A strain of Priesteria gigantea, bacterial agent, preparation method and application thereof

Abstract

The present invention belongs to the technical field of microorganisms and biological products, and specifically relates to a strain of Priesteria gigantea, a bacterial agent, a preparation method and its application in preventing and treating swine contact infectious pleuropneumonia. The Priesteria gigantea is Priesteria gigantea ( Priestia megaterium )2203LBZ02, deposit number: CGMCC No.24901, deposit date: May 18, 2022, deposit unit: General Microbiology Center of China Microbiological Culture Collection Administration, deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the biological bacterial agent prepared based on this giant Priesteria not only has a strong antibacterial effect on APP, but also has a strong antibacterial effect on multidrug-resistant APP, and its preparation method is simple, easy to use, no antibiotic residue, efficient and safe.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and biological products, and specifically relates to a strain of Priesteria gigantea, a bacterial agent, a preparation method and application thereof in preventing and treating swine contact infectious pleuropneumonia. Background Art

[0002] Swine contact contagious pleuropneumonia is an acute respiratory infectious disease caused by Actinobacillus pleuropneumoniae. The mortality rate of acute cases is high, causing huge economic losses to large-scale pig farms. After pleuropneumonia occurs in pig farms, a variety of measures can be taken to prevent and treat contagious pleuropneumonia, such as antibiotic therapy, inactivated vaccine immunization, subunit vaccine immunization, etc. However, long-term antibiotic treatment will aggravate the drug resistance of Actinobacillus, and multi-drug resistant strains and super-drug resistant strains will appear. If effective prevention and control is not carried out in time, it will seriously affect the growth rate of the pig herd and cause a higher mortality rate.

[0003] In order to reduce the impact of Actinobacillus pleuropneumoniae on the pig herd, the most convenient, economical and effective way of prevention and control is to insist on self-breeding and self-feeding and introduce pigs without the pathogen. We should strengthen pig quarantine, do a good job of pig bacteria detection, introduce pigs from disease-free areas, and prevent pigs with bacteria from mixing into qualified pigs when introducing. However, due to the high cost of pig resources and limited detection methods, the most commonly used, fastest and most direct way of prevention and control is antibiotic prevention and control. Antibiotic prevention and control has reduced the occurrence of pleuropneumonia to a certain extent, but at the same time, the problems of veterinary drug residues, food safety, drug-resistant strains, environmental pollution, ecological balance destruction and pathogen resistance caused by excessive and frequent use of antibiotics and fungicides have also largely restricted the use of antibiotics.

[0004] Based on the above situation, with the development of healthy breeding, people are calling for pollution-free, pollution-free green livestock products. Biological control has gradually become a research hotspot for the prevention and control of bacterial diseases in animals. It is well compatible with the future development direction of mankind in terms of production methods, veterinary drug residues, food safety, nutritional health, ecological balance and biodiversity.

[0005] At present, biocontrol bacteria, as an important part of biological control, are also a hot topic in the research of biological control of animal bacterial diseases. Due to the wide variety of biocontrol bacteria, fungi, bacteria, actinomycetes and other types of biocontrol bacteria are widely used in production. However, there are no reports on endophytic microorganisms of medicinal plants as biocontrol bacteria. Therefore, it is urgent to develop new endophytic microbial biocontrol strains of medicinal plants, so as to play an important role in the process of controlling animal bacterial diseases, harmonious coexistence between man and nature, maintaining ecological balance and promoting sustainable agricultural development. Summary of the invention

[0006] In view of the defects and problems of poor control effect and difficulty in control of multidrug-resistant porcine Actinobacillus pleuropneumoniae by current biological bacteria control, the present invention provides a strain of Priesteria gigantea 2203LBZ02, a bacterial agent and its application on multidrug-resistant porcine Actinobacillus pleuropneumoniae.

