CN112680388B

CN112680388B - Bacillus subtilis planted in nasal cavity of pig and application of bacillus subtilis in antiviral activity

Info

Publication number: CN112680388B
Application number: CN202110273828.4A
Authority: CN
Inventors: 杨倩; 李云锋; 李昱辰
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2021-03-15
Filing date: 2021-03-15
Publication date: 2021-06-29
Anticipated expiration: 2041-03-15
Also published as: CN112680388A

Abstract

The invention discloses a bacillus subtilis fixedly planted in a nasal cavity of a pig and application of the bacillus subtilis in antiviral activity. The swine bacillus subtilis NS15 has the preservation number of CGMCC No. 20344. The application of the bacillus subtilis NS15 in preparing a nasal administration preparation for treating and/or preventing porcine epidemic diarrhea virus. The application of the bacillus subtilis NS15 in preparing a probiotic preparation for enhancing the congenital immunity of the nasal mucosa of the pig. In vitro and in vivo experiments firstly prove that the bacillus subtilis NS15 has better adhesion and colonization capacity in nasal mucosa of pigs, can adhere to PEDV by secreting extracellular matrix components, prevents viruses from invading host cells, and can induce piglets to generate high-efficiency nasal mucosa innate immune response. In conclusion, the bacillus subtilis strain is expected to be developed into a probiotic preparation for resisting virus from invading and infecting organisms through nose.

Description

Bacillus subtilis planted in nasal cavity of pig and application of bacillus subtilis in antiviral activity

Technical Field

The invention relates to a bacillus subtilis fixedly planted in a nasal cavity of a pig and application of the bacillus subtilis in antiviral activity, and belongs to the technical field of biological prevention and control.

Background

1. Current research situation of infectious pathogen through nasal infection of pig

Cause pigsPathogenic microorganisms of infectious diseases invade through the mucous membranes of the respiratory tract and the digestive tract, wherein pathogens invading the body through the respiratory tract, especially through the nasal cavity, have attracted much attention in recent years in China, such as Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine pseudorabies virus (PRV), Swine Influenza Virus (SIV), mycoplasma hyopneumoniae (SIV)Mycoplasma pneumoniae of swine, MPS), Porcine Epidemic Diarrhea Virus (PEDV), and Haemophilus parasuis (Haemophilus parasuis)Haemophilus parasuisHp), etc. The pathogenic microorganisms can be spread by spray and air, explosive epidemic situation is easily formed in swinery, great challenge is brought to prevention and control, and development of pig industry in China is seriously hindered.

2. Research status of nasal mucosa immunity of pigs

The nasal mucosa is used as the part of the upper respiratory tract which is directly communicated with the outside, and is an important entrance for invasion and infection of a plurality of pathogens. Can directly cut off the invasion way of pathogenic microorganisms by inducing mucosal immunoreaction in the nasal cavity, and has important significance for effectively preventing respiratory infectious diseases. Researches show that the nasal cavity immunity can effectively induce local mucosal immunoreaction of respiratory tract, and has the advantages of small antigen usage amount, low immunoreaction threshold, small stress response to animals, no digestive enzyme degradation and the like, so that the nasal cavity immunity has wide attention in prevention and control of animal epidemic diseases in recent years, and the research reports of preventing porcine respiratory infectious diseases such as PRRSV, PRV, SIV and the like by applying the nasal cavity mucosal immunity are reported abroad at present.

The nasal mucosa of the pig forms stronger congenital immunity of the mucosa under the long-term evolution, and can resist the invasion of various pathogens. However, recent research shows that the congenital immunity of the nasal mucosa of the pig gradually degrades with the development of large-scale intensive breeding industry, and the capability of resisting pathogen invasion is obviously reduced. In contrast, the ecologically stocked herds still maintain a high nasal mucosal immunity level, which is highly resistant to a variety of pathogenic infections. The method is mainly characterized in that various complex microorganisms are distributed in field soil, and pigs raised in the field frequently contact with the microorganisms in the natural environment, so that more beneficial microorganisms enter and are planted in nasal vestibules, and the nasal mucosa is induced to generate high-level innate immunity protection.

3. Research status and application of bacillus subtilis as probiotics

Bacillus subtilis (A), (B) and (C)Bacillus subtilis) Is a dominant biological population widely existing in soil and plants, belonging to Bacillaceae and Bacillus. The bacillus subtilis has stronger stress resistance and viability, and can be widely used as a feed additive because the bacillus subtilis can obviously enhance the immunity of intestinal mucosa. However, due to the existence of physical barriers of nasal mucosa, such as mucus secretion, some bacillus subtilis is difficult to colonize on the mucosa after entering the nasal cavity, and the long-acting immune enhancement effect cannot be maintained.

4. Research status and application of pig nasal cavity epithelial cell in-vitro culture model

The respiratory tract epithelial cells are cultured in vitro under the traditional cell monolayer culture method, the difference between the in vivo state and the in vivo state is very large, and the real situation of the cells infected with viruses cannot be reflected, so that the accuracy of the experimental result is influenced. For example, respiratory epithelial cells NuLi-1 and CuFi have low susceptibility to new respiratory viruses, Human bocavirus type I virus (Human bocavirus 1, Hbov 1) in normal adherent culture, but if the two cells are subjected to gas-liquid interface culture, the susceptibility to Hbov1 is greatly improved. In the air-liquid interface (ALI) culture, respiratory epithelial cells obtained by digestion are laid on a Transwell bracket, and after the respiratory epithelial cells are converged, an upper chamber culture medium is discarded, so that the top ends of the respiratory epithelial cells are directly contacted with air. In the culture state, the differentiation and polarization states of the respiratory epithelium are very similar to those of the respiratory epithelium under the real condition in vivo, and the in vitro culture model of the respiratory epithelium established by applying the method at present is widely applied to the related researches of the pathogenic mechanism and the immune control of various respiratory pathogens.

