A biological preparation for porcine actinobacillus pleuropneumoniae disease

Abstract

The present invention provides a biological preparation for porcine Actinobacillus pleuropneumoniae disease, belonging to the technical field of porcine Actinobacillus pleuropneumoniae disease treatment. The present invention finds that Inula grandiflora can effectively inhibit Porcine Actinobacillus pleuropneumoniae, and finds that it can break the biofilm of Porcine Actinobacillus pleuropneumoniae and inhibit the adhesion of Porcine Actinobacillus pleuropneumoniae to porcine lung epithelial cells. Therefore, Inula grandiflora can be prepared into a biological preparation for treating Porcine Actinobacillus pleuropneumoniae disease.

Description

[0001] This case is a divisional application. The name of the invention of the original application is: Use of Inula grandiflora in the preparation of drugs for the treatment of porcine actinomycetemcomitum pleuropneumoniae disease. The application date of the original application is: 2020-11-06, and the application number of the original application is: CN202011227605.6. Technical Field

[0002] The invention relates to the technical field of treating porcine actinobacillus pleuropneumoniae disease, and in particular to a biological preparation for treating porcine actinobacillus pleuropneumoniae disease. Background Art

[0003] With the widespread use of antibiotics in clinical practice, more and more pathogens have developed resistance to antibiotics. The resistance of pathogens has caused serious losses to livestock and poultry farming. How to effectively fight bacterial infections has become a common concern of countries around the world, and alternative strategies to antibiotics have also attracted widespread attention from humans.

[0004] Porcine Actinobacillus pleuropneumoniae disease, also known as porcine contagious pleuropneumoniae, is one of the respiratory diseases caused by Actinobacillus hyopneumoniae that harms intensive pig farms. It is characterized by symptoms and lesions of pleurisy and pneumonia. Since the disease usually develops acutely, it will quickly cause breathing difficulties in infected pigs and even cause rapid death of pigs, thus causing huge economic losses to the pig farming industry. At present, the treatment of porcine Actinobacillus pleuropneumoniae disease is mostly treated with antibiotics, which can easily cause bacteria to develop drug resistance, so it is particularly important to choose drugs that can replace antibiotics.

[0005] Inula grandiflora is a natural plant extract extracted from Inula grandiflora. At present, the research on Inula grandiflora is relatively small, and the biological functions that have been discovered mainly include anti-inflammatory, anti-allergic, and anti-tumor effects. However, there are no reports on the inhibition of Inula grandiflora on Actinobacillus pleuropneumoniae. Summary of the invention

[0006] The purpose of the present invention is to provide the use of Inula grandiflora in preparing a medicine for treating porcine Actinobacillus pleuropneumoniae disease.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The invention provides application of Inula grandiflora in inhibiting Actinobacillus pleuropneumoniae.

[0009] Preferably, the minimum inhibitory concentration of the Inula macrophylla tincture against Actinobacillus pleuropneumoniae is 62.5 ug / ml.

[0010] Preferably, the minimum bactericidal concentration of the Inula macrophyllae tincture against Actinobacillus pleuropneumoniae is 125 ug / ml.

[0011] In addition, the present invention provides the use of Inula grandiflora in the preparation of a fungicide for inhibiting Actinobacillus pleuropneumoniae in porcine.

[0012] In addition, the present invention provides a fungicide for inhibiting porcine Actinobacillus pleuropneumoniae, wherein the core active ingredient of the fungicide is Inula grandiflora.

[0013] In addition, the present invention provides the use of Inula grandiflora in the preparation of a bactericide for destroying the biofilm of Actinobacillus pleuropneumoniae.

[0014] In addition, the present invention provides the use of Inula grandiflora in the preparation of a bactericide for reducing the adhesion of Actinobacillus pleuropneumoniae bacteria.

[0015] In addition, the present invention provides the use of Inula grandiflora in the preparation of a medicine for treating porcine Actinobacillus pleuropneumoniae disease.

[0016] The beneficial effects of the present invention are:

[0017] The present invention first discovered that Inula grandiflora can effectively inhibit Actinobacillus pleuropneumoniae.

[0018] At the same time, the present invention first discovered that Inula grandiflora extract can destroy the biofilm of Actinobacillus pleuropneumoniae.

[0019] In addition, the present invention first discovered that Inula grandiflora can inhibit the adhesion of Actinobacillus pleuropneumoniae to pig lung epithelial cells. Therefore, Inula grandiflora can be used to prepare a drug for treating Actinobacillus pleuropneumoniae disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Crystal violet staining results of Actinobacillus pleuropneumoniae biofilm

[0021] Figure 2 Actinobacillus pleuropneumoniae biofilm absorbance results

[0022] Figure 3 Results of inhibition rate of adhesion of Actinobacillus pleuropneumoniae DETAILED DESCRIPTION

[0023] In order to clearly illustrate the technical features of this solution, this solution is described below through a specific implementation method.