[0007] The solution adopted by the present invention to solve the technical problem is: a strain of Priesteria gigantea, wherein the Priesteria gigantea is Priesteria gigantea ( Priestia megaterium )2203LBZ02, deposit number: CGMCCNo.24901, deposit date: May 18, 2022, deposit unit: General Microbiology Center of China Culture Collection Administration, deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The above-mentioned Priesteria gigantea, the Priesteria gigantea ( Priestia megaterium )2203LBZ02 was screened from the Chinese medicinal seed Fenugreek.

[0009] The present invention also provides a Priesteria gigantea biological agent, comprising the Priesteria gigantea 2203LBZ02 and / or a metabolite of the Priesteria gigantea 2203LBZ02.

[0010] The present invention also provides a method for preparing a Priesteria gigantea biological agent, comprising the following steps:

[0011] Step 1, inoculate Priesteria gigantea 2203LBZ02 onto a tryptic soy agar solid medium and culture at 28-32°C for 24-48 hours;

[0012] Step 2: Take the cultured bacteria in step 1 and inoculate it into tryptic soy liquid culture medium, and culture it at 28-32°C for 12-24 hours to obtain seed liquid;

[0013] Step 3: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 3-5%, ferment and culture for 24-72 hours at 28-32° C., rotation speed 200-240 rpm, and pH 7.0-7.5, and collect the fermentation liquid to obtain the microbial agent.

[0014] The preparation method of the Priesteria gigantea biological agent, wherein the live bacteria concentration of the Priesteria gigantea biological agent is 9-12×10 9 cfu / mL.

[0015] The invention also provides application of a strain of Priesteria gigantea or a biological bacterial agent prepared therefrom in preventing and treating swine contact infectious pleuropneumonia.

[0016] The above application is its application in preventing and treating porcine contact infectious pleuropneumonia caused by common porcine Actinobacillus pleuropneumoniae and multidrug-resistant porcine Actinobacillus pleuropneumoniae.

[0017] Beneficial effects of the present invention:

[0018] The Priesteria gigantea 2203LBZ02 separated and screened from fenugreek seeds and the biological bacterial agent prepared based on the Priesteria gigantea not only have extremely strong antibacterial effects on APP, but also have extremely strong antibacterial effects on multi-drug resistant APP.

[0019] The biological bacterial agent of the present invention has a simple preparation method, can achieve good therapeutic effect on swine contagious pleuropneumonia by any mode of oral administration, accompanying feed or drinking water, is easy to use, has no antibiotic residue, and is highly effective and safe. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The antibacterial activity of Priesteria megaterium 2203LBZ02 against Actinobacillus pleuropneumoniae.

[0021] Figure 2 The antibacterial activity of Priesteria gigantea 2203LBZ02 against multidrug-resistant Actinobacillus pleuropneumoniae.

[0022] Figure 3 The inhibition zone method is used to measure the antibacterial activity of Priesteria gigantea 2203LBZ02 against Actinobacillus pleuropneumoniae.

[0023] Figure 4 The inhibition zone method is used to test the antibacterial activity of Priesteria gigantea 2203LBZ02 against multidrug-resistant Actinobacillus pleuropneumoniae.

[0024] Figure 5 These are the colony morphological characteristics of Priesteria gigantea 2203LBZ02.

[0025] Figure 6 This is the bacterial morphology of Priesteria gigantea 2203LBZ02 under a microscope after Gram staining.

[0026] Figure 7 This is the electrophoresis pattern of PCR amplification using 16S rRNA.

[0027] Figure 8 This is the electrophoresis pattern of PCR amplification using 23S rRNA.

[0028] Fig. 9 This is the phylogenetic tree analysis diagram of Priesteria gigantea 2203LBZ02.

