Use of akkermansia in the preparation of a medicine for preventing and treating avian infectious bronchitis virus

Abstract

This invention discloses the application of Akkermansia in the preparation of drugs for the prevention and treatment of avian infectious bronchitis virus (AIB). Through research, this invention has found that Akkermansia can improve the survival rate of chickens infected with AIB and inhibit the replication of AIB. This demonstrates that Akkermansia has important applications in the preparation of drugs for the prevention and / or treatment of AIB infection, providing a new solution for the treatment of AIB infection, and can be specifically applied in drugs or feed additives for combating AIB infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Akkermansia in the preparation of drugs for the prevention and treatment of avian infectious bronchitis virus. Background Technology

[0002] In recent years, probiotics have been widely used in disease prevention and treatment, especially as immune enhancers in poultry farming. Probiotics refer to a class of active microorganisms, cultures, and other substances that regulate the balance of the gastrointestinal microecological community in animals to prevent disease, promote animal growth, and improve feed utilization. Studies have shown that Lactobacillus and Bifidobacterium can appropriately activate inflammasomes and regulate respiratory mucosal immunity in animal models of respiratory virus infection. Furthermore, compound probiotics can promote intestinal development in Newcastle disease-infected chickens and promote IgA production in the intestinal immune system, thereby delaying disease progression and reducing disease damage. In the process of hepatitis B virus infection, the use of probiotics combined with antiviral intervention can also improve the therapeutic effect of antiviral drugs. Akkermansia muciniphila, a star in the intestinal microbiota, has been a research hotspot in recent years. It is one of the very few known enterobacteria that can still induce T-cell-dependent immunity under intestinal homeostasis, suggesting that it may participate in the normal intestinal immune tolerance process. However, there are no reports on Akkermansia muciniphila's role in the prevention and treatment of avian infectious bronchitis. Summary of the Invention

[0003] The purpose of this invention is to provide the use of Akkermansia and / or its cultures in the preparation of products for the prevention and / or treatment of avian infectious bronchitis virus infection.

[0004] The technical solution adopted in this invention is:

[0005] This invention provides the use of Akkermansia and / or its cultures in the preparation of products for the prevention and / or treatment of avian infectious bronchitis virus infection.

[0006] Preferably, the Akkermansia and / or its cultures improve the survival rate of chickens infected with avian infectious bronchitis virus.

[0007] Preferably, the Akkermansia and / or its cultures inhibit the replication of avian infectious bronchitis virus.

[0008] Preferably, the Akkermania species is of the Akkermania genus.

[0009] Preferably, the Akkermania is Akermania glutinis.

[0010] Preferably, the preservation number of the *Ackermania myxophilus* includes ATCC BAA-835; however, it is not limited to a specific strain. Those skilled in the art will understand that other strains identified as *Ackermania myxophilus* can also achieve the technical effects of the present invention and all fall within the protection scope of the claims of the present invention.

[0011] Preferably, the culture includes culture medium, culture medium extract, whole bacteria, whole bacteria extract, fermentation broth, and fermentation broth extract.

[0012] Preferably, the product includes a drug, an inhibitor, or a feed additive.

[0013] Preferably, the product comprises: (a) a safe and effective amount of Akkermania and / or its culture, and (b) a feed-acceptable excipient or a pharmaceutically acceptable excipient.

[0014] Preferably, the product is a probiotic product.

[0015] Preferably, the product also includes other probiotics and / or prebiotics.

[0016] Preferably, the excipients include at least one of the following: diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, cosolvent, solubilizer, preservative, pH adjuster, osmotic pressure adjuster, surfactant, coating material, antioxidant, antibacterial agent, or buffer.

[0017] Preferably, the product dosage form includes at least one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, oral preparations, suppositories, enemas, aerosols, patches, or drops.

[0018] Preferably, the route of administration of the product includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, gavage, oral administration, sublingual administration, nasal administration, nebulized administration, or transdermal administration.

[0019] The beneficial effects of this invention are:

[0020] This invention has shown that Akkermania can improve the survival rate of chickens infected with avian infectious bronchitis virus (AIV) and inhibit the replication of AIV. This indicates that Akkermania has important applications in the preparation of drugs for the prevention and / or treatment of AIV infection, and can provide a new solution for the treatment of AIV infection. It can be specifically applied in drugs or feed additives for the treatment of AIV infection. Attached Figure Description

[0021] Figure 1This is the analysis result of the gut microbiota of pullets before challenge with IBV (0 dpi) and on days 3, 5, and 7 after challenge (3, 5, and 7 dpi). In this table, A represents the top 10 most abundant bacteria in the gut microbiota of pullets at 0, 3, 5, and 7 dpi; B represents the fold change of these 10 bacteria at 3, 5, and 7 dpi relative to 0 dpi.

[0022] Figure 2 This is an experimental design and survival curve diagram for challenging chicks with IBV after transplanting Akkermansia into their intestines. A is the animal experiment flowchart for Akkermansia transplantation and IBV challenge, and B is the survival curve diagram for 15 consecutive days after challenge.

