Clostridium butyricum and application thereof

Abstract

The application discloses Clostridium butyricum and application thereof, the Clostridium butyricum is isolated from the fresh content of the intestine of chicken resistant to IBV infection, has the effect of resisting broad-spectrum avian infectious bronchitis virus infection, has good protective efficacy to the infection of avian infectious bronchitis virus QX type strain and Mass genotype strain, and simultaneously, the application shows through experiments that the isolated Clostridium butyricum can activate chicken I-type interferon expression, realizes good protection effect to the infection of different serotypes of strains by improving I-type interferon expression.The Clostridium butyricum of the application solves the problem of low cross-protection efficacy of vaccine, greatly shortens the response time when the breeding industry encounters sudden IBV disease outbreak, and simultaneously, IBV prevention and control can be carried out by adding in drinking water, so that the use convenience is greatly improved, and the prevention and control work efficiency is increased.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Clostridium butyricum and its applications. Background Technology

[0002] Avian infectious bronchitis virus (IBV) belongs to the gamma coronavirus family and is a highly contagious respiratory disease that primarily infects the respiratory, digestive, and urogenital systems of chickens. This virus spreads rapidly, has a high morbidity rate, and can cause growth retardation in chicks, decreased egg production in adult chickens, and secondary infections, leading to high mortality rates and significant economic losses to poultry farming worldwide.

[0003] Because IBV is a coronavirus, like other coronaviruses, it is prone to mutation. Different serotypes or genotypes of the virus can continuously emerge in different regions and at different times. Each epidemic period has a dominant genotype, and serotypes change simultaneously with genotype changes, making prevention and control extremely difficult. Therefore, among common infectious diseases in poultry, this virus poses a significant threat to poultry production.

[0004] Currently, prevention and control primarily rely on vaccination, especially live attenuated vaccines, which offer good protection against viruses of the same serotype. However, cross-protection against different serotypes is very low. Furthermore, live attenuated vaccines are made from artificially attenuated chicken infectious bronchitis virus, typically using chicken embryo passage, a process that is lengthy. Therefore, there is an urgent need to develop alternative prevention and control strategies. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a Clostridium butyricum strain and its applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides Clostridium butyricum su001.

[0007] In some embodiments of the present invention, the Clostridium butyricum su001 is taxonomically named Clostridium butyricum It was deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) on January 12, 2026, with accession number GDMCC NO: 67631.

[0008] In some embodiments of the present invention, the Clostridium butyricum su001 is isolated from fresh intestinal contents of chickens resistant to IBV infection.

[0009] In a second aspect, the present invention provides a culture of Clostridium butyricum as described above.

[0010] In some embodiments of the present invention, the culture includes its metabolites and / or active substances isolated from the Clostridium butyricum.

[0011] In some embodiments of the present invention, the culture is selected from any one of the following: (1) Live bacteria, attenuated bacteria, inactivated bacteria, or freeze-dried bacteria; (2) The fermentation broth of the Clostridium butyricum; (3) The supernatant of the fermentation broth of the Clostridium butyricum; (4) The inactivated fermentation broth of the Clostridium butyricum; (5) The fermentation broth, fermentation broth supernatant and / or the concentrated or dried product of the inactivated fermentation broth in (2)-(4).

[0012] In this invention, "supernatant" or "supernatant" refers to the culture supernatant of a bacterial strain according to the invention, optionally containing compounds and / or cell debris of the strain, and / or metabolites and / or molecules secreted by the strain.

[0013] A third aspect of the present invention provides a microbial agent comprising Clostridium butyricum and / or its culture as described above.

[0014] In some embodiments of the present invention, the microbial agent is provided in liquid or solid form.

[0015] In some embodiments of the present invention, the Clostridium butyricum in the bacterial agent is an attenuated bacterium, a killed bacterium, a freeze-dried bacterium, or an irradiated bacterium, for example, it can be a heat-inactivated bacterium, preferably pasteurized.

[0016] A fourth aspect of the present invention provides the use of at least one of the above-described Clostridium butyricum, a culture of Clostridium butyricum, or a bacterial agent in the preparation of a product for preventing avian infectious bronchitis virus infection.

[0017] A fifth aspect of the invention provides the use of at least one of the above-described Clostridium butyricum, a culture of Clostridium butyricum, or a bacterial agent in the preparation of a product for the treatment or adjunctive treatment of diseases caused by avian infectious bronchitis virus infection.

[0018] In some embodiments of the present invention, the avian infectious bronchitis virus includes: Mass type strain, QX type strain, Conn type strain, OX type strain, LDT3-A type and LDT3-B type strain.

