Swine bacillus subtilis TZ1202 and application thereof

Abstract

The invention discloses swine bacillus subtilis TZ1202 and application thereof, and belongs to the technical field of genetic engineering. The preservation number of the bacillus subtilis TZ1202 is CGMCC (China General Microbiological Culture Collection Center) No.33665, and the 16S rRNA (Ribose Nucleic Acid) sequence of the bacillus subtilis TZ1202 is shown as SEQ ID NO.1. The bacillus subtilis TZ1202 carries toxin genes such as hblA, hblC and the like, and is sensitive to 20 antibiotics and resistant to oxacillin. Animal experiments prove that the strain has pathogenicity / lethality to piglets and mice. The invention also provides a preparation method of an inactivated vaccine of the strain. An efficacy test shows that 0.25 mL of the strain can completely protect mice. In conclusion, the strain provided by the invention can be used for development of porcine epidemic disease quarantine reference substances and vaccines, and the detection accuracy and the prevention and treatment effect are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a porcine-derived Bacillus subtilis TZ1202 and applications thereof. Background Art

[0002] Bacillus subtilis is a Gram-positive aerobic or facultative anaerobic bacterium belonging to the Bacillaceae family. Its rod-shaped body is motile and possesses flagella, but lacks a capsule. Bacillus subtilis can grow in diverse environments, including soil, plant roots, and the gastrointestinal tract of animals. It is widely found in nature and easily isolated and cultured. Bacillus subtilis is highly heat-resistant, surviving for 20 minutes at 120°C and remaining active in acidic environments. Due to its rapid reproduction rate and the numerous antibiotics and enzymes it produces, Bacillus subtilis possesses broad-spectrum antimicrobial activity and stress resistance, making it widely used in food, livestock feed additives, and plant disease control. However, there are also reports that some strains of Bacillus subtilis may harbor vomitoxin, hemolytic and non-hemolytic enterotoxins, and potentially transmissible antibiotic resistance genes.

[0003] Currently, there are significant gaps in rapid detection methods, drug resistance analysis, and effective prevention and control measures for pathogenic Bacillus subtilis. Existing feed additives or vaccines are primarily developed based on their probiotic properties and lack targeted control of virulence genes. Therefore, isolating and characterizing pathogenic strains, and developing specific detection tools and immunization strategies, are of urgent importance for ensuring safe production in the livestock industry and reducing economic losses from disease outbreaks. Summary of the Invention

[0004] The present invention aims to provide a porcine-derived Bacillus subtilis TZ1202 and its application to solve the problems of the prior art. Animal experiments have confirmed that the present invention has pathogenic / lethal properties in piglets and mice through animal regression tests.

[0005] In order to solve the above problems, the present invention provides the following solutions:

[0006] Technical solution 1: A Bacillus subtilis TZ1202, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with a deposit number of CGMCC No.33665.

[0007] Furthermore, its 16S rRNA sequence is shown as SEQ ID NO.1.

[0008] Technical Solution 2: A method for preparing an inactivated vaccine, comprising the steps of culturing the Bacillus subtilis TZ1202 and inactivating it with formaldehyde.

[0009] Preferably, the steps are specifically as follows: culturing the Bacillus subtilis TZ1202 to the logarithmic growth phase, adding 1% formaldehyde solution to inactivate for 24 hours to obtain an inactivated bacterial solution, and mixing the inactivated bacterial solution with an aluminum gel adjuvant in a volume ratio of 1:3 to obtain the inactivated vaccine.

[0010] Technical solution three: inactivated vaccine prepared by the preparation method.

[0011] Technical Solution 4: An application of the Bacillus subtilis TZ1202 in preparing a swine disease quarantine control sample.

[0012] Technical Solution 5: A swine epidemic disease detection kit, comprising primers for amplifying the Bacillus subtilis TZ1202, the sequences of the primers being shown in SEQ ID NO.22 and SEQ ID NO.23.