A certain number of dendritic cells are distributed under the epithelium of the nasal mucosa of the pig, have a strong immune monitoring function and are an important bridge for connecting natural immunity and acquired immunity. When pathogenic microorganisms invade, dendritic cells under the nasal mucosa can quickly sense 'danger signals' and extend out of trans-epithelial dendrites to capture invaded pathogens, then the dendritic cells self-start a maturation process, antigen information is presented to lymphocytes, and mucosal immune response is started. In the process of screening the high-efficiency nasal mucosa immunopotentiator, the activation capability of the immunopotentiator on the submucosal dendritic cells is often used as an important evaluation standard. The nasal cavity epithelial cells for establishing the gas-liquid interface and the dendritic cells obtained by separating the nasal cavity mucosa are co-cultured, so that the in vivo environment can be fully simulated, the bacillus subtilis capable of activating the dendritic cells under the nasal cavity mucosa can be efficiently screened and obtained, and a theoretical basis is provided for further development of the bacillus subtilis into a nasal cavity mucosa immunopotentiator.

At present, no report about the separation of bacillus subtilis with nasal mucosa colonization ability from nasal cavities of ecological stocking swinery exists; nor has any report on the establishment of a bacillus subtilis-porcine nasal epithelial cell-dendritic cell co-culture model; the bacillus subtilis is not used for researching the antiviral function of the bacillus subtilis and enhancing the immunity of nasal mucosa.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the first technical problem of providing bacillus subtilis NS15 with nasal cavity colonization capacity. The Bacillus subtilis NS15 can adhere to PEDV by secreting extracellular matrix components and inhibit infection of piglets via nasal cavity. Besides the function of antivirus, the bacillus subtilis NS15 has stronger activation capability to dendritic cells under the nasal mucosa, and the bacillus subtilis NS15 can induce high-efficiency local immune response of the nasal mucosa.

The second technical problem to be solved by the invention is to provide a construction method of a co-culture model of bacillus subtilis-nasal epithelial cells-dendritic cells and an application of the co-culture model in screening and evaluating probiotic preparations capable of enhancing the congenital immunity of nasal mucosa.

The technical scheme is as follows: in order to solve the technical problem, the inventionThe technical scheme adopted by the invention is as follows: the invention relates to identification and application of bacillus subtilis NS15 separated from nasal cavity of field stocking pig, wherein the classification name of the bacillus subtilis NS15 is bacillus subtilis (Bacillus subtilis)Bacillus subtilis) And is preserved in China general microbiological culture Collection center (CGMCC) at 13.07.2020, with the preservation number of CGMCC No.20344 and the address: west road No. 1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101.

from said Bacillus subtilis (A), (B), (C), (Bacillus subtilis) NS 15.

The application of the bacillus subtilis NS15 in preparing a nasal administration preparation for treating and/or preventing porcine epidemic diarrhea virus.

As a preferred aspect of the present invention, the nasal administration preparation is one of spray or powder.

The application of the bacillus subtilis NS15 in preparing a nasal administration preparation for adhering porcine epidemic diarrhea virus and preventing the virus from invading epithelial cells of a nasal cavity.

Preferably, the nasal administration preparation is one of spray and powder.

The application of the bacillus subtilis NS15 in preparing a probiotic preparation for enhancing the congenital immunity of the nasal mucosa of the pig.

As a preferable aspect of the present invention, the probiotic preparation is a nasal administration preparation, preferably one of spray and powder.

The invention further comprises an in vitro bacillus subtilis-nasal epithelial cell-dendritic cell co-culture model, wherein the co-culture model is constructed by establishing a gas-liquid culture interface for the separated nasal epithelial cells, then co-culturing the nasal epithelial cells and dendritic cells, and finally adding the bacillus subtilis NS 15.

The invention also discloses a construction method of the in vitro bacillus subtilis-nasal epithelial cell-dendritic cell co-culture model, which comprises the following steps:

1) establishing a gas-liquid model of the epithelial cells of the nasal cavity of the pig;

2) carrying out isolated culture on dendritic cells of the nasal mucosa of the pig;

3) and (3) establishing a bacillus subtilis-nasal epithelial cell-dendritic cell co-culture model.

The invention also comprises the application of the bacillus subtilis NS15 or the in vitro bacillus subtilis-nasal cavity epithelial cell-dendritic cell co-culture model in preparing probiotic preparations for enhancing the congenital immune response of nasal cavity mucous membranes and preventing respiratory tract pathogen invasion.

Wherein the respiratory tract pathogen is porcine epidemic diarrhea virus.

Wherein, the probiotic preparation is one of spray or powder.

Has the advantages that:

in-vivo and in-vitro tests prove that the bacillus subtilis NS15 has better adhesion and colonization capacity in the nasal mucosa of the pig; secondly, the strain is found to be capable of adhering Porcine Epidemic Diarrhea Virus (PEDV) by secreting extracellular matrix components to prevent the virus from invading host cells, and surfactant components in the extracellular matrix can inhibit the membrane fusion of virus cyst membrane and nasal cavity epithelial cell membrane; further, a nasal spray test proves that the bacillus subtilis NS15 can induce the piglets to generate high-efficiency nasal mucosa innate immunity reaction, and finally, a co-culture model of bacillus subtilis-porcine nasal cavity epithelial cells-dendritic cells is constructed, so that the mechanism of the strain for enhancing the nasal mucosa innate immunity is found to be that the strain can promote the formation of trans-epithelial dendritic active antigen uptake by activating the dendritic cells under the nasal mucosa. In conclusion, the bacillus subtilis strain is expected to be developed into a probiotic preparation for resisting virus, particularly PEDV virus, from infecting organisms through nasal invasion.