[0029] Fig.10The antibacterial activity of the fermentation supernatant of Priesteria gigantea 2203LBZ02 against multidrug-resistant APP. DETAILED DESCRIPTION

[0030] In order to explain the specific content of the present invention in detail, the present invention is further described below in conjunction with the drawings and embodiments. It should be noted that: the instruments and equipment involved in the following embodiments are all conventional instruments and equipment unless otherwise specified; the reagents and culture media involved are all conventionally commercially available unless otherwise specified; the test methods involved are all conventional methods unless otherwise specified. The multidrug-resistant APP used in the test is the multidrug-resistant porcine pleuropneumoniae used in the "In vitro antibacterial test and antibacterial effect study of Chinese herbal medicine extracts on multidrug-resistant porcine pleuropneumoniae Actinobacillus pleuropneumoniae". Chinese Journal of Veterinary Drugs, 2022, 56 (2): 70-77.

[0031] Example 1: Isolation and screening of Priesteria gigantea 2203LBZ02

[0032] (1) Isolation and purification of Priesteria megaterium 2203LBZ02

[0033] The seeds of Semen Lucidum (purchased from the seed store of Yuzhou Chinese Medicinal Materials Market in Henan Province, the seeds are flat) were surface disinfected and the ectoderm was separated under a microscope, then inoculated on PDA and LB medium, cultured at 30°C for 3-7 days, and colonies of different morphologies were picked and purified on TSA medium, and then the same strains were eliminated according to the morphological characteristics of the colonies under a microscope. The purified strains were stored at -80°C with 10% skim milk powder.

[0034] (2) Screening of antibacterial endophytes

[0035] Endophytes stored at -80℃ were selected and inoculated into TSA (10% fetal bovine serum + 10% NAD) culture plates, and the antibacterial activity was tested by streak method. A line was drawn in the center of two TSA (10% fetal bovine serum + 10% NAD) culture plates, and the endophytes were inoculated; then APP and multidrug-resistant APP (the strain information of multidrug-resistant APP is shown in Table 1) were inoculated on both sides of the streaked endophytes on the two culture plates, and the plates were placed in a 30℃ incubator for 24 hours to observe the antibacterial activity of the endophytes on APP and multidrug-resistant APP, as shown in Table 1. Figure 1 and Figure 2 .

[0036]

[0037] Depend on Figure 1 and Figure 2 It can be seen that the extracted endophytes have a certain antibacterial effect on APP and multidrug-resistant APP.

[0038] In order to further obtain the strength of the inhibition, the size of the inhibition zone was measured using the inhibition zone method. Endophytic bacteria stored at -80 °C were selected and inoculated into the center of the TSA (10% fetal bovine serum + 10% NAD) plate, and then 1×10 9 APP bacterial solution and 1×10 9 The multidrug-resistant APP bacterial solution was spread around the endophytes and placed in a 37 ℃ incubator for 24 h. The antibacterial effect of the endophytes on APP was observed and the size of the inhibition zone was measured. If there is a transparent inhibition zone around the endophytes, it means that this strain of endophytes has an inhibitory effect on APP. The strength of the inhibition effect is indicated by the size of the inhibition zone. The diameter of the inhibition zone > 20 mm indicates a strong inhibition effect; the inhibition zone < 20 mm and > 10 mm indicates a moderate inhibition effect; the inhibition zone < 10 mm indicates a weak inhibition effect; see Figure 3 and Figure 4 ,in Figure 3 The size of the middle inhibition zone is 45 mm. Figure 4 The size of the middle inhibition zone was 44 mm.

[0039] Depend on Figure 3 and Figure 4 It can be seen that the diameters of the inhibition zones of the obtained endophytes against APP and multidrug-resistant APP were both >20 mm, indicating that the obtained endophytes had a strong antibacterial effect against both APP and multidrug-resistant APP.

[0040] (3) Observation of endophytic bacteria morphology

[0041] Endophytes were inoculated on TSA culture plates and placed in a 30 ℃ incubator for 24 h. The colony morphology of endophytes was observed. Figure 5 .

[0042] Pick a single colony of endophytes and spread it on a slide, stain it with Gram stain, and then observe the bacterial morphology of the endophytes under an oil microscope. Figure 6 .