[0019] In some embodiments of the present invention, the avian infectious bronchitis virus includes or is: Mass type strain or QX type strain.

[0020] In some embodiments of the present invention, the product includes feed, feed additives, or pharmaceuticals.

[0021] In some embodiments of the present invention, the product also contains excipients.

[0022] In some embodiments of the present invention, the excipients include pharmaceutically acceptable excipients or animal nutrients.

[0023] In some embodiments of the present invention, the pharmaceutically acceptable excipients include, but are not limited to, one or more of the following: buffers, lyophilization protectants, preservatives, stabilizers, binders, compactors, lubricants, dispersants, disintegrants, antioxidants, flavoring agents, sweeteners, and colorants.

[0024] In some embodiments of the present invention, the product further comprises one or more other active agents.

[0025] In some embodiments of the present invention, the other active agents may be one or more of probiotics and prebiotics, or a combination thereof.

[0026] In some embodiments of the present invention, the Clostridium butyricum in the product may be in solid or liquid form.

[0027] In some embodiments of the present invention, the dosage form of the medicine includes powder, granules, capsules, tablets, pills, or oral liquid.

[0028] In some embodiments of the present invention, the product is administered orally.

[0029] In some embodiments of the invention, the oral administration comprises adding 1×10 to the drinking water of each poultry. 9 -5×10 9 CFU products containing Clostridium butyricum.

[0030] In some embodiments of the present invention, the poultry includes: chickens.

[0031] The beneficial effects of this invention are: This invention provides a *Clostridium butyricum* strain and its applications. The *Clostridium butyricum* strain isolated from the fresh intestinal contents of chickens resistant to IBV infection exhibits broad-spectrum anti-avian infectious bronchitis virus (IBV) infection, showing good protective efficacy against both the QX and Mass genotypes of IBV. The protection rate against the QX genotype reaches 75%, and against the Mass genotype reaches 85%, with a mortality rate of 0% in challenge experiments against both genotypes. Furthermore, this invention demonstrates through experiments that the isolated *Clostridium butyricum* strain can activate type I interferon expression in chickens, achieving good protective effects against infection by different serotypes of the virus by increasing type I interferon expression.

[0032] This invention, *Clostridium butyricum*, solves the problem of low cross-protective efficacy of vaccines. It can be formulated into probiotic preparations, greatly shortening the response time for sudden IBV outbreaks in the aquaculture industry. At the same time, IBV can be controlled by adding it to drinking water, greatly improving ease of use and increasing the efficiency of prevention and control work. Attached Figure Description

[0033] Figure 1 The image shows the morphological identification results of Clostridium butyricum su001.

[0034] Figure 2 This is a diagram showing the genome analysis results of Clostridium butyricum su001.

[0035] Figure 3 This is a schematic diagram of the animal experiment process in an embodiment of the present invention.

[0036] Figure 4 The graph shows the results of detecting the type I interferon transcription level in chickens by Clostridium butyricum. The horizontal axis represents the grouping by the number of days after challenge, and the vertical axis represents the relative expression level of type I interferon mRNA. Detailed Implementation

[0037] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the testing or experimental methods in the present invention are conventional methods in the art.

[0038] In this invention, the statistical analysis of the embodiments was performed using GraphPad Prism 8.0.2 software and one-way ANOVA analysis of variance. If P>0.05, it represents no significant difference and is marked with "ns"; if P<0.05, it represents a significant difference and is marked with "*"; if P<0.01, it is marked with "**"; if P<0.001, it is marked with "***"; if P<0.0001, it is marked with "****".

[0039] Example 1 This embodiment provides the isolation and identification of a strain of Clostridium butyricum. The specific experimental steps are as follows.

[0040] Take 1 g of fresh intestinal contents from chickens infected with IBV, add 9 mL of 0.5% physiological saline, and perform serial dilutions. Take 10 g of each of these diluted samples. 1 10 2 10 3 10 4 10 5 10 6 Spread 100 mL of each dilution solution onto nutrient broth agar plates, with 3 plates for each dilution. Incubate the plates at 37°C under anaerobic and aerobic conditions for 24 h, respectively. Pick single colonies of different morphologies from the plates for microscopic examination and purify them using conventional methods to obtain pure bacteria.

[0041] The purified bacteria obtained from the culture were mixed into the drinking water of chickens at a CFU level, and then the chickens were challenged with IBV. The IBV strains used for challenge were QX type IBV and Mass type IBV, both of which were purchased. The QX type IBV was strain HSJ-2016 (database number: GenBank MG544176), and the Mass type IBV was strain M41, both from the China Institute of Veterinary Drug Control (IVDC).