Drawings

FIG. 1: colony morphology and gram stain results for bacillus subtilis NS 15;

FIG. 2: establishing and evaluating a pig nasal epithelial cell gas-liquid culture model; a: digesting to obtain the optical microscope observation of the nasal cavity epithelial cells; b: observing the epithelial cells of the nasal cavity by an optical microscope after establishing a gas-liquid culture interface; c: immunofluorescence detects the expression of keratin 18 (red) of nasal epithelial cells, and cell nuclei are stained by DAPI (blue); d: expression of zonulin ZO-1 (red); e: scanning electron microscopy observation of NECs on day 7 of gas-liquid culture, scale: 5 μm; f: confocal observations of the distribution of APN receptors (red) in the NECs culture model, nuclei stained with DAPI (blue), scale: 20 μm;

FIG. 3: evaluating the adhesion capacity of the bacillus subtilis NS15 to the porcine nasal epithelial cells; a: comparing the adhesion capacity of different bacillus strains to the nasal cavity epithelial cell gas-liquid culture model; 168: standard strains of Bacillus subtilis; NS15, NS23, NS 24: bacillus subtilis nasal isolate; BM: bacillus megaterium; BVS: bacillus belgii; b: observing the adhesion of the bacillus subtilis NS15 to the porcine nasal epithelial cells by a fluorescence microscope;

FIG. 4: the bacillus subtilis NS15 can be planted in the mucous membrane of the nasal cavity of the pig; a: collecting nasal swab 7 days after spraying to nose to detect Bacillus subtilis NS 15; b: collecting nasal swab 14 days after spraying the nose to detect bacillus subtilis NS 15;

FIG. 5: bacillus subtilis NS15 is capable of adhering to PEDV; a: comparing the adhesion ability of bacillus subtilis NS15 to different MOI viruses; PEDV: virus control; NC: bacillus subtilis NS15 thallus control; b: comparing the ability of different strains to adhere to PEDV; 168: standard strains of Bacillus subtilis; NS15, NS23, NS 24: bacillus subtilis nasal isolate; SQR 9: bacillus amyloliquefaciens; BM: bacillus megaterium; BVS: bacillus belgii; NC: bacillus subtilis NS15 thallus control; c: the ability of bacillus subtilis NS15 to adhere to PEDV under different treatment conditions; 1: normal control; 2: inactivating NS 15; 3: buffer PH = 9; 4: buffer PH = 9; 5: incubating at 4 ℃; d: adherence of bacillus subtilis NS15 to PEDV in nasal mucus at different dilution ratios;

FIG. 6: observation of adherence of bacillus subtilis NS15 to PEDV; a: confocal microscopy of adhesion of bacillus subtilis NS15 (red) to PEDV (green); b: transmission electron microscopy observation of Bacillus subtilis NS15 adherence to PEDV, black arrow indicated PEDV viral particle, scale: 5 μm;

FIG. 7: bacillus subtilis NS15 inhibits PEDV infection of nasal epithelial cells; a: incubating NS15 and PEDV for 1h, then directly inoculating the incubated NS15 and PEDV to a nasal cavity epithelial cell gas-liquid culture model for 24h, and detecting the virus replication level by Western blot; b: incubating NS15 and PEDV for 1h, centrifuging, taking the supernatant, inoculating the supernatant into a nasal cavity epithelial cell gas-liquid culture model for 24h, and detecting the virus replication level by Western-blot; c: quantitatively detecting the antiviral effect of the bacillus subtilis by fluorescence; d: a plaque inhibition test is used for detecting the antiviral effect of the bacillus subtilis NS 15;

FIG. 8: extracting, identifying and evaluating the toxicity of a surfactin (surfactin) component of bacillus subtilis NS 15; a: the bacillus subtilis surfactant component is precipitated by acid and dissolved in a methanol solvent; b: and (3) performing mass spectrum analysis on the purified product of the bacillus subtilis NS15 surfactant. The abscissa is the charge-to-mass ratio and the ordinate is the signal intensity. The main peak is marked with the molecular weight and the corresponding surfactin particle form;

FIG. 9: bacillus subtilis NS15 surfactin (surfactin) inhibits PEDV infection of host cells; a plaque inhibition test (a), a Western blot (b) and an RT-qPCR (c) are respectively used for detecting the inhibitory effect of Surfactin with concentration gradient on PEDV;

FIG. 10: detecting the integrity of the nasal epithelial cell barrier in the co-culture model;

FIG. 11: the effect of bacillus subtilis NS15 on the molecular phenotype of dendritic cells under the nasal epithelium;

FIG. 12: the effect of bacillus subtilis NS15 on the expression of dendritic cell cytokines under the nasal epithelium;

FIG. 13: the effect of bacillus subtilis NS15 on the stimulation of lymphocyte proliferation by dendritic cells under the nasal epithelia;

FIG. 14: the effect of bacillus subtilis NS15 on the protrusion of dendrites from dendritic cells under the nasal epithelia; mock: negative control, culturing in normal culture medium without any treatment; NS15 group: adding 1X 10 per hole⁴CFU Bacillus subtilis NS15(ii) a NS15 (IA) group: adding 1X 10 per hole⁴CFU inactivated Bacillus subtilis NS 15; 168 groups are as follows: adding 1X 10 per hole⁴CFU Bacillus subtilis standard strain 168; group 168 (IA): adding 1X 10 per hole⁴CFU inactivated bacillus subtilis standard strain 168;

FIG. 15: morphological observation of nasal mucosa after spraying of bacillus subtilis NS 15; in the drawings, I, II and III represent the front, middle and rear parts of the nasal cavity of a pig, respectively;

FIG. 16: bacillus subtilis NS15 can enhance innate immunity protection of nasal mucosa of pig; a: the influence of the bacillus subtilis NS15 on the secretion of cell factors of nasal mucosa of pigs; b: bacillus subtilis NS15 for treating swine nasal mucosa CD3⁺Effects of T cells and IgA secreting cells; c: the effect of bacillus subtilis NS15 on dendritic cells in the nasal mucosa of pigs.