[0043] Depend on Figure 5 It can be seen that strain 2203LBZ02 forms round milky white colonies on solid culture medium, the surface of the colonies is smooth and moist, and Gram staining is positive.

[0044] Depend on Figure 6 It can be seen that the endophyte is in the shape of a thick rod, with blunt or truncated ends. It is a Gram-positive large bacillus, 1.0~1.2 µm×3~5 µm, and the spore is oval and located in the center of the bacterium. The bacterium is single or in 2~5 short chains connected, and the connected ends are truncated to form a bamboo node. Combined with the morphological observation results of the colony and the bacterium, it is preliminarily determined that the endophyte is Priesteria gigantea.

[0045] (4) Molecular identification of endophytes

[0046] 16S rRNA and 23 S rRNA were used for PCR amplification and sequencing identification. The primers used are shown in Table 2.

[0047]

[0048] The DNA of endophytes was used as template and amplified in 50 μL amplification system. The amplified product was first electrophoresed on 1% agarose gel to determine the size and position of the bands. The results are shown in Figure 7 and Figure 8 .

[0049] The PCR product was then sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing, and the sequence is shown in SEQ ID NO.1.

[0050] CCATTCTGTCCACTTAGGCGGCTAGCTCCTTACGGTTACTCCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGTTTTATGGGATTGGCTTGACCTCGCGGTCTTGCAGCCCTTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTAAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGTCCCCCGAAGGGGAACGCTCTATCTCTAGAGTTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAAAAAGCCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCGCTTTTCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGATAACGCTTGCCACCTACGTATTA(SEQ ID NO.1)

[0051] The sequencing results were analyzed and compared in the NCBI database, and the phylogenetic tree was constructed using the Neighbor Joining method in MEGA 5.0 software. Fig. 9 The sequence was analyzed for homology with the sequence of Priesteria gigantea, see Table 3.

[0052]

[0053] As can be seen from Table 3, there are 19 strains with high homology to 2203LBZ02, with homology reaching more than 99%, of which the homology to MG937742.1 and MK318790.1 reached 100%. According to the sequencing results, the 2203LBZ02 strain was determined to be Priesteria gigantea.

[0054] (5) Activity detection of endophytic bacterial fermentation broth

[0055] In order to further detect the antibacterial effect of endophytic fermentation broth, the endophytic strains were fermented and the activity of the fermentation broth was detected. The endophytic bacteria stored at -80℃ were selected and inoculated into NB medium, placed in a 30℃ incubator for 24 hours, centrifuged at 12,000rpm / min for 10 minutes, and the supernatant was filtered and sterilized with a 0.22µm sterile filter membrane. The antibacterial effect was then measured using the Oxford cup method. 100µL of endophytic fermentation broth was added to each well, and 4 replicates were performed. The size of the inhibition zone was measured and the average value was taken. The results are shown in Fig.10 , Fig.10 The size of the middle inhibition zone is 27 mm.

[0056] Example 2: Preparation of Priesteria gigantea 2203LBZ02 bacterial agent

[0057] (1) Strain culture and preparation

[0058] P. megaterium 2203LBZ02 was inoculated onto a tryptone soy agar plate and cultured at 28-32°C for 24-48 hours;

[0059] (2) Preparation of seed solution

[0060] The bacteria cultured in step (1) are inoculated into a tryptone soybean liquid culture medium and cultured at 28-32° C. for 12-24 hours to obtain a seed solution;

[0061] (3) Liquid fermentation production

[0062] Take the seed liquid and inoculate it into the fermentation medium at an inoculation rate of 3-5%. After fermentation and cultivation for 24-48 hours at 28-32°C, rotation speed 200-240rpm, pH 7.0-7.5, collect the fermentation liquid to obtain the microbial agent. The live bacteria concentration of the agent is 9-12×10 9cfu / mL.

