Separation and identification culture medium for actinobacillus pleuropneumoniae in pig clinical disease material and preparation method of separation and identification culture medium
By developing a specific selective culture medium, using specific components and additives to inhibit the growth of bacteria, the problem of difficult to quickly and accurately isolate and identify Actinobacter pleuropneumoniae in the clinical disease of pigs in the prior art, achieving higher isolation accuracy and faster diagnosis time.
Patent Information
- Application Number
- CN202510091779.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-21
- Publication Date
- 2025-04-11
AI Technical Summary
The prior art is difficult to quickly and accurately isolate and identify Actinobacter pleuropneumoniae from pig clinical data, resulting in an extended clinical diagnosis and treatment time.
Develop a selective culture medium including tryptone, soy peptone, lactose and other components to inhibit the growth of other bacteria through specific component ratios and additives (such as Coenzyme I, vancomycin, amphotericin B, etc.), and significantly improve the accuracy of isolation and identification of Actinobacter pleuropneumoniae.
This culture medium can significantly reduce the impact of other miscellaneous bacteria, improve the isolation accuracy of Actinobacter pleuropneumoniae, and significantly shorten the diagnosis and treatment time.
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Abstract
Description
Technical Field
[0001] The invention relates to a separation and identification culture medium for Actinobacillus pleuropneumoniae in pig clinical disease materials and a preparation method thereof, and belongs to the field of microbial culture. Summary of the invention
[0002] Actinobacillus pleuropneumoniae (APP) can cause porcine contagious pleuropneumonia and is a Gram-negative bacterium. APP is spherical or short rod-shaped under a microscope and has pili, flagella and capsule. According to the different components of the capsule polysaccharide, the bacterium can be divided into 15 serotypes. There are different virulence differences between different serotypes. Among them, the serotype with the strongest ability to synthesize type I and type III Apx proteins shows the highest virulence level.
[0003] Porcine contagious pleuropneumonia can present in a variety of clinical manifestations. The acute type is characterized by high mortality and sudden death. Its clinical manifestations include high fever (41°C), difficulty breathing and other symptoms. The typical symptom is that the animal dies suddenly without any signs, and bloody foam flows out of the mouth and nose. The subacute type shows milder symptoms and a lower mortality rate. Animals that have recovered after the acute type turn into the chronic type without clinical manifestations, but most animals that have the acute type have sequelae. In addition, there are cases where there are no acute symptoms and they directly turn into the chronic type.
[0004] Rapid isolation and identification of Actinobacillus pleuropneumoniae from clinically ill pig disease tissues is of great significance for the prevention and control of porcine contagious pleuropneumoniae. Currently, common culture media, such as ordinary TSA medium or modified TSA medium, and TSAYE agar medium, are mostly used for clinical isolation of Actinobacillus pleuropneumoniae. These culture media are difficult to quickly separate Actinobacillus pleuropneumoniae from other respiratory infection bacteria. Judging by the naked eye and streaking cultures multiple times greatly delays the time for clinical diagnosis and effective treatment. Therefore, it is necessary to develop a selective culture medium that can quickly identify Actinobacillus pleuropneumoniae from clinical pig disease materials.
[0005] In the article "Isolation and Identification of Actinobacillus pleuropneumoniae Serum Type 1 in a Large-scale Pig Farm in Fujian Province" published in the July 2024 issue of "Chinese Journal of Animal Husbandry and Veterinary Medicine", the aseptically collected lung tissue was streaked and inoculated in modified TSA medium (containing 1.5% NAD and 5% fetal bovine serum) and ordinary TSA medium (containing 5% fetal bovine serum) at 37°C and 5% CO 2Incubate in the incubator for 18 to 24 hours, and judge its dependence on NAD based on the growth of the colonies. Pick a single colony and purify it on ordinary TSA or modified TSA medium until the colony color and shape are uniform. It is difficult to separate it from other pathogens at one time by using ordinary TSA or modified TSA. The separation steps are cumbersome because it is cultured by multiple streaking.