Biological material preservation information

NS15, classified and named as Bacillus subtilis (B.) (Bacillus subtilis) The microbial inoculum is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.20344 and the address: the date of preservation is 2020, 7 months and 13 days in Beijing, Xilu No. 1, Beijing, Chaoyang, Beijing, and China academy of sciences.

Detailed Description

The invention is further illustrated by the following specific examples and figures. The methods used in the following examples are conventional reagents and conventional methods unless otherwise specified.

Example 1 isolation and identification of Bacillus subtilis NS15 of porcine origin

1.1 isolation of the origin of the sample from Bacillus subtilis

The nasal swab sample is from a certain ecological pig farm in northwest China, and the collection method is as follows: after the pigs were housed, the nasal cavities were harvested by rotating the sterilized cotton swabs at least 3 times in the nasal cavities of the pigs. Dipping secretion, immediately putting the swab into a sterilized 1.5mL centrifuge tube, cutting off the part exposed out of the tube orifice, tightly covering the centrifuge tube cover, marking, sealing and storing at low temperature.

With reference to GB/T1.1-2009, Bacillus subtilis in a pig nasal cavity swab is separated, and the method specifically comprises the following steps: 1) aseptically opening a centrifuge tube filled with a nasal swab in a super clean bench, adding 500 mu L of PBS solution into each tube, repeatedly oscillating the centrifuge tube for three times, oscillating for 15s each time, and standing in a refrigerator at 4 ℃ for 15 min; 2) opening the centrifugal tube, squeezing the residual leachate in the swab into the centrifugal tube by using a pair of tweezers, and heating in a water bath at 80 ℃ for 15 min; 3) sucking 100 μ L of the leaching solution, transferring to LB liquid, culturing at 37 deg.C and 200 rpm overnight; 4) after the bacterial liquid is subjected to gradient dilution, the bacterial liquid is sucked into an LB solid plate culture medium, after the bacterial liquid is uniformly coated, the bacterial liquid is inverted and cultured in an incubator at 37 ℃ for 12 hours until a single colony grows out.

The bacillus subtilis NS15 forms a large bacterial colony which is round or oval, milky white and wrinkled on the surface of an LB plate, a suspected bacterial colony is selected for gram staining, and a blue-purple long-rod bacterium can be seen under a light mirror (figure 1).

Biochemical identification of Strain NS15

The biochemical reaction results of the strain NS15 are shown in Table 1, and according to Bergey's Manual of bacteria identification and Manual of common bacteria System identification, the strain NS15 is preliminarily identified as Bacillus subtilis.

TABLE 1 Biochemical reaction results of Strain B.S N15

Biochemical reaction	Results
		Oxidase enzyme	+
VP assay	+
		Xylose	+
L-arabinose	+
		Mannitol	+
Production of gas by glucose	-
		Starch hydrolysis	+
Liquefaction of gelatin	-

"+" indicates a positive result and "-" indicates a negative result.

1.3 Bacillus subtilis specific PCR identification

According to the gram staining and biochemical identification results, suspected single colonies of the bacillus subtilis are picked, a bacterial genome DNA extraction kit (Tiangen, China) is used for extracting bacterial genome, 16S rDNA is amplified by PCR, and after a sequence obtained by sequencing of a PCR product is compared by NCBI Blast, the sequence is found to be similar to a plurality of classical strains of the bacillus subtilis such as: the homology of Bacillus subtilis strain HTI 2316S ribosomal RNA gene, partial sequence (GenBank: MK521066.1), Bacillus subtilis strain KBL 316S ribosomal RNA gene and partial sequence (MG 576156.1) is up to 99%. The strain NS15 is determined by combining the physiological and biochemical characteristics of the strain NS15 and the analysis result of the 16S rDNA sequence, is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 20344.

Example 2 establishment of a pig Nasal cavity epithelial cell culture in gas and liquid (NECAM) model

2.1 isolation of nasal mucosal epithelial cells

Negative piglets of 3 weeks of age were selected, anesthetized with thiopentan sodium at 12.5mg/kg body weight, bled and sacrificed. The head was removed from the carcass and the nose (the anterior portion of the eye) was sawed from the skull. Nasal concha and nasal septum on the ventral surface of the nasal explant are carefully dissected away using a surgical blade and 16mm is cut²Size of respiratory mucosa.

(1) Nasal Epithelial Cells (NECs) were obtained by digestion:

(2) termination of digestion: adding 1.5mL of FBS into each tube, namely adding 10% FBS to terminate digestion; transferring the digestive juice and the short section of nasal mucosa epithelium into another new centrifugal tube, and reversing for 10 times (taking care that the liquid can not be bubbled by violent shaking); the liquid in the tube and the short segment of the trachea are poured into a flat dish, the short segment of the trachea is picked up by hemostatic forceps or tweezers, the digestive juice is sucked into a 10mL centrifuge tube by a tip head with a cut sharp corner, the digestive juice passes through a 70-mesh disposable sieve, the digestive juice is centrifuged at 180g (880 r/min) at 4 ℃ for 8 minutes to collect cells, and the nasal epithelial cells with cilium structures are remained at the visible part under a light microscope (figure 2 a).

(3) Differential adherent fibroblast removal: centrifugation was followed by discarding the supernatant, resuspending the cells with DMEM (5% FBS), pooling in a 25mL flask, adding DMEM (5% FBS + double antibody) to 5mL, mixing, placing in a 37 ℃ incubator while discarding the total collagen solution in the chamber, air drying the collagen in a clean bench and washing twice with HBSS before plating the cells.