[0013] Technical Solution 6: Application of the Bacillus subtilis TZ1202 in establishing an animal pathogenicity model.

[0014] Furthermore, the animal pathogenicity model is used for pathogenic mechanism research or drug screening.

[0015] The present invention discloses the following technical effects:

[0016] The present invention isolates pathogenic Bacillus subtilis TZ1202 (deposit number: CGMCC No. 33665), which carries a unique combination of toxin genes. Animal regression tests have demonstrated that the bacterium is pathogenic / lethal to piglets and mice, overturning traditional understanding of probiotics. Drug sensitivity tests have confirmed the strain's sensitivity to 20 antibiotics and its resistance to oxacillin (it is sensitive to neomycin, enrofloxacin, doxycycline, ciprofloxacin, florfenicol, kanamycin, azithromycin, amoxicillin, tetracycline, clindamycin, minocycline, co-trimoxazole, carbenicillin, polymyxin B, piperacillin, amikacin, penicillin, cephalexin, cefadroxil, ceftriaxone, and cefoperazone, moderately sensitive to erythromycin and ampicillin, and resistant to oxacillin and ceftazidime), providing a scientific basis for the rational use of drugs in the aquaculture industry. In addition, the present invention further developed an inactivated vaccine based on TZ1202, which can completely protect mice from virus infection at a dose of 0.25 mL / mouse, providing an efficient solution for the prevention and control of swine diseases. The present invention provides reference samples and epidemic prevention supplies for the prevention and treatment of fatal diseases in pigs, which can significantly speed up the diagnosis time of the disease, reduce the economic losses caused by swine diseases, and promote the steady development of the pig farming industry. The disclosure of the strain of the present invention fills the gap in the research of pathogenic Bacillus subtilis, and provides important theoretical support and application value for the prevention and control of animal diseases and production practices. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 PCR test results for ASFV, CSFV, PRRSV, PRV, and PCV2;

[0019] Figure 2 For microscopic examination of bacterial morphology (40×);

[0020] Figure 3 The image of parasporal crystals was examined under a microscope (100× oil objective lens);

[0021] Figure 4 This is the gel image of the strain 16S rRNA;

[0022] Figure 5 is the 16S rRNA gene phylogenetic tree;

[0023] Figure 6 The lesions of the autopsied mice (A, B, C) and weaned piglets (D, E, F) were observed. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] Example 1

[0030] After screening for common viral diseases such as African swine fever, classical swine fever, and porcine reproductive and respiratory syndrome in a pig farm (Tongliao City, Inner Mongolia) with sick and dead piglets, the present invention conducted bacterial culture on the blood of the sick and dead piglets and found colony growth, and isolated and identified the grown bacteria through biological identification.

[0031] 1. Isolation and identification of strains

[0032] The strain was initially cultured in ordinary LB agar medium in a 37°C incubator overnight and became opaque grayish white. When picked up with an inoculation loop, it showed a stringy phenomenon. Microscopic examination showed that the bacteria were short rods with blunt ends and were Gram-positive bacteria ( Figure 2 After special staining, it was placed under a microscope and no parasporal crystals were observed ( Figure 3 ). Named TZ1202.

[0033] 16S rRNA sequence analysis of TZ1202: The strain was inoculated into LB liquid medium and cultured at 37°C, 200 rpm, for 12 hours. 1 mL of the bacterial culture was collected by centrifugation and DNA was extracted.

[0034] Bacterial 16S rRNA universal primers (F: 5′-TACGGYTACCTTG-TTACGACT-3′ (SEQ ID NO. 22); R: 5′-AGAGTTTGATCM-TGGCTCAG-3′ (SEQ ID NO. 23)) were designed for PCR experiments. PCR conditions were as follows: 95°C pre-denaturation for 5 min: 94°C denaturation for 1 min, 52°C annealing for 1 min, 72°C extension for 1 min 30 s, 35 cycles; 72°C extension for 10 min. Electrophoresis analysis was then performed, and a band of approximately 1500 bp was obtained ( Figure 4 The target band was recovered from the gel and sent to a biological company for sequencing to obtain its nucleic acid sequence (the sequence is shown in SEQ ID NO.1).