[0006] In the "Isolation, Identification and Biological Characteristics of Xinjiang Strain of Actinobacillus pleuropneumoniae" published in the December 2023 issue of the Journal of Tarim University, the diseased material was aseptically inoculated on TSAYE agar medium containing 10μg / mL NAD and TSA-YE agar medium, chocolate agar medium, and sheep blood agar medium without NAD, and cultured at 37℃ for 24-48h. The growth of streaked bacteria and the morphology of colonies were observed. The results showed that small turbid white colonies with round protrusions, smooth, translucent, and a diameter of 1-2mm were observed on TSA-YE medium containing 10μg / mL NAD, chocolate agar medium, and sheep blood agar medium, while the bacteria grew poorly on TSA-YE medium without NAD, and even no colonies grew. The above culture medium studied the biological characteristics of the Xinjiang strain of Actinobacillus pleuropneumoniae, but its colony determination was still mainly based on naked eye determination, which is prone to errors.
[0007] Based on this, the present invention is proposed. Summary of the invention
[0009] In the prior art, the culture medium used for clinical separation of Actinobacillus pleuropneumoniae is mostly conventional ordinary TSA or modified TSA culture medium, which is difficult to accurately separate Actinobacillus pleuropneumoniae from other respiratory infection bacteria and is not suitable for clinical separation and identification of Actinobacillus pleuropneumoniae. In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a culture medium for separation and identification of Actinobacillus pleuropneumoniae in clinical pig disease materials and a preparation method thereof. The culture medium includes components such as tryptone, soy peptone, lactose, etc. When used for the separation of pathogens in clinical pig respiratory disease materials, Actinobacillus pleuropneumoniae can be accurately separated and identified, and the influence of other miscellaneous bacteria is significantly reduced, which is suitable for promotion and application in clinical practice.
[0010] The present invention achieves the above technical effects through the following technical solutions:
[0011] A culture medium for separating and identifying Actinobacillus pleuropneumoniae in clinical pig disease materials comprises the following components: 12.0-18.0 g / L of tryptone, 3.0-7.0 g / L of soy peptone, 4.0-6.0 g / L of sodium chloride, 2.0-4.0 g / L of lactose, 0.2-0.4 g / L of pyruvic acid, 40-60 ml / L of calf serum, 12.0-18.0 g / L of agar, 0.02-0.04 g / L of coenzyme I, 15-25 mg / L of vancomycin, 3-5 mg / L of amphotericin B, and 0.05-0.15 g / L of 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside, and the volume is fixed to 1 L with pure water.
[0012] As a preferred embodiment, the isolation and identification medium for Actinobacillus pleuropneumoniae in pig clinical samples is composed of the following components: 15.0 g / L tryptone, 5.0 g / L soy peptone, 5.0 g / L sodium chloride, 3.0 g / L lactose, 0.3 g / L pyruvate, 50 ml / L calf serum, 15.0 g / L agar, 0.03 g / L coenzyme I, 20 mg / L vancomycin, 4 mg / L amphotericin B, 0.10 g / L 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside, and the volume is made up to 1 L with pure water.
[0013] As another preferred embodiment, the culture medium for isolating and identifying Actinobacillus pleuropneumoniae in pig clinical samples is composed of the following components: 12.0 g / L tryptone, 7.0 g / L soy peptone, 4.0 g / L sodium chloride, 4.0 g / L lactose, 0.2 g / L pyruvate, 60 ml / L calf serum, 12.0 g / L agar, 0.04 g / L coenzyme I, 25 mg / L vancomycin, 3 mg / L amphotericin B, 0.15 g / L 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside, and the volume is made up to 1 L with pure water.
[0014] As another preferred embodiment, the culture medium for isolating and identifying Actinobacillus pleuropneumoniae in pig clinical samples is composed of the following components: 18.0 g / L tryptone, 3.0 g / L soy peptone, 6.0 g / L sodium chloride, 2.0 g / L lactose, 0.4 g / L pyruvate, 40 ml / L calf serum, 18.0 g / L agar, 0.02 g / L coenzyme I, 15 mg / L vancomycin, 5 mg / L amphotericin B, 0.05 g / L 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside, and the volume is made up to 1 L with pure water.