Plating nasal mucosa epithelial cells and establishing a gas-liquid culture interface

Epithelial cells were collected and plated: after 3 h of culture, the cells were removed, the cell suspension was transferred to a 10mL centrifuge tube (adherent cells were heterocytes), and 120g (880 r/min) was centrifuged for 5-6 minutes. Cells were resuspended using human bronchial epithelial cell culture medium BEGM (containing 1% double antibody). The cells were counted using a cell counting plate at 1X 10 per well⁶The upper chamber of a Transwell chamber of 3 μm in number of cells was inoculated, the lower chamber was inoculated with 500uL of a medium, the medium was cultured at 37 ℃ for 24 hours, and the resistance value was measured using a resistance meter> 200 Ω·cm^-2Discarding the upper chamber liquid, and continuing culturingAfter 3 days of incubation, the chamber was dry air interface with no liquid leakage by visual inspection (FIG. 2 b). To demonstrate that the isolated primary cells were indeed NECs, indirect immunofluorescent staining was performed using CK18, an important marker for epithelial cells. From fig. 2c we can see that most cells are marked red by CK18 antibody.

To further understand the polarization state of the culture model of NECs, the morphological distribution of ZO-1, a cellular Claudin, in NECAM, was observed by confocal microscopy at day 7 of culture, showing complete and continuous expression of ZO-1 at the cell junction (FIG. 2 d). Scanning electron microscopy results also demonstrated the successful establishment of NECAM, and we can see from fig. 2e that 7 days of ALI cultured NECs appeared as ciliated epithelial cells with a multilayer structure including both ciliated epithelial cells and piliferous columnar epithelial cells and goblet cells arranged in alternating rows. We also examined the distribution of the PEDV functional receptor aminopeptidase N (APN) in NECAM using confocal microscopy, which showed that the APN receptor was abundantly distributed at the top of NECAM (fig. 2 f). The above results indicate that the NECs cultured in gas-liquid culture at day 7, which were well connected and in a better polarization state, could satisfy the requirements of the subsequent experiments.

Example 3 adhesion and colonization of porcine nasal mucosal epithelium by Bacillus subtilis NS15

3.1 detection of the adhesive Capacity of Bacillus subtilis to nasal epithelial cells

Fresh bacillus subtilis NS15 suspensions were prepared and counted, and other nasal isolates and bacillus subtilis standard strain 168 were used as controls. Bacterial adhesion tests were performed using NECs cultured to day 7 after establishing a gas-liquid interface, and after the upper chamber of the chamber was washed once with 500. mu.L of PBS solution, 500. mu.L of Bacillus subtilis suspension (1X 10) was added per well⁶CFU/well). After the chamber was cultured in an incubator at 37 ℃ for 6 hours, the upper chamber culture medium was discarded, and the upper chamber of the chamber was washed with a blank DMEM medium for 3 times. After adding 0.25% pancreatin for digestion and resuspension, the mixture is diluted by multiple times (10)^-4) Plates were painted and counted. The results showed that NS15 has the strongest adhesion to nasal epithelial cells (fig. 3 a). Further, in the present invention,nasal epithelial cells were plated onto cell-slide overnight and cell adhesion assays were performed using CFSE-labeled bacillus subtilis NS15, which was shown in fig. 3b to demonstrate adhesion of bacillus subtilis NS15 strain to the nasal epithelial cell surface.

Planting characteristics of bacillus subtilis NS15 in nasal mucosa

The experimental animals were 6 new born 15-day-old triple hybrid piglets, Duroc, Changbai and Dabai, and purchased from Bailv animal husbandry, Inc. in Lianchong, Jiangsu province. Sows are singly housed before piglets live, probiotics such as bacillus subtilis and the like are not contained in the feed, and bacillus subtilis detection in the anus swab and the nasal cavity swab of the sows is negative. After the piglets suckle for 15 days old and the nasal swab is proved to be negative by the bacillus subtilis, the bacillus subtilis NS15 (2 multiplied by 10) is sprayed to the nasal cavity⁸CFU/mL) 500 muL/head, collecting nasal cavity swabs after spraying the nose for 7 days (figure 4 a) and 14 days (figure 4 b), detecting the colonization condition of bacillus subtilis in the nasal cavity swabs, and the result shows that the bacillus subtilis is detected in the nasal cavity swabs until 14 days, and the bacillus subtilis is detected to be negative in the nasal cavity swabs in the PBS group.

Example 4 Bacillus subtilis NS15 prevention of viral invasion of nasal epithelial cells by adhesion to PEDV

4.1 evaluation of the Effect of Bacillus subtilis NS15 adherence to PEDV

To examine the adherence of Bacillus subtilis to PEDV, 10 was first prepared⁷cfu/mL of Bacillus subtilis NS15 suspension, PEDV virus was added at MOI of 0.01, 0.1 and 0.2, and incubated at 37 ℃ for 2 h; then, centrifugation is carried out at 7000 rpm for 10 min, the precipitated thalli are resuspended by using 0.01M PBS and centrifuged again, then the operation is repeated, and the thalli are washed for 3 times; and finally, adding 100 mu of LRIPA protein lysate, collecting total protein, and detecting the content of PEDV in the thallus by using the PEDV-N protein as the target protein. The positive control and the negative control are respectively PEDV and Bacillus subtilis alone, and meanwhile, the Bacillus subtilis standard strain 168 is used as a thallus control, and Western blot results (figure 5 a) show that the Bacillus subtilis NS15 has better adhesion effect on the PEDV, and the amount of adhered viruses is increased along with the increase of the virus content.