[0035] In addition, the present invention also refers to the methods of documents 1-3 to perform PCR detection on African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV) and porcine circovirus type 2 (PCV2), and the test results show that there are no target bands, which can rule out the possibility of viral diseases ( Figure 1 ).

[0036] Reference 1: Li Ling. Isolation of African swine fever virus and study on its adaptability to passaged cells[D]. Gansu Agricultural University, 2023.DOI:10.27025 / d.cnki.ggsnu.2023.000278.

[0037] Reference 2: Wang Liping, Jin Qianyue, Liu Xingyou, et al. Preliminary application of PCR-ELISA detection method for classical swine fever virus [J]. Journal of Animal Ecology, 2023, 44(07): 64-71. DOI: 10.3969 / j.issn.1673-1182.2023.07.010.

[0038] Reference 3: Zhang Yuxin, Wu Yan, Zhang Wei, et al. Establishment of a quadruple PCR detection method for porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine circovirus type 2 and swine influenza virus [J]. Advances in Veterinary Medicine, 2023, 44(08): 22-27. DOI: 10.16437 / j.cnki.1007-5038.2023.08.026.

[0039] SEQ ID NO.1:

[0040]

[0041] The sequencing results of TZ1202 were compared by BLAST, and the gene sequence of this strain had a high homology with the gene sequence of Bacillus subtilis in the GenBank database ( Figure 5 ).

[0042] According to references 4-6, primers for the toxin gene and housekeeping gene of Bacillus subtilis were designed for PCR experiments (toxin primers and annealing temperatures are shown in Table 1).

[0043] Reference 4: Gu Ying. Isolation, identification, preservation and mechanism of action of safe and efficient Bacillus subtilis inhibitory against Spirogyra[D]. Shanghai Ocean University, 2023. DOI: 10.27314 / d.cnki.gsscu.2023.000220.

[0044] Reference 5: Cao Haipeng, Gu Ying, Sun Miaomiao, et al. Safety analysis of Bacillus subtilis A4 used in Chinese mitten crab aquaculture [J]. Freshwater Fisheries, 2023, 53(05): 104-112. DOI: 10.13721 / j.cnki.dsyy.2023.05.006.

[0045] Reference 6: Roberts MS, Nakamura LK, Cohan FM. Bacillus mojavensis sp. nov., distinguishable from Bacillus subtilis by sexual isolation, divergencein DNA sequence, and differences in fatty acid composition. Int J SystBacteriol. 1994Apr;44(2):256-64.doi:10.1099 / 00207713-44-2-256.PMID:8186089.

[0046] The results showed that the TZ1202 strain contained enterotoxin genes (hblA, hbC, hblD, nheA, bceT, entFM), cytotoxin gene (cytK) and housekeeping genes (ropB, gyrA).

[0047] Table 1 Toxin primers and annealing temperature

[0048]

[0049]

[0050] The present invention compares TZ1202 with the standard strain WB800N in a biochemical test. The results show that TZ1202 is consistent with the biochemical results of the standard strain WB800N. Both can decompose glucose, sucrose, lysine, ornithine and amino acid controls, but cannot decompose lactose, maltose, mannitol, hydrogen sulfide, phenylalanine, gluconate, indole, methyl red, citrate, urea, raffinose, sorbitol and styraxol. Bacillus subtilis has flagella and can move. The semi-solid agar (power) test is positive, does not produce gas, and glucose (gas production) is negative. It can be seen that TZ1202 can decompose sucrose, glucose and mannitol, but cannot decompose lactose and maltose. According to Bergey's Bacterial Identification Manual and Common Bacterial System Identification Manual, the biochemical characteristics of TZ1202 are consistent with those of Bacillus. The results of the biochemical tests are shown in Table 2.