[0015] The present invention also provides a method for preparing a culture medium for separating and identifying Actinobacillus pleuropneumoniae in the above-mentioned pig clinical disease materials, which comprises the following steps:
[0016] 1) Weigh or measure the prescribed amount of tryptone, soy peptone, sodium chloride, lactose, pyruvic acid and agar, add them to distilled water, stir to dissolve, adjust the pH value to 7.2±0.2, and sterilize at 121°C for 15 minutes to obtain a culture medium mother solution;
[0017] 2) Weigh coenzyme I and dissolve it in pure water to prepare a 30 mg / ml coenzyme I solution, weigh vancomycin and dissolve it in pure water to prepare a 20 mg / ml vancomycin solution, weigh 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside and amphotericin B and dissolve them in DMSO to obtain a 0.16 g / ml 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside solution and a 40 mg / ml amphotericin B solution, and filter and sterilize the prepared solutions for later use;
[0018] 3) Cool the culture medium to below 55°C, add the prescribed amount of 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside solution, coenzyme I solution, vancomycin solution, amphotericin B solution and calf serum, stir to mix and pour into a plate.
[0019] The present invention also claims the use of a culture medium for isolating and identifying Actinobacillus pleuropneumoniae in pig clinical disease materials. Specifically, the present invention claims the use of the culture medium in isolating and identifying Actinobacillus pleuropneumoniae in pig respiratory tract lesions.
[0020] Preferably, in the use described above, the porcine respiratory tract diseased tissue is lower respiratory tract disease material of porcine respiratory disease or porcine diseased lung tissue material.
[0021] Compared with the existing separation culture medium, the present invention has high separation accuracy and can reduce the growth interference of other common respiratory flora. Specifically, it has the following technical advantages compared with the technology:
[0022] 1) The detection effects of different culture media on common bacteria in Example 4 of the present invention show that respiratory flora such as Streptococcus suis, Enterococcus faecium, and Staphylococcus aureus still grow well on conventional blood plates and BHI solid culture media, but cannot grow on the selective culture media described in the present invention. The selective culture media described in the present invention can grow Actinobacillus pleuropneumoniae, Pasteurella multocida, and Bordetella bronchiseptica. Compared with conventional blood plates and BHI solid culture media, the color development of the two bacteria on the culture plates is obviously different. Picking the blue colonies on the plate can obtain a pure culture of Actinobacillus pleuropneumoniae.
[0023] 2) Example 4 of the present invention shows that the number of strains grown on the selective culture medium of the present invention is significantly reduced compared with the commercially available culture medium, and the accuracy of the color culture determined by sequencing comparison shows that most of the strains are Actinobacillus pleuropneumoniae. This shows that the separation and identification culture medium of the present invention can not only significantly reduce the growth interference of other bacteria, but also make Actinobacillus pleuropneumoniae present specific blue colonies, and its accuracy is significantly better than the existing selective culture medium. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 The figures are the streak culture results of Actinobacillus pleuropneumoniae (clinical isolate 1), Actinobacillus pleuropneumoniae (clinical isolate 2), Streptococcus suis (clinical isolate), and Bordetella bronchiseptica (clinical isolate) on different culture media, wherein a is the blood plate culture result, b is the BHI solid culture result, C is the chromogenic culture result of Example 1, and d is a streak schematic diagram.
[0025] Figure 2 The figures are the streak culture results of Pasteurella multocida (clinical isolate), Staphylococcus aureus (clinical isolate), Enterococcus faecium (clinical isolate), and Gramseria parasuis (clinical isolate) on different culture media, wherein a is the blood plate culture result, b is the BHI solid culture result, C is the chromogenic culture result of Example 1, and d is a streak schematic diagram.
[0026] Figure 3 These are the streaking culture results of the lower respiratory tract tracheal swab sample 1 in TSA++ medium and the chromogenic medium of the present invention, wherein a is the culture result of TSA++ medium, and b is the culture result of chromogenic medium of Example 1.
[0027] Figure 4 These are the streaking culture results of the lower respiratory tract tracheal swab sample 2 in TSA++ medium and the chromogenic medium of the present invention, wherein a is the culture result of TSA++ medium, and b is the culture result of the chromogenic medium of Example 1.
[0028] Figure 5 These are the streaking culture results of the lower respiratory tract tracheal swab sample 3 in TSA++ medium and the chromogenic medium of the present invention, wherein a is the culture result of TSA++ medium, and b is the culture result of chromogenic medium in Example 1.