Further, according to 10⁷cfu/mL suspensions of different Bacillus strains (including Bacillus subtilis isolates NS15, NS23, NS24 and Standard strain 168; Bacillus amyloliquefaciens SQR 9; Bacillus beleister and Bacillus megaterium) were prepared and tested for their ability to adhere to PEDV by adding PEDV virus at MOI = 0.2, showing that NS15 adheres significantly more to PEDV than to other Bacillus strains (FIG. 5 b).

Furthermore, we examined the ability of NS15 to adhere to PEDV under different treatment conditions, and showed that NS15 adhered to PEDV depending on the PH of its buffer, and that the ability to adhere to PEDV after inactivation was significantly reduced, but was not significantly affected by NS15 at 4 ℃ (fig. 5 c).

Finally, we also explored the effect of porcine nasal mucus on the ability of bacillus subtilis NS15 to adhere to virus, and the results showed that bacillus subtilis NS15 still retained strong virus adhesion in dilutions of nasal mucus and had a significant dose-dependent profile (fig. 5 d).

Immunofluorescence and electron microscope observation of adhesion of bacillus subtilis NS15 to PEDV

Adherence of Bacillus subtilis NS15 to PEDV was observed using confocal microscopy by first preparing a CFSE label concentration of 10⁷cfu/mL suspension of Bacillus subtilis NS15, and DyLight 633 fluorescent dye (Thermo Fisher Scientific, USA) labeled PEDV suspension after mixing DyLight 633 labeled PEDV with fluorescently labeled Bacillus subtilis NS15 at MOI 0.2, incubating at 37 ℃ for 2h, centrifuging at 3000 rpm for 15min, resuspending the precipitated mycelia with 0.01M PBS and centrifuging again, and repeating this operation 3 times, then pipetting the pellet onto a glass slide, observing adhesion of Bacillus subtilis to PEDV virus by confocal microscopy, as shown in FIG. 6a, PEDV particles (green fluorescent label) adhered around Bacillus subtilis NS15 (red fluorescent label).

Adhesion of Bacillus subtilis NS15 to PEDV was observed using transmission electron microscopy, first preparing 10⁷cfu/mL of Bacillus subtilis NS15 suspension, PED was added at MOI 0.2V virus, after incubating for 2h at 37 ℃, centrifuging for 15min at 3000 rpm, resuspending the precipitated thallus with sterile deionized water, repeating the operation, cleaning the thallus for 2 times, then dropping the bacterial liquid on a copper net, counterstaining with phosphotungstic acid, and observing by a transmission electron microscope, wherein coronavirus-like particles with the diameter of 100 plus 120nm are adhered to the extracellular matrix components around the Bacillus subtilis NS15 (FIG. 6 b).

Bacillus subtilis NS15 for inhibiting PEDV from invading epithelial cells of nasal cavity

First, 10 is prepared⁶cfu/mL of bacillus subtilis NS15 suspension, adding PEDV virus according to MOI 0.01 by taking the inactivated thallus of the bacillus subtilis NS15, the Bacillus belgii (incapable of adhering to PEDV) and the inactivated thallus of the bacillus belgii with the same bacterial quantity as a reference, incubating for 1h at 37 ℃, respectively inoculating the thallus and the supernatant to a pig nasal cavity epithelial cell gas-liquid culture model cultured for 7 days, incubating for 1h at 37 ℃, discarding the supernatant, adding a cell maintenance solution containing 1% of double antibody, culturing for 24h at 37 ℃, collecting a cell sample, detecting the protein content of the PEDV by a Western-blot method, and taking pig GAPDH as an internal reference; western-blot results show that the bacillus subtilis NS15 can obviously inhibit the bacillus subtilis from infecting porcine nasal epithelial cells after being adhered to PEDV (figure 7 a), and the content of residual viruses in the supernatant is also obviously reduced due to the adhesion effect of the bacillus subtilis NS15 (figure 7 b); further preparation 10⁵，10⁶And 10⁷Adding PEDV virus into cfu/mL bacillus subtilis NS15 suspension according to MOI 0.1 and 0.5 respectively, incubating for 1h at 37 ℃, centrifuging to remove non-adhered virus, inoculating thalli to PEDV susceptible cells Vero E6, incubating for 1h, and performing the following 2 treatments: 1) adding cell maintenance solution containing 1% double antibody, culturing at 37 deg.C for 24 hr, and collecting cell sample for detecting nucleic acid level of virus. After RNA extraction and reverse transcription of a cell sample, carrying out fluorescence quantitative detection on the N protein of PEDV by taking pig GAPDH as an internal reference; 2) 1% Low melting agar DMEM was added, and after the agar solidified at room temperature, the culture was carried out for 48 hours. 1mL of 4% paraformaldehyde was added to each well and fixed for 1 hour, and after agar was washed off with running water, crystal violet was added and stained for 1 hour. Washing away excessive crystal violet, air drying and taking a picture. Counting the number of plaques per well. The results of the fluorescence quantification (fig. 7 c) and plaque inhibition assays (fig. 7 d) further demonstrate that bacillus subtilis NS15 is able to inhibit PEDV invasion into host cells and has a significant dose-dependent effect, i.e. the higher the concentration of bacillus subtilis, the lower the level of viral infection.