[0023] Figure 3 This graph shows the results of IBV virus copy number detection in chicken kidneys and trachea collected at 3, 5, and 7 dpi after challenge. The Akkermansia transplantation and challenge results shown in this graph are consistent with... Figure 2 The values ​​are the same. Where A represents the IBV viral copy number in the kidneys at 3, 5, and 7 dpi after challenge; and B represents the IBV viral copy number in the trachea at 3, 5, and 7 dpi after challenge. Detailed Implementation

[0024] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0025] The experimental procedure in this embodiment is summarized as follows:

[0026] 1. First, pullets were challenged with IBV, and fecal swabs were collected before challenge and on days 3, 5, and 7 post-challenge for 16S sequencing. Bioinformatics analysis identified bacteria with high abundance and significant changes in abundance as potential agents that could help the host resist IBV infection. Ultimately, Akkermansia muciniphila was identified as the bacterium that showed the highest upregulation on days 5 and 7 post-infection.

[0027] 2. After transplanting Akkermansia into the intestines of chicks, challenge them with IBV. Control groups such as blank group and positive group were set up. The chicks were observed for 15 consecutive days and the mortality rate was recorded.

[0028] 3. After transplanting Akkermansia into the intestines of chicks, they were challenged with IBV. Control groups such as blank and positive groups were set up. The chicks were euthanized on days 3, 5 and 7 after challenge, and their kidneys and tracheas were collected. The viral load in the tissues was detected by RT-qPCR.

[0029] The materials used in this experiment are as follows:

[0030] (1) Animals and Viruses

[0031] Both pullet chickens and SPF chickens were purchased from the Experimental Animal Center of Guangdong Wenshi Dahua Agricultural Biotechnology Co., Ltd.; the IBV strain was a routine strain isolated, purified and preserved in our laboratory.

[0032] (2) Main reagents

[0033] Akkermansia muciniphila was purchased from the American Type Culture Collection (ATCC BAA-835). In this example, the Akkermansia muciniphila culture medium formula was: BHI 37g; mucin 2g; L-cysteine ​​5mg; ddH2O to a final volume of 1L. The Akkermansia muciniphila culture conditions were: optimal growth pH 6.5, temperature 37℃, 80% N2, 10% CO2, 10% H2 for 2-3 days.

[0034] The EZ-press RNA Purification Kit was purchased from EZBioscience. The VC58P2 Multiplex One Step RT-qPCR Probe Kit (UDG Plus) was purchased from Yisheng Biotechnology; other reagents were prepared using conventional methods in the field.

[0035] Example 1: Detection of changes in gut microbiota in pullets before and after IBV challenge.

[0036] Growing chickens exhibit high resistance to IBV, and their gut microbiota may play an important antiviral role. Using IBV at a concentration of 10... 5 EID 50 Sixty-day-old Sanhuang chickens were challenged with a dose of 16S per bird via eye drops and nasal drops. Intestinal fecal swabs were collected before challenge (0 dpi) and on days 3, 5, and 7 after challenge (3 dpi, 5 dpi, and 7 dpi) for 16S sequencing.

[0037] The top 10 most abundant bacteria, such as Figure 1As shown in Figure A, the abundance of four bacterial species was significantly upregulated at different days after challenge. Among them, *Akkermansia muciniphila* showed significant upregulation on both days 5 and 7 post-challenge, and its upregulation fold was the highest among all bacterial groups. Figure 1 As shown in B, the levels were increased by 6.3-fold and 6.6-fold on days 5 and 7 after challenge, respectively, significantly higher than other strains, indicating that it may have the potential to help pullets resist IBV infection.

[0038] Example 2: Effect of Akkermansia transplantation on the survival rate of chicks challenged with IBV

[0039] One hundred and twenty one-day-old SPF chickens were randomly divided into four groups: a blank control group (Mock), a challenge control group (PC), an antibiotic treatment and challenge control group (Abx-PC), and an Akkermansia transplantation and challenge group (Akk). To verify whether Akkermansia muciniphila has the ability to help the host resist IBV infection, Akkermansia was transplanted into the chicks and challenged in the same manner as in Example 1. The experimental procedure is as follows: Figure 2 As shown in A, the specific conditions are as follows:

[0040] 1. Abx-PC group: One-day-old SPF chickens were fed drinking water containing a combination of antibiotics (ampicillin 1 mg / mL, metronidazole 1 mg / mL, neomycin 1 mg / mL, vancomycin 0.5 mg / mL). After 4 days of antibiotic treatment, normal drinking water was provided on day 5, and then on day 10, antibiotics were administered at a concentration of 10 mg / mL. 5 EID 50 IBV challenge was performed at a dose of / animal, using eye drops or nasal drops as the challenge method;

[0041] 2. Akk Group: One-day-old SPF chickens were fed drinking water containing a combination of antibiotics (ampicillin 1 mg / mL, metronidazole 1 mg / mL, neomycin 1 mg / mL, vancomycin 0.5 mg / mL). After four days of antibiotic treatment, normal drinking water was provided on day 5, followed by Akkermansia muciniphila strain transplantation. The transplantation was performed on days 6 and 9 at a dose of 10 mg / mL. 7 The bacteria were administered via gavage at a dose of CFU Akk / animal, and the strain was transplanted twice in total; on day 10, 10 5 EID 50 IBV challenge was performed at a dose of / ani, using eye drops or nasal drops; antibiotic treatment was to eliminate the influence of the original intestinal flora.