[0029] Figure 6 These are the streaking culture results of lower respiratory tract tracheal swab sample 4 in TSA++ medium and the chromogenic medium of the present invention, wherein a is the culture result of TSA++ medium, and b is the culture result of chromogenic medium of Example 1.
[0030] Figure 7These are the streaking culture results of the diseased lung tissue sample 1 in TSA++ medium and the chromogenic medium of the present invention, wherein a is the culture result in TSA++ medium, and b is the culture result in chromogenic medium of Example 1.
[0031] Figure 8 These are the streaking culture results of the diseased lung tissue sample 2 in TSA++ medium and the chromogenic medium of the present invention, wherein a is the culture result of TSA++ medium, and b is the culture result of chromogenic medium of Example 1.
[0032] Fig. 9 These are the streaking culture results of the diseased lung tissue sample 3 in TSA++ medium and the chromogenic medium of the present invention, wherein a is the culture result of TSA++ medium, and b is the culture result of chromogenic medium of Example 1.
[0033] Fig.10 These are the streaking culture results of the diseased lung tissue sample 4 in TSA++ medium and the chromogenic medium of the present invention, wherein a is the culture result of TSA++ medium, and b is the culture result of chromogenic medium of Example 1. DETAILED DESCRIPTION
[0034] The present invention is further described below through specific implementations, but those skilled in the art should be aware that the embodiments do not limit the scope of protection of this patent in any way. The scope of patent protection is based on the scope defined by the claims of the present invention, and equivalent replacements for the technical solution of the present invention are included in the scope of protection of the patent of the present invention.
[0035] Example 1 Isolation and identification culture medium for Actinobacillus pleuropneumoniae in clinical pig disease materials of the present invention and preparation method thereof
[0036] The culture medium for the isolation and identification of Actinobacillus pleuropneumoniae in clinical tissues was prepared according to the following ratio:
[0037]
[0038] The preparation method of the culture medium for the isolation and identification of Actinobacillus pleuropneumoniae in the above clinical tissues is as follows:
[0039] 1. Weigh tryptone, soy peptone, sodium chloride, lactose, pyruvic acid and agar according to the formula, put them into a container and add 950 ml of distilled water, stir to dissolve, adjust the pH value to 7.2±0.2, and sterilize at 121℃ for 15 minutes;
[0040] 2. Weigh 0.03g of coenzyme I, dissolve it in 1ml of pure water to prepare a 30mg / ml coenzyme I solution, filter and sterilize for later use; weigh 0.02g of vancomycin, dissolve it in 1ml of pure water to prepare a 20mg / ml vancomycin solution, filter and sterilize for later use; weigh 0.16g of 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside, dissolve it in 1ml of DMSO to prepare a 0.16g / ml 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside solution, filter and sterilize for later use; weigh 0.04g of amphotericin B, dissolve it in 1ml of DMSO to prepare a 40mg / ml amphotericin B solution, filter and sterilize for later use;
[0041] 3. When the culture medium is cooled to below 55°C, add the prepared 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside solution, coenzyme I solution, vancomycin solution, amphotericin B solution and calf serum to the solid culture medium; mix well and pour into a plate to prepare a thickness of 3-4 mm / dish.
[0042] Example 2 Isolation and identification culture medium for Actinobacillus pleuropneumoniae in clinical pig disease materials of the present invention and preparation method thereof
[0043] The culture medium for the isolation and identification of Actinobacillus pleuropneumoniae in clinical tissues was prepared according to the following ratio
[0044]
[0045] The preparation method of the above selective culture medium is the same as that in Example 1.
[0046] Example 3 Isolation and identification culture medium for Actinobacillus pleuropneumoniae in clinical pig samples of the present invention and preparation method thereof
[0047] The culture medium for the isolation and identification of Actinobacillus pleuropneumoniae in clinical tissues was prepared according to the following ratio
[0048]
[0049] The preparation method of the above selective culture medium is the same as that in Example 1.