[0051] Table 2 Biochemical test results

[0052] project WB800N Results TZ1202 Results project WB800N Results TZ1202 Results glucose + + Citrate — — lactose — — urea — — maltose — — Semisolid agar (power) + + Mannitol — — Glucose (gas production) — — sucrose + + Lysine + + hydrogen sulfide — — Ornithine + + Phenylalanine — — Raffinose — — Gluconate — — sorbitol — — Indole test — — Adonitol — — Methyl red — — Xylose ± ± Voges-Proskauer ± ± Amino acid control + +

[0053] From the above, it can be concluded that TZ1202 belongs to Bacillus subtilis. TZ1202 has been deposited in the General Microbiology Center of China Culture Collection Administration, with a deposit date of February 26, 2025, a deposit number of CGMCC No. 33665, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0054] The present invention selected multiple drugs to perform drug sensitivity tests on strain TZ1202. The results showed that the strain was sensitive to neomycin, enrofloxacin, doxycycline, ciprofloxacin, florfenicol, kanamycin, azithromycin, amoxicillin, tetracycline, clindamycin, minocycline, co-trimoxazole, carbenicillin, polymyxin B, piperacillin, amikacin, penicillin, cephalexin, cefadroxil, ceftriaxone and cefoperazone, moderately sensitive to erythromycin and ampicillin, and resistant to oxacillin and ceftazidime (see Table 3 for details).

[0055] Table 3 Drug sensitivity test

[0056]

[0057]

[0058] The present invention also conducted a pathogenicity test on the TZ1202 strain: 30 adult mice weighing 20-25g (half male and half female, never mated and conceived) and 15 weaned piglets weighing 9.76±0.06kg were selected, and after grouping, they were all injected intraperitoneally for infection. The specific operation is as follows: the mice were restrained on the dorsal side, with the tail clamped between the little finger and ring finger of the left hand, the neck skin between the two ears was pinched with the index finger and thumb, and the abdomen was turned upward with the organs inclined to the front cavity, and the injection was performed on the right side of the midline of the lower abdomen; the piglets were restrained in an upright position, with the two front limbs of the piglets lifted to make their organs inclined to the front cavity, and the injection was performed on the posterior side of the thigh. There should be no resistance during the injection and the skin should not bulge. After the injection, the clinical status and autopsy pathological conditions of the mice and weaned piglets were observed within 7 days, and the number of deaths was recorded (see Tables 4 and 4 for details). Figure 6 ).

[0059] Table 4 Animal regression test

[0060]

[0061] From Table 4 and Figure 6 It can be seen that the Bacillus subtilis TZ1202 strain isolated by the present invention is pathogenic / lethal to pigs, which is significantly different from previous cognition. Previously, Bacillus subtilis has always been considered a probiotic that is harmless to humans and animals. However, this study revealed the potential pathogenicity of the bacterium, reminding us that the safety of Bacillus subtilis needs to be re-evaluated in practical applications, and research on this strain needs to be strengthened. The strain isolated by the present invention provides an important theoretical basis for the prevention and treatment of swine diseases. The Bacillus subtilis TZ1202 isolated by the present invention can be used as a control sample for swine disease quarantine, or as a strain for a swine disease prevention vaccine. The obtained strain and its related information can be used as standard reference substances for pig quarantine, providing an important reference for quarantine work. These reference substances can be used for laboratory testing, proficiency testing and the formulation of relevant technical regulations to ensure the accuracy and reliability of the quarantine process.