[0042] 3. The PC group was at 10 on the 10th day. 5 EID 50 The Mock group was challenged with a dose of / animal, and the Mock group was inoculated with an equal amount of PBS. The challenge method was eye drops or nasal drops.

[0043] Observe the chickens for 15 consecutive days after the viral challenge and record the mortality rate.

[0044] The results are as follows Figure 2 As shown in Figure B, at the experimental endpoint, the survival rate of the PC group was 45%, while the survival rate of the Akk group reached 90%, significantly higher than that of the PC group. This indicates that Akk transplantation significantly improved the survival rate of chicks and helped them resist IBV infection.

[0045] Example 3: Effect of Akkermansia transplantation on IBV viral load in the kidneys and trachea of ​​chicks

[0046] One hundred and twenty one-day-old SPF chickens were randomly divided into four groups, as described in Example 2: a blank control group (Mock), a challenge control group (PC), an antibiotic treatment and challenge control group (Abx-PC), and an Akkermansia transplantation and challenge group (Akk). Chickens were euthanized at 3, 5, and 7 dpi post-challenge, and their kidneys and tracheas were collected. Viral load was detected by RT-qPCR, as detailed below:

[0047] 1. 0.1g each of tracheal and kidney samples collected at 3, 5, and 7 dpi after challenge were used to extract RNA and reverse transcribe it. The viral load was then quantitatively quantified using real-time PCR. The EZ-press RNA Purification Kit was used to extract RNA according to the instructions, and then... (The sentence is incomplete and requires more context to translate accurately.) Perform RT-qPCR using the VC58P2 Multiplex OneStep RT-qPCR Probe Kit (UDG Plus) instruction manual. Calculate IBV viral copy number based on the standard curve and detect IBV-N mRNA expression level in tissues. The primer sequences used are as follows:

[0048] IBV-NF CAA GAG CTT GCT GCATAT CGT AAA (SEQ ID NO. 1);

[0049] IBV-NR GCG CTT CCT TAT ACATAG TGT CAT AGC (SEQ ID NO. 2).

[0050] The results are as follows Figure 3 A and Figure 3 As shown in Figure B. The results indicated that on day 5 post-infection, the kidneys of chickens in the Akk transplant group ( Figure 3 A) and in the trachea ( Figure 3The viral load in B) was significantly lower than that in the control group. In conclusion, transplantation of Akkermansia muciniphila into the intestines of chicks can significantly improve their resistance to IBV infection. Therefore, Akkermansia muciniphila has the potential to prepare anti-IBV virus infection microecological preparations.

[0051] The above detailed embodiments have provided a comprehensive description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. The use of Akkermansia and / or its cultures in the preparation of products for the prevention and / or treatment of avian infectious bronchitis virus infection, wherein the Akkermansia is Akkermansia myxophilus with accession number ATCC BAA-835. Akkermansia muciniphila The culture is a culture medium or whole bacteria.

2. The application according to claim 1, characterized in that, The Akkermansia and / or its cultures improve the survival rate of chickens infected with avian infectious bronchitis virus.

3. The application according to claim 1, characterized in that, The Akkermansia and / or its cultures inhibit the replication of avian infectious bronchitis virus.

4. The application according to claim 1, characterized in that, The products include pharmaceuticals or feed additives.

5. The application according to claim 1, characterized in that, The products include: (a) Akkermania and / or its cultures, and (b) feed-acceptable or pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The excipients include at least one of the following: diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, cosolvent, solubilizer, preservative, pH adjuster, osmotic pressure adjuster, surfactant, coating material, antioxidant, or buffer.

7. The application according to any one of claims 1 to 6, characterized in that, The product in question is a probiotic product.

8. The application according to claim 7, characterized in that, The product also includes other probiotics and / or prebiotics.

9. The application according to claim 1, characterized in that, The dosage form of the product includes at least one of the following: suspension, granules, capsules, powders, tablets, emulsions, pellets, injections, suppositories, enemas, aerosols, patches, or drops.

10. The application according to claim 1, characterized in that, The dosage form of the product includes solutions.

11. The application according to claim 1, characterized in that, The product can be in oral dosage form.

12. The application according to any one of claims 9 to 11, characterized in that, The product can be administered via at least one of the following routes: intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, gavage, oral administration, sublingual administration, nasal administration, nebulization, or transdermal administration.

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