[0050] Example 4 Comparison of the culture medium for the isolation and identification of Actinobacillus pleuropneumoniae of the present invention and other common culture media for the cultivation of swine respiratory tract infection materials
[0051] Blood agar and BHI solid medium (BHI agar) were used as controls, and the culture medium of the present invention was used to culture common respiratory bacteria at 37°C for 18-24 hours. The blood agar and BHI solid medium used were purchased from Qingdao Haibo Biotechnology Co., Ltd. and prepared according to their instructions. The test results of common bacteria in common swine respiratory tract infections in blood agar and BHI solid medium are as follows: Figure 1 and Figure 2 As shown in Table 1, the comparison of the detection results of common bacteria causing porcine respiratory tract infection using different culture media.
[0052] Table 1 Comparison of the detection effects of different culture media on common bacteria in pig respiratory tract infection materials
[0053]
[0054] The number of "+" indicates bacterial growth, "++++" indicates good growth, and 1-3 "+" indicates that the growth is inhibited to varying degrees. "-" indicates no growth. The strains listed in the table were all isolated from pathological lung tissue samples of pigs with respiratory diseases.
[0055] From the detection effects of the above-mentioned different culture media on common bacteria, it can be seen that respiratory flora such as Streptococcus suis, Enterococcus faecium, Staphylococcus aureus, etc. still grow well on conventional blood plates and BHI solid culture media, but cannot grow on the selective culture media described in the present invention. The separation and identification culture media described in the present invention can grow Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, and Pasteurella multocida. Compared with conventional blood plates and BHI solid culture media, the three bacteria have obvious differences in color development on the separation and identification culture media described in the present invention, which shows that the culture media described in the present invention can significantly distinguish common pathogens in porcine respiratory bacterial diseases from Actinobacillus pleuropneumoniae, effectively separate and identify Actinobacillus pleuropneumoniae, and pick the blue colonies on the plate to obtain pure culture of Actinobacillus pleuropneumoniae.
[0056] Example 5 Isolation of Actinobacillus pleuropneumoniae from clinical samples using TSA++ medium and the separation and identification medium of the present invention
[0057] Actual sample detection: Take clinically collected pig lower respiratory tract tracheal swabs (4) and diseased lung tissue samples (4), rinse the swabs or diseased tissues thoroughly with 500ul sterile saline, take 20ul and drop it on the separation and identification culture medium of Example 1, and inoculate it in 3-4 zones, culture it at 37°C for 18-24h, and observe the growth of the colonies; pick the red colonies, expand the culture, use the bacterial 16S rRNA gene universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'), PCR amplify the purified isolated bacteria, send the amplicon to the sequencing company for sequencing, and after obtaining the sequence, conduct sequence comparison online to identify the bacterial species and compare the accuracy of the color development culture. At the same time, a culture medium commonly used for isolating porcine respiratory pathogens (TSA + 5% calf serum + 0.003% NAD +, TSA++) was used as a control culture medium. The TSA++ basic culture medium used was purchased from Qingdao Haibo Biotechnology Co., Ltd. and prepared according to its instructions.
[0058] Table 2 Comparison of detection effects of the isolation and identification culture medium of the present invention and commercially available culture medium in clinical samples
[0059]
[0060] "-" indicates no growth. One colony of each morphology was selected and species identification was performed using 16s rRNA sequencing.
[0061] The results of streaking culture of lower respiratory tract tracheal swab sample 1, lower respiratory tract tracheal swab sample 2, lower respiratory tract tracheal swab sample 3, lower respiratory tract tracheal swab sample 4, diseased lung tissue sample 1, diseased lung tissue sample 2, diseased lung tissue sample 3, and diseased lung tissue sample 4 in TSA++ medium and the chromogenic medium of the present invention are as follows: Figure 3-Figure 10 As shown, a is the result of TSA++ culture medium, and b is the result of color culture medium culture in Example 1. The experimental results show that the number of strains grown on the culture medium using the selective culture medium of the present invention is significantly reduced compared with the commercially available culture medium, and the accuracy of the color culture determined by sequencing comparison shows that most of the strains are Actinobacillus pleuropneumoniae. This shows that the separation and identification culture medium of the present invention can not only significantly reduce the growth interference of other bacteria, but also can significantly distinguish between common pathogens in porcine respiratory bacterial diseases and Actinobacillus pleuropneumoniae, and its accuracy is significantly better than the existing selective culture medium.