Example 5 secretion of surfactant by Bacillus subtilis NS15 to inhibit invasion of nasal epithelial cells by PEDV

5.1 purification and characterization of Bacillus subtilis NS15 surfactant

Bacillus subtilis NS15 was inoculated into LB medium, cultured at 37 ℃ and 200 rpm for 24 hours. The culture medium is inoculated into 120 mL LB culture solution according to the volume ratio of 1%, and Landy culture solution is inoculated into fresh bacterial liquid according to the volume ratio of 3%. Incubated at 30 ℃ for 36 hours at 200 rpm. Centrifugation (11 ℃, 8000 g, 10 minutes) is carried out, and the supernatant is taken to avoid the contamination of the thalli as much as possible. Hydrochloric acid was added to adjust the pH to 2.0 and the reaction was allowed to proceed overnight at 4 ℃. Centrifugation (11 ℃, 8000 g, 10 min) was carried out and the supernatant discarded. A small amount of concentrated sodium hydroxide solution was added dropwise to the precipitate to dissolve the precipitate and adjust the pH to 7.0, to obtain a crude extract of surfactant (FIG. 8 a). And (5) drying by rotary evaporation, weighing, and subpackaging for later use.

The methanol solution of the Bacillus subtilis NS15 surfactant sample was analyzed by LC-MS. mu.L of the sample was injected into an ultra high performance liquid chromatography column at a flow rate of 0.4 mL/min. Buffer a (0.1% formic acid in water) and Buffer B (0.1% formic acid in acetonitrile) were chromatographed at 5% Buffer B for 2 minutes, 5% to 95% Buffer B for 15 minutes, 95% Buffer B for 2 minutes. The mass spectrum part adopts a positive charge MSe acquisition mode, and the charge-to-mass ratio range is 50-1200. The ionization parameters were as follows: the capillary voltage was 2.5 kV, the collision energy was 40 eV, the source temperature was 120 ℃ and the desolvation gas temperature was 400 ℃. Data acquisition and processing was performed using Masslynx 4.1.

The surfactant standards used in the control group had a purity of 98% and almost all of the mass spectrum peaks corresponded to various homologs of surfactant. The molecular weight of the expressed protein homologues in the purified product was identical to that of the standard, but the ratio of homologues was different. The surfactant concentration in the purified product was obtained by comparing the integrated peak area in the sample with the standard. The dry weight of the surfactant sample is measured, and the calculated purity reaches over 75 percent.

5.2 evaluation of cytotoxicity of Bacillus subtilis NS15 secretion of surfactant

The cytotoxicity test was carried out by the CCK8 method. NECs cultured to day 7 after establishing gas-liquid interface were seeded in 96-well plates. Samples of different concentrations were added and after 12 hours of incubation, 0.8 mg/mL CCK8 reagent was added and incubation continued for 4 hours. OD560 was read with a microplate reader. Results are expressed as a percentage of the test and blank data. More than 20 μ g/mL of surfactant will decrease the cell viability of the NECs (FIG. 8 b). Lysophosphatidylcholine (LPC) was used as a positive control, showing similar cytotoxicity. Dimyristoyl glycerophosphocholine (DEPE) as a negative control showed no cytotoxicity in the soluble concentration range.

5.3 secretion of surfactant by Bacillus subtilis NS15 to inhibit invasion of nasal epithelial cells by PEDV

The PEDV suspension was diluted to contain about 100 PFU per 10. mu.L. mu.L of PEDV dilution was mixed with equal volume series of concentrations of surfactant (0.5, 1, 5 and 10. mu.g/mL) or their reagents and incubated at 37 ℃ for 1 h. Diluting the incubation product by 20 times, adding the diluted incubation product into the upper chamber of a nasal cavity epithelial cell gas-liquid culture model, culturing for 1 hour at 37 ℃, removing the supernatant, and performing the following 2 treatments: (1) adding a maintenance liquid to the mixture and culturing the mixture at 37 ℃ for 24 hours, and collecting cell samples for detecting the nucleic acid, protein and plaque levels of the virus. After RNA extraction and reverse transcription are carried out on a cell sample, carrying out fluorescence quantitative detection on the N protein of PEDV, taking pig GAPDH as an internal reference, detecting the N protein of PEDV in the cell sample by a Western-blot method, and taking pig GAPDH as the internal reference; (2) adding 1% low melting point agar DMEM (1L packaged DMEM dry powder is added with sodium bicarbonate according to the specification and then dissolved in 400 mL of triple distilled water, dropwise adding hydrochloric acid to adjust the pH to 7.4, fixing the volume to 500 mL, filtering and sterilizing to a sterilization bottle, weighing 2 g of low melting point agar for 2 × DMEM, adding 100 mL of triple distilled water, keeping for use after high pressure, heating and melting the 2% low melting point agar before use, mixing with the same amount of 2 × DMEM uniformly, balancing at 37 ℃ for 1 hour, and culturing for 48 hours after the agar is solidified at room temperature. 1mL of 4% paraformaldehyde was added to each well and fixed for 1 hour, and after agar was washed off with running water, crystal violet was added and stained for 1 hour. Washing away excessive crystal violet, air drying and taking a picture. The number of plaques per well was counted. The results showed that the concentration of surfactant 10. mu.g/mL almost completely inhibited the invasion of the virus to be detected, and the antiviral effect gradually decreased with decreasing surfactant concentration (FIGS. 9a-9 c). Therefore, the surfactant can exert a potent antiviral effect in a concentration range of 2 to 10. mu.g/mL without causing damage to cells.

Example 6 establishment of Bacillus subtilis-nasal epithelia-dendritic cell Co-culture model

6.1 isolated culture of porcine bone marrow-derived dendritic cells

The pig dendritic cells are obtained by in vitro induction culture of mononuclear cells derived from pig bone marrow.