[0042] Based on the results of animal challenge protection experiments, a strain with protective efficacy against IBV was screened.

[0043] The obtained strains were cultured on Clostridium-enriched medium plates for 12 h and then examined under a microscope after Gram staining.

[0044] The obtained anaerobic bacteria were sent to the Guangdong Provincial Microbial Culture Collection Center (GDMCC) for whole-genome sequencing and sequence analysis, and API anaerobic identification was performed to determine the species classification.

[0045] The results are as follows Figure 1 and Figure 2 As shown.

[0046] Figure 1 Gram-stained microscopic images of anaerobic bacteria that are protective against IBV were obtained. Figure 2 This is a diagram showing the genomic analysis results of the isolated anaerobic bacteria. Combining the genomic analysis results with the API anaerobic identification experiment results, the obtained anaerobic bacteria can be identified as Clostridium butyricum.

[0047] The butyric acid bacterium was named su001, and its taxonomic name was... Clostridium butyricumIt was deposited on January 12, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The accession number is GDMCC NO: 67631.

[0048] Example 2 Avian infectious bronchitis virus QX genotype (QX type IBV) is currently the dominant circulating IBV strain in China. This example provides a test of the protective efficacy of Clostridium butyricum su001 isolated in Example 1 against avian infectious bronchitis virus QX genotype.

[0049] The specific testing method is as follows.

[0050] On days 1, 6, and 9 after hatching, a preventative supplement of 4 × 10⁻⁶ mg / L was added to the drinking water of the SPF chickens in the experimental group. 9 CFU / bacterium butyricum su001 was administered; neither the challenge group nor the negative control group received any additional Clostridium butyricum. On day 10 post-incubation, the challenge group and the experimental group received a dose of 4 × 10⁻⁶ CFU / bacterium. 5 EID 50 / bird infectious bronchitis virus QX genotype strain was used to challenge SPF chickens via eye drop and nasal drop, and the morbidity and mortality rates of the chickens were monitored within 21 days after challenge.

[0051] The experimental results are shown in the table below.

[0052] Table 1. Protective efficacy of Clostridium butyricum against QX genotype strain of avian infectious bronchitis virus in Example 1.

[0053] It was observed that, up to 21 days after challenge, the morbidity and mortality rates of chickens supplemented with Clostridium butyricum (experimental group) were significantly lower than those of chickens in the challenge group (with normal drinking water). The screened Clostridium butyricum su001 achieved a protection rate of 75% against QX genotype IBV, demonstrating good protective efficacy.

[0054] Example 3 The Mass genotype of avian infectious bronchitis virus is also a major circulating strain widely distributed globally. This embodiment provides a test of the protective efficacy of Clostridium butyricum su001 isolated in Example 1 against the Mass genotype of avian infectious bronchitis virus.

[0055] The specific testing method is as follows.

[0056] Following the method described in the above embodiments, on days 1, 6, and 9 after hatching, a preventative addition of 4 × 10⁻⁶ mg / L to the drinking water of the SPF chickens in the experimental group was made. 9CFU / bacterium butyricum su001 was administered; neither the challenge group nor the negative control group received any additional Clostridium butyricum. On day 10 of the experiment, both the challenge group and the experimental group received a dose of 4 × 10⁻⁶ CFU / bacterium. 5 EID 50 Mass type IBV strain was used to challenge SPF chickens with the virus via eye drops and nasal drops. The morbidity and mortality rates of the chickens were monitored within 21 days after the challenge.

[0057] The results are shown in the table below.

[0058] Table 2. Protective efficacy of Clostridium butyricum against Mass genotype strains of avian infectious bronchitis virus.

[0059] Until 21 days after challenge, the morbidity and mortality rates of chickens with Clostridium butyricum added to their drinking water (experimental group) were significantly lower than those of chickens in the control group (normal drinking water). The Clostridium butyricum isolated in Example 1 achieved an 85% protection rate against Mass genotype IBV, demonstrating good protective efficacy.

[0060] Example 4 This embodiment uses Clostridium butyricum su001 obtained in Example 1 to conduct a challenge experiment to determine the optimal control method against IBV. The experimental flowchart is shown below. Figure 3 As shown, the specific experimental steps are as follows.

[0061] After activating the Clostridium butyricum su001 obtained from the screening in Example 1, it was subjected to routine culture. On days 1, 6, and 9 after chick hatching, it was added to the drinking water at a concentration of 4 × 10⁻⁶. 9 Add CFU / only the amount of Clostridium butyricum su001 and maintain a normal diet.