[0062] The separation and identification culture medium of the present invention in Example 4 and Example 5 can be replaced with the culture medium described in Example 2 or Example 3, which can achieve the same technical effect.
[0063] In summary, the separation and identification culture medium of the present invention can effectively inhibit the growth of miscellaneous bacteria and distinguish the target bacteria-Actinobacillus pleuropneumoniae through specific color development, which can improve the diagnosis of Actinobacillus pleuropneumoniae infection in clinical samples and its separation and identification.
Claims
1. A culture medium for the isolation and identification of Actinobacillus pleuropneumoniae in clinical swine disease materials, which is composed of the following components: Tryptone 12.0-18.0g / L, soy peptone 3.0-7.0g / L, sodium chloride 4.0-6.0g / L, lactose 2.0-4.0g / L, pyruvate 0.2-0.4g / L, calf serum 40-60ml / L, agar 12.0-18.0g / L, coenzyme I 0.02-0.04g / L, vancomycin 15-25mg / L, amphotericin B 3-5mg / L, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside 0.05-0.15g / L, dilute to 1L with pure water.
2. The culture medium for separating and identifying Actinobacillus pleuropneumoniae in clinical samples of pigs according to claim 1, characterized in that: It is composed of the following components: tryptone 15.0 g / L, soy peptone 5.0 g / L, sodium chloride 5.0 g / L, lactose 3.0 g / L, pyruvic acid 0.3 g / L, calf serum 50 ml / L, agar 15.0 g / L, coenzyme I 0.03 g / L, vancomycin 20 mg / L, amphotericin B 4 mg / L, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside 0.10 g / L, and the volume is made up to 1 L with pure water.
3. The culture medium for separating and identifying Actinobacillus pleuropneumoniae in pig clinical disease materials according to claim 1, characterized in that: It is composed of the following components: tryptone 12.0 g / L, soy peptone 7.0 g / L, sodium chloride 4.0 g / L, lactose 4.0 g / L, pyruvic acid 0.2 g / L, calf serum 60 ml / L, agar 12.0 g / L, coenzyme I 0.04 g / L, vancomycin 25 mg / L, amphotericin B 3 mg / L, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside 0.15 g / L, and the volume is made up to 1 L with pure water.
4. The culture medium for separating and identifying Actinobacillus pleuropneumoniae in pig clinical disease materials according to claim 1, characterized in that: It is composed of the following components: tryptone 18.0 g / L, soy peptone 3.0 g / L, sodium chloride 6.0 g / L, lactose 2.0 g / L, pyruvic acid 0.4 g / L, calf serum 40 ml / L, agar 18.0 g / L, coenzyme I 0.02 g / L, vancomycin 15 mg / L, amphotericin B 5 mg / L, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside 0.05 g / L, and the volume is made up to 1 L with pure water.
5. A method for preparing a culture medium for separating and identifying Actinobacillus pleuropneumoniae in clinical samples of pigs according to any one of claims 1 to 4, comprising the following steps: 1) Weigh or measure the prescribed amount of tryptone, soy peptone, sodium chloride, lactose, pyruvic acid and agar, add them to distilled water, stir to dissolve, adjust the pH value to 7.2±0.2, and sterilize at 121°C for 15 minutes to obtain a culture medium mother solution; 2) Weigh coenzyme I and dissolve it in pure water to prepare a 30 mg / ml coenzyme I solution, weigh vancomycin and dissolve it in pure water to prepare a 20 mg / ml vancomycin solution, weigh 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside and amphotericin B and dissolve them in DMSO to obtain a 0.16 g / ml 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside solution and a 40 mg / ml amphotericin B solution, and filter and sterilize the prepared solutions for later use; 3) Cool the culture medium to below 55°C, add the prescribed amount of 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside solution, coenzyme I solution, vancomycin solution, amphotericin B solution and calf serum, stir to mix and pour into a plate.
6. Use of the separation and identification culture medium according to any one of claims 1 to 4 in isolating and identifying Actinobacillus pleuropneumoniae in lesional respiratory tissues of pigs.
7. The use of the separation and identification medium according to claim 6, characterized in that: The respiratory tract diseased tissue is lower respiratory tract disease material of pig respiratory disease or pig diseased lung tissue material.