[0063] Example 3: Experimental verification of the Priesteria gigantea 2203LBZ02 bacterial agent (the bacterial solution cultured in Example 2) in the prevention and treatment of swine contagious pleuropneumonia

[0064] This experimental verification was conducted in the experimental animal room of Henan Academy of Agricultural Sciences in March 2022. Florfenicol and blank were used as controls.

[0065] The specific steps are as follows:

[0066] (1) 60 fattening pigs of about 60 kg were selected and divided into 5 groups, each with 12 pigs, including different bacterial agent experimental groups, thiamphenicol control group and blank control group. The pigs were pre-fed for 3 days.

[0067] (2) Select porcine Actinobacillus pleuropneumoniae stored at -80°C and inoculate it into a liquid culture medium of tryptic soy broth (supplemented with 10% fetal bovine serum + 10% NAD) to make a 1×10 9 Bacterial liquid was used for the challenge test. Each pig was injected with 0.5 mL.

[0068] (3) After inoculation, the disease was observed every day. After clinical symptoms appeared, the bacteria Priesteria gigantea 2203LBZ02 and fluoroquinolone were used for treatment, and the treatment effect was observed and statistically analyzed. The results of the treatment experiment are shown in Table 4:

[0069]

[0070] It can be seen from Table 4 that the 5× bacterial agent experimental group and the 10× bacterial agent experimental group both have a 100% preventive and therapeutic effect on swine contagious pleuropneumonia.

[0071] Example 4: Experimental verification of the Priesteria gigantea 2203LBZ02 bacterial agent (the bacterial solution cultured in Example 2) in the prevention and treatment of swine contagious pleuropneumonia

[0072] The test was conducted in a pig farm in Yuanyang County, Henan Province in June 2022. The pigs weighing about 75 kg in the fattening farm had elevated body temperatures, coughing, depression, loss of appetite, lying on the ground, and unwillingness to move; they also became emaciated, grew slowly, and the dead pigs had bleeding from the mouth and nose. After clinical symptom observation, autopsy changes, and laboratory diagnosis, it was confirmed to be porcine contact infectious pleuropneumonia.

[0073] A 5× bacterial agent was used to conduct a feed-accompanying therapeutic test on sick pigs. During the treatment, the clinical symptoms of the sick pigs gradually alleviated, the body temperature gradually returned to normal, the appetite increased, and the coughing and wheezing symptoms alleviated and returned to normal; all sick pigs were cured after 3 days of treatment.

[0074] In summary, the experimental results prove that the biological bacterial agent prepared by Priesteria gigantea 2203LBZ02 obtained in the present invention has a good preventive and therapeutic effect on porcine contagious pleuropneumonia, and can effectively solve the problem of prevention and treatment of porcine contagious pleuropneumonia.

[0075] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Priestia megaterium 2203LBZ02, characterized in that: Deposit number: CGMCC No.24901, deposit date: May 18, 2022, deposit unit: General Microbiology Center of China Culture Collection Administration, deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

2. A Priesteria gigantea biological agent, characterized in that: The invention comprises the Priesteria megaterium 2203LBZ02 as claimed in claim 1 and the metabolites of the Priesteria megaterium 2203LBZ02.

3. A method for preparing the Priesteria gigantea biological agent as claimed in claim 2, characterized in that: The following steps are involved: Step 1, inoculate Priesteria gigantea 2203LBZ02 onto a tryptic soy agar solid medium and culture at 28-32°C for 24-48 hours; Step 2: Take the cultured bacteria in step 1 and inoculate them into tryptic soy liquid culture medium, and culture them at 28-32°C for 12-24h to obtain seed liquid; Step 3: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 3-5%, ferment and culture for 24-72 hours at 28-32° C., rotation speed 200-240 rpm, and pH 7.0-7.5, and collect the fermentation liquid to obtain the microbial agent.

4. The method for preparing the Priesteria gigantea biological agent according to claim 3, characterized in that: The live bacteria concentration of the Priesteria gigantea biological agent is 9-12×10 9 cfu / mL.

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