosin on the lawsonia intracellularis under different antibacterial effects, substituting the pharmacodynamic target values corresponding to different antibacterial effects of tilmicosin into a dose calculation equation, calculating to obtain the administration dose under the conditions of bacteriostasis, sterilization and eradication, establishing a PK-PD synchronous model of the tilmicosin on the lawsonia intracellularis, and making a reasonable administration scheme.
2. The method of establishing a PK-PD synchrony model of tilmicosin on lawsonia intracellularis according to claim 1, wherein said lawsonia intracellularis is a strain isolated from a suspected ileal pathology of porcine proliferative enteritis, and said strain is qualitatively detected by PCR and the concentration of said strain is quantitatively detected by fluorescent quantitative PCR.
3. The method of establishing a PK-PD synchrony model of tilmicosin on lawsonia intracellularis in pigs according to claim 1, wherein the obtaining of pharmacokinetic parameters of tilmicosin on lawsonia intracellularis in step 1 comprises the steps of: establishing a piglet fistula model, setting an experimental group and a control group, using the separated lawsonia intracellularis strain to detoxify the experimental group, establishing a fistula model for artificially infecting lawsonia intracellularis, intragastrically administering two groups of animals according to body weight, taking plasma and ileum contents at different time points after administration, and detecting tilmicosin drug concentration in the plasma and the ileum contents by using a high performance liquid chromatography.
4. The method for establishing the PK-PD synchronous model of tilmicosin on lawsonia intracellularis of pigs as claimed in claim 1, wherein the mutation-preventing concentration is determined by taking the determined minimum intracellular inhibitory concentration of tilmicosin on lawsonia intracellularis of pigs as a reference, preparing cell culture media containing tilmicosin with different concentrations, and observing for 5 days to obtain the minimum drug concentration without colony formationTo an initial antimutagenic concentration MPCprThen using MPCprThe final MPC was prepared by linearly decreasing the concentration of the antibacterial agent by 20% as a baseline, preparing cell culture media of tilmicosin at different concentrations, and taking the lowest drug concentration at which colonies did not appear after 5 days.
5. The method for establishing the PK-PD synchronous model of tilmicosin for lawsonia intracellularis according to claim 1, wherein said post-antibacterial effect is based on the measured minimum intracellular inhibitory concentration of tilmicosin for lawsonia intracellularis, preparing cell culture medium containing tilmicosin with different concentrations, inducing the production of PAE, setting two control groups containing IPEC-J2 cells and lawsonia intracellularis, removing and reconstructing drugs for the experimental group and the control group, measuring the lawsonia intracellularis content at different time points by using fluorescence quantitative PCR, establishing the kinetic curve of growth recovery after reconstruction of the control group and the experimental group, and calculating PAE.
6. The method of establishing a PK-PD synchrony model of tilmicosin on lawsonia intracellularis in pigs according to claim 1, wherein appropriate PK-PD parameters, i.e. (AUC/MIC) are selected according to the measured half in vivo sterilization curve of tilmicosin on lawsonia intracellularis in pigsexSimulation of Sigmoid E using Winnolin softwaremaxA PK-PD model equation, calculating pharmacodynamic target values of tilmicosin under different antibacterial effects on lawsonia intracellularis of pigs, and corresponding (AUC/MIC) to the tilmicosin with different antibacterial effectsexSubstituting the values into a dose calculation equation I to calculate the required dose of tilmicosin for achieving different antibacterial effects:
wherein, in formula I, Dose represents the administered Dose; (AUC/MIC)exThe expression refers to the PK-PD parameter values for different therapeutic effects (bactericidal, bacteriostatic and eradication); CL refers to the clearance rate of tilmicosin in the pig digestive tract; f is bioavailability; MIC is the intracellular MIC value of lawsonia intracellularis;fu represents the ratio of the free drug concentration.