[0062] Subsequently, chickens were challenged with the aforementioned QX-type IBV, specifically, at a dose of 4 × 10⁻⁶. 5 EID 50 / bird, challenged with the virus by eye drops and nasal drops on day 10 of the experiment. Spleens of chickens in each group were collected on days 3 and 7 post-infection (dpi), RNA was extracted using a commercially available kit, and the transcriptional level of type I interferon IFN-α was detected by RT-qPCR.

[0063] The RT-qPCR primers are: Primer F: 5'-ATCCTGCTGCTCACGCTCCTTCT-3' (SEQ ID NO:1); Primer R: 5'-GGTGTTGCTGGTGTCCAGGATG-3' (SEQ ID NO:2).

[0064] The reaction system is shown below: 10 μL of qPCR Mix (containing enzyme), 0.4 μL each of primers F / R (both at 10 μM), and RNase-Free water to a final volume of 20 μL.

[0065] The reaction procedure was: 95°C for 30 seconds; then 95°C for 5 seconds, 60°C for 30 seconds (40 cycles).

[0066] Chickens challenged with the virus and drinking water free of Clostridium butyricum su001 were used as positive controls.

[0067] The results are as follows Figure 4 As shown.

[0068] Experimental results show that 4×10⁻⁶ tons of oral fluid can be ingested through drinking water. 9 CFU / bacterial Clostridium butyricum su001 can effectively activate the expression of type I interferon, thereby producing an anti-IBV effect.

[0069] Example 5 In this embodiment, the efficacy of Clostridium butyricum su001 and existing commercially available Clostridium butyricum in the prevention and control of IBV was compared.

[0070] The specific experiment is as follows.

[0071] Following the method described in the above embodiments, on days 1, 6, and 9 after hatching, a preventative addition of 4 × 10⁻⁶ mg / L to the drinking water of the SPF chickens in the experimental group was made. 9 Commercially available Clostridium butyricum (CFU / vial, purchased from Beina Biotechnology, catalog number BNCC337239) was administered to both the challenge and negative control groups without the addition of Clostridium butyricum. On day 10 of the experiment, the dose administered to both the challenge and experimental groups was 4 × 10⁻⁶ CFU / vial. 5 EID 50 SPF chickens were challenged with QX or Mass IBV strains via eye drops or nasal drops, and the morbidity and mortality rates were monitored within 21 days post-challenge.

[0072] The results are shown in the table below.

[0073] Table 3. Protective efficacy of Clostridium butyricum against QX genotype strains of avian infectious bronchitis virus.

[0074] Table 4. Protective efficacy of Clostridium butyricum against Mass genotype strains of avian infectious bronchitis virus.

[0075] It can be observed that commercially available Clostridium butyricum does not provide effective broad-spectrum protection against avian infectious bronchitis virus. This indicates that not all existing Clostridium butyricum strains can be used for the prevention and treatment of avian infectious bronchitis virus.

[0076] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Clostridium butyricum su001, characterized in that, The taxonomic name of Clostridium butyricum su001 is... Clostridium butyricum It was deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) on January 12, 2026, with accession number GDMCC NO: 67631.

2. The culture of Clostridium butyricum su001 as described in claim 1.

3. A microbial agent, characterized in that, The microbial agent comprises Clostridium butyricum su001 as described in claim 1 and / or the culture as described in claim 2.

4. The use of at least one of the Clostridium butyricum Su001 of claim 1, the culture of claim 2, or the bacterial agent of claim 3 in the preparation of a product for the prevention of avian infectious bronchitis virus infection.

5. The use of at least one of the Clostridium butyricum Su001 of claim 1, the culture of claim 2, or the bacterial agent of claim 3 in the preparation of a product for the treatment or adjunctive treatment of diseases caused by avian infectious bronchitis virus infection.

6. The application according to claim 4 or 5, characterized in that, The avian infectious bronchitis virus includes: Mass type strain, QX type strain, Conn type strain, OX type strain, LDT3-A type and LDT3-B type strain.

7. The application according to claim 4 or 5, characterized in that, The products include feed, feed additives, or pharmaceuticals.

8. The application according to claim 7, characterized in that, The dosage forms of the medicine include powder, granules, capsules, tablets, pills, or oral liquid.

9. The application according to claim 4 or 5, characterized in that, The product also contains auxiliary materials; Preferably, the excipients include pharmaceutically acceptable excipients or animal nutrients.

10. The application according to claim 4 or 5, characterized in that, The product contains Clostridium butyricum in either solid or liquid form; Preferably, the Clostridium butyricum is in the form of lyophilized powder, bacterial solution, or granular inoculation agent.

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