[0062] In addition, the TZ1202 strain isolated in this invention provides key basic data for vaccine research and development. This data covers the biological characteristics of the strain, providing an important scientific basis for vaccine development and production. The present invention specifically uses the preparation of an inactivated vaccine as an example:

[0063] 1. Preparation of inactivated vaccines

[0064] Bacterial culture: Streak TZ1202 onto a regular agar plate and incubate overnight at 37°C. Subsequently, single colonies were picked from the plate and inoculated into LB liquid medium, incubated at 37°C for 12 hours. After incubation, 1% formaldehyde solution was added to the bacterial suspension, mixed thoroughly, and incubated at 37°C for another 24 hours to complete the inactivation treatment. The inactivated bacterial suspension was mixed evenly with aluminum gel adjuvant in a 1:3 volume ratio and stored at 4°C until ready for use.

[0065] 2. Quality inspection of inactivated vaccines

[0066] (1) Sterility test: Inoculate the inactivated vaccine into nutrient agar medium and observe the bacterial growth after culture to determine whether the vaccine meets the sterility standard.

[0067] (2) Safety Study: Twenty Kunming mice were randomly divided into two groups, 10 in each. The first group received the alum-adjuvanted vaccine by intramuscular injection, while the second group received an equal volume of saline as a negative control. The inoculation dose was 0.5 mL per mouse. All mice were housed under the same conditions and observed, and clinical manifestations were recorded within 10 days.

[0068] 3. Quality inspection results

[0069] No colonies were observed on nutrient agar after formaldehyde-inactivation of the bacteria, demonstrating a significant and thorough inactivation effect. Furthermore, the skin at the injection site remained normal in both groups of mice, with no redness, swelling, or other abnormal reactions. The mice were in good health and showed no obvious clinical symptoms.

[0070] 4. Efficacy testing of inactivated vaccines

[0071] Sixty Kunming mice were selected and randomly divided into six groups, with 10 mice in each group. Groups 1 to 5 were injected intramuscularly with inactivated vaccine at doses of 0.1 mL, 0.15 mL, 0.2 mL, 0.25 mL, and 0.3 mL, respectively; Group 6 (should be Group 7) was injected intramuscularly with PBS as a negative control. On the 15th day after immunization, all mice were challenged with Bacillus subtilis intraperitoneally at a volume of 0.25 mL / mouse, with a live bacterial concentration of 1×10 8 CFU / mL. The mice were then observed for clinical manifestations.

[0072] The results of the efficacy test are shown in Table 5. The results show that a small dose (0.1 mL / mouse) of the vaccine cannot provide immune protection to mice after immunization. When the immunization dose is increased to 0.15 mL / mouse-0.2 mL / mouse, although a certain immune protection effect can be produced, complete protection cannot be achieved. Only when the vaccine dose reaches 0.25 mL / mouse can complete immune protection be provided to mice. However, further increasing the vaccine dose (such as 0.3 mL / mouse) does not further enhance the immune protection effect, but only maintains at a certain level.

[0073] Table 5 Efficacy test of inactivated vaccine

[0074]

[0075] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A Bacillus subtilis TZ1202, characterized in that It is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No.33665.

2. The Bacillus subtilis TZ1202 according to claim 1, characterized in that Its 16S rRNA sequence is shown in SEQ ID NO.

1.

3. A method for preparing an inactivated vaccine, characterized in that: The method comprises the steps of culturing the Bacillus subtilis TZ1202 according to claim 1 or 2 and inactivating the same with formaldehyde.

4. The inactivated vaccine prepared by the preparation method according to claim 3.

5. Use of the Bacillus subtilis TZ1202 according to claim 1 or 2 in preparing a swine disease quarantine control sample.

6. A swine disease detection kit, characterized in that: The method comprises primers for amplifying the Bacillus subtilis TZ1202 according to claim 1 or 2, wherein the sequences of the primers are shown in SEQ ID NO. 22 and SEQ ID NO.

23.

7. Use of the Bacillus subtilis TZ1202 according to claim 1 or 2 in establishing an animal pathogenicity model.

8. The use according to claim 7, characterized in that The animal pathogenicity model is used for pathogenic mechanism research or drug screening.

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