7. The method for establishing a PK-PD synchronization model of tilmicosin to L.suis as claimed in any one of claims 1 to 6, wherein a dosage regimen of tilmicosin to L.suis is formulated.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010409968.5A CN111549156A (en) | 2020-05-15 | 2020-05-15 | Method for establishing PK-PD (PK-PD) synchronous model of tilmicosin on lawsonia intracellularis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010409968.5A CN111549156A (en) | 2020-05-15 | 2020-05-15 | Method for establishing PK-PD (PK-PD) synchronous model of tilmicosin on lawsonia intracellularis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111549156A true CN111549156A (en) | 2020-08-18 |
Family
ID=72006416
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010409968.5A Pending CN111549156A (en) | 2020-05-15 | 2020-05-15 | Method for establishing PK-PD (PK-PD) synchronous model of tilmicosin on lawsonia intracellularis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111549156A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877397A (en) * | 2021-01-26 | 2021-06-01 | 北京大学 | Method for evaluating efficacy of antibacterial agent for resisting intracellular bacterial infection and application thereof |
| CN113215292A (en) * | 2021-06-05 | 2021-08-06 | 山东新希望六和农牧科技有限公司 | TaqMan-LNA (low noise amplifier) kit and method for detecting Lawsonia intracellularis of porcine proliferative enteropathy pathogenic bacteria |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019152791A1 (en) * | 2018-02-02 | 2019-08-08 | Novozymes A/S | Management of pathogenic lawsonia |
| CN110223734A (en) * | 2019-07-22 | 2019-09-10 | 华中农业大学 | A kind of construction method of antibacterials Ceftiofur PK-PD model and its application |
| CN110607344A (en) * | 2019-09-03 | 2019-12-24 | 华中农业大学 | Construction method and application of veterinary antibacterial drug cefquinome PK/PD model |
-
2020
- 2020-05-15 CN CN202010409968.5A patent/CN111549156A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019152791A1 (en) * | 2018-02-02 | 2019-08-08 | Novozymes A/S | Management of pathogenic lawsonia |
| CN110223734A (en) * | 2019-07-22 | 2019-09-10 | 华中农业大学 | A kind of construction method of antibacterials Ceftiofur PK-PD model and its application |
| CN110607344A (en) * | 2019-09-03 | 2019-12-24 | 华中农业大学 | Construction method and application of veterinary antibacterial drug cefquinome PK/PD model |
Non-Patent Citations (6)
| Title |
|---|
| WANHE LUO等: "Pharmacokinetics/Pharmacodynamics models of veterinary antimicrobial agents", 《J VET SCI》 * |
| 南佳惠: "乙酰吉他霉素对猪产气荚膜梭菌病的PK-PD同步模型研究", 《万方数据知识服务平台》 * |
| 周芳等: "影响左氧氟沙星胞内抗菌活性的靶细胞PK/PD研究", 《第十届全国药物和化学异物代谢学术会议暨第三届国际ISSX/CSSX联合学术会议论文集》 * |
| 罗万和等: "胞内劳森菌检测方法研究进展", 《中兽医医药杂志》 * |
| 韩冰: "猪回肠炎的诊治", 《养猪》 * |
| 马景霞等: "猪胞内劳森菌定量PCR检测方法的建立与初步应用", 《畜牧与兽医》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877397A (en) * | 2021-01-26 | 2021-06-01 | 北京大学 | Method for evaluating efficacy of antibacterial agent for resisting intracellular bacterial infection and application thereof |
| CN112877397B (en) * | 2021-01-26 | 2022-04-22 | 北京大学 | Method for evaluating efficacy of antibacterial agent for resisting intracellular bacterial infection and application thereof |
| CN113215292A (en) * | 2021-06-05 | 2021-08-06 | 山东新希望六和农牧科技有限公司 | TaqMan-LNA (low noise amplifier) kit and method for detecting Lawsonia intracellularis of porcine proliferative enteropathy pathogenic bacteria |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111549156A (en) | Method for establishing PK-PD (PK-PD) synchronous model of tilmicosin on lawsonia intracellularis | |
| JP2022516984A (en) | Use in the manufacture of drugs to treat human papillomavirus infections of the isolated Rhodococcus louver cell wall skeleton | |
| WO2022156119A1 (en) | Enterococcus faecium, drug for preventing or treating tumor, and application | |
| CN112760247A (en) | Lactobacillus rhamnosus for preventing and/or treating diseases caused by genital tract flora disorder and/or bone loss | |
| CN114891677B (en) | Separation and application of strain with anticancer effect | |
| CN114344338B (en) | Novel application of bacteroides fragilis and/or zwitterionic capsular polysaccharide thereof | |
| Illambas et al. | Pharmacodynamics of marbofloxacin for calf pneumonia pathogens | |
| CN116350662A (en) | Microorganism strain of chaetoviridae, medicament for preventing or treating tumor and application thereof | |
| CN113797232B (en) | Composition with insulin resistance relieving function and application thereof | |
| CN110607344A (en) | Construction method and application of veterinary antibacterial drug cefquinome PK/PD model | |
| Zhang et al. | Immunomodulatory and antitumor activities of the exopolysaccharide produced by potential probiotic Lactobacillus plantarum YW11 in a HT-29 tumor-burdened nude mouse model | |
| CN111647525B (en) | Composite probiotics, microbial inoculum and application of composite probiotics and microbial inoculum in preparation of microbial inoculum for preventing and treating candida albicans vaginitis | |
| CN111529707B (en) | Application of GSDMD inhibitor in preparation of medicine for treating helicobacter pylori infection | |
| KR101498942B1 (en) | Pharmaceutical Composition for Treatment of Tuberculosis | |
| CN113616652A (en) | Application of SMS2 inhibitor in preparation of medicine for treating highly invasive breast cancer | |
| CN111419829A (en) | Application of honokiol in inhibiting streptococcus suis or biofilm thereof | |
| WO2011157802A1 (en) | Pharmaceutical preparation comprising actinomycin d for the treatment of b-cll in patients having del (17) | |
| CN110946862B (en) | Application of sanguinarine in inhibition of growth of multiple drug-resistant enterobacter hopcalis | |
| CN114796284A (en) | Microbial strain, medicine for preventing or treating tumors and application | |
| CN109161501B (en) | Feeding bacillus licheniformis and application thereof | |
| CN104403996A (en) | Human gastric cancer cell line with 5-fluorouracil resistance and establishing method and application thereof | |
| CN110172524A (en) | Chicken C.perfringens is formulated to the method for the drug resistance criterion of avilamycin | |
| CN115105491B (en) | Application of serine in preparation of medicines for inhibiting streptococcus suis | |
| CN111888467B (en) | Application of bacillus subtilis plasmin in medicines for treating silicosis | |
| CN114886939A (en) | Application of scutellaria baicalensis extract in resisting haemophilus parasuis disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200818 |