[0024] Example 1

[0025] Detection of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Inula grandiflora

[0026] Minimum inhibitory concentration test

[0027] (1) The experimental groups were 500ug / ml, 250ug / ml, 125ug / ml, 62.5ug / ml, 31.25ug / ml, 15.60ug / ml of Inula japonica extract and the negative control group ddH20;

[0028] (2) Adjust the number of Actinobacillus pleuropneumoniae to 2×10 5 CFU / ml were inoculated in a 96-well plate, and Inula grandiflora was added according to the experimental groups (each concentration was repeated 3 times), and the 96-well plate was placed in an incubator and cultured for 24 h;

[0029] (3) The wells without turbidity are the minimum inhibitory concentration of Inula grandiflora;

[0030] Minimum bactericidal concentration test

[0031] (1) Continue to culture the bacteria for 24 hours, aspirate the bacterial solution from the wells without turbidity, evenly spread it on the agar plate, place it in a 37°C incubator, culture it for 36 hours, and observe the results;

[0032] (2) The concentration of Inula macrophylla in the wells where the number of bacterial colonies does not exceed 5 is the minimum bactericidal concentration.

[0033] The experimental results showed that the minimum inhibitory concentration of Inula grandiflora was 62.5ug / ml, and the minimum bactericidal concentration was 125ug / ml.

[0034] Example 2

[0035] Detection of the bactericidal effect of Inula grandiflora

[0036] (1) culturing porcine Actinobacillus pleuropneumoniae in a liquid culture medium until the OD600 is 1;

[0037] (2) The cells were grouped into Inula grandiflora (125ug / ml), positive control group (100ug / ml penicillin), and negative control group ddH20;

[0038] (3) Culture the culture in a 37°C shaking incubator and measure the OD600 value of the bacterial solution at 2 h, 4 h, 6 h, 12 h, and 24 h.

[0039]

[0040] As can be seen from the table, Inula grandiflora has a significant bactericidal effect on Actinobacillus pleuropneumoniae, and the difference is statistically significant.

[0041] Example 3

[0042] Inhibitory effect of Inula grandiflora on biofilm of Actinobacillus pleuropneumoniae

[0043] (1) Adding Actinobacillus pleuropneumoniae in the logarithmic phase to a 96-well polystyrene microplate;

[0044] (2) Experimental groups: 125ug / ml Inula grandiflora, control group ddH2O;

[0045] (3) Treat porcine Actinobacillus pleuropneumoniae according to the experimental groups, place the microplate in an incubator and culture for 24 h, remove the culture medium, and add sterile PBS for washing;

[0046] (4) Fix with 70% methanol for 30 min, remove the fixative, and add crystal violet for staining;

[0047] (5) After staining for 5 minutes, remove the staining solution, wash with water, and take a photo;

[0048] (6) Add 70% ethanol to dissolve the biofilm and measure the absorbance at OD570.

[0049] The experimental results are as follows Figure 1 and Figure 2 As shown in the figure, it can be seen that Inula grandiflora can significantly inhibit the biofilm of porcine Actinomycetes pleuropneumoniae, and the difference is statistically significant (the control group is 2.117±0.156, and the Inula grandiflora is 0.113±0.055, P<0.0001).

[0050] Example 4

[0051] Inhibitory effect of Inula grandiflora on bacterial adhesion of Actinobacillus pleuropneumoniae

[0052] (1) Porcine lung epithelial cells SJPL were inoculated into 24-well plates, and 62.5ug / ml of Inula grandiflora, 125ug / ml of Inula grandiflora, and a blank group were added (three replicates were set for each treatment);

[0053] (2) Add 1×10 8 CFU / ml Actinobacillus pleuropneumoniae, placed in a cell culture incubator and cultured for 6 h;

[0054] (3) Remove the supernatant and gently wash the wells three times with PBS to remove non-adherent cells;

[0055] (4) Add 200 μl of 0.1% Triton-X100 to each well, shake gently to mix, and leave at room temperature for 15 min to allow the cells to fully lyse, and collect the liquid in the well;

[0056] (5) Count the colonies on the plate after gradient dilution with saline;

[0057] (6) Adhesion inhibition rate (%) = (bacterial colony count in blank group - bacterial colony count in experimental group) / bacterial colony count in blank group × 100%.

[0058] The experimental results are as follows Figure 3 As shown in the figure, it can be seen that the inhibitory effect of 62.5ug / ml scutellarin on the adhesion of porcine Actinobacillus pleuropneumoniae to porcine lung epithelial cells SJPI is 40.67±1.528; the adhesion inhibition effect of 125ug / ml scutellarin is 78.33±2.517. The above results show that scutellarin can significantly inhibit the adhesion of porcine Actinobacillus pleuropneumoniae to porcine lung epithelial cells, thereby reducing the probability of pigs being infected with porcine Actinobacillus pleuropneumoniae disease.

[0059] Technical features not described in the present invention can be achieved through or by adopting existing technologies and will not be described in detail here. Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by ordinary technicians in this technical field within the essential scope of the present invention should also fall within the scope of protection of the present invention.

Claims

1. The use of Inula grandiflora in preparing a drug for treating porcine Actinobacillus pleuropneumoniae, characterized in that: The Inula grandiflora extract can treat Actinobacillus pleuropneumoniae disease by reducing the adhesion of Actinobacillus pleuropneumoniae bacteria to pig epithelial cells. The concentration of the Inula macrophylla in the medicine is 62.5 ug / ml-125 ug / ml.

Images