6.2 Co-culture of Bacillus subtilis-nasal epithelia-dendritic cells

Inoculating the dendritic cells from the pig bone marrow to the back side of a pig nasal cavity epithelial cell culture model forming a good gas-liquid interface, carrying out co-culture, and verifying the barrier integrity of the co-culture model through tight protein staining and resistance measurement. The transmembrane resistance is stabilized at 300 omega cm²In the above, the Transwell chamber was first placed upside down in a 12-well plate, and then the immature dendritic cells (5X 10)⁵And) inoculating to the back side of the membrane of the chamber, attaching dendritic cells to the back side of nasal epithelial cells by siphoning with a plate cover covered with a 12-well plate, culturing at 37 ℃ for 4h, washing off non-adhered dendritic cells, and placing the chamber in a 24-well cell plate. 1X 10 additions per well⁶CFU Bacillus subtilis NS15, and a co-culture model of in vitro Bacillus subtilis-nasal epithelial cell-dendritic cell is constructed.

6.4 evaluation of Co-culture model of Bacillus subtilis-nasal epithelia-dendritic cells

The effect of co-culture on the integrity of the NECs barrier was evaluated by measuring the transcellular resistance. Resistance measurements showed that dendritic cells did not affect the transmembrane resistance of nasal epithelia, and in addition, inoculation of bacillus subtilis NS15 on the free side of the coculture model epithelial cells did not affect the integrity of the NECs barrier. However, when treated with EDTA, the NECs tight junction continuity was significantly disrupted and the resistance was also significantly reduced (fig. 10).

Example 7 detection of the activation of dendritic cells in a Co-culture model by Bacillus subtilis NS15

7.1 Effect of Bacillus subtilis NS15 on dendritic cell phenotype

The surface markers CD1a, MHCII and SWC3a can reflect the maturation and presentation conditions of dendritic cells, in order to detect the influence of the Bacillus subtilis NS15 on the maturation and presentation capabilities of the dendritic cells in the co-culture model, the Bacillus subtilis NS15 with different concentrations is inoculated to the upper chamber of the nasal epithelial cells, after 22 hours of culture, the dendritic cells in the co-culture model are collected, and the change conditions of the surface markers of the dendritic cells after being treated by the Bacillus subtilis NS15 are detected in a flow mode. Statistical results show that after the bacillus subtilis NS15 treatment, the expression levels of SWC3a, CD1a and MHCII of dendritic cell surface markers are all increased remarkably (FIG. 11). The above results demonstrate that Bacillus subtilis NS15 stimulates phenotypic maturation of dendritic cells in a co-culture model.

7.2 Effect of Bacillus subtilis NS15 on dendritic cytokine secretion and transepithelial dendritic formation

The release of dendritic cell cytokines is an important manifestation of the maturation of dendritic cell functions. In order to further detect the influence of NS15 on the secretion of dendritic cell cytokines in the co-culture system, different concentrations of Bacillus subtilis NS15 were inoculated into the upper chamber of nasal epithelial cells, and after 22 h of culture, dendritic cells and lower chamber culture medium in the co-culture model were collected. On one hand, an ELISA kit (Shanghai Biotechnology Co., Ltd.) is used for detecting the secretion of IL-6, IFN-gamma and TNF-alpha in the supernatant, and the test method is mainly carried out according to the operation instruction of the ELISA kit; on the other hand, total RNA of dendritic cells is extracted, and the transcription levels of IL-6, IFN-gamma and TNF-alpha mRNA are detected by fluorescent quantitative PCR. As shown in figure 12, Bacillus subtilis NS15 can remarkably promote the secretion of inflammatory cytokines such as IL-6, IFN-gamma and TNF-alpha in dendritic cells. After being activated, the dendritic cells can elongate the dendrites and cross the nasal epithelium, actively take in the intracavity antigens, and through observation of a confocal microscope, the bacillus subtilis NS15 can effectively recruit DCs on the basal side of the nasal epithelium after being treated and promote the DCs to form transepithelial dendrites (figure 13).

7.3 Effect of Bacillus subtilis NS15 on the proliferative Capacity of dendritic cells to activate T lymphocytes

The most important task of dendritic cells is to present antigen to downstream T lymphocytes, and therefore a mixed lymphocyte reaction assay is used to evaluate further functional maturation of dendritic cells. Firstly, separating T lymphocytes of porcine mesenteric lymph nodes, and comprises the following steps: one porcine mesenteric lymph node is taken aseptically, soaked in 75% alcohol for 5 minutes and then soaked in 2% double-resistant PBS for 5 minutes. The lymph nodes are cut off by sterile scissors, the surrounding connective tissues are removed completely, and the lymph nodes are cut into small pieces. Grinding lymph nodes with a 200-mesh cell sieve, collecting cell suspension filtered by the mesh sieve, and sorting by magnetic beads to obtain CD3⁺T lymphocytes; the T lymphocytes obtained from the above isolation were then labeled with CFSE live cell dye according to the kit instructions provided by Invitrogen, and after labeling, mixed with collected sets of dendritic cells (as in 7.2, in a CO-culture system, basal-lateral dendritic cells were collected) and plated into 24-well cell plates for reaction at cell ratios of 1:2 and 1:5, 37 ℃, 5% CO₂After 3 days in the incubator, cells were harvested, washed 2 times with PBS, and the proliferation of CFSE-labeled T cells was detected by flow. As shown in FIG. 14, when the ratio of dendritic cells to lymphocytes was 1:2 and 1:5, the proliferation ability of T lymphocytes activated by the dendritic cells of the group treated with Bacillus subtilis NS15 was significantly enhanced, and the effect of cell concentration dependence was significant.

Example 8 Effect of Bacillus subtilis NS15 nasal spray on local mucosal immune level in nasal cavity of piglet

In order to further explore the influence of the bacillus subtilis NS15 on the local congenital immunity of the nasal mucosa of the pig after the planting, a piglet nose spraying test is carried out. Selecting 14-day-old newborn three-element hybrid piglets Duroc, Changbai and Dabai, and limited greenish grazing industry purchased in Lianhong Kong of Jiangsu provinceA company. The experimental groups are divided into two groups, namely 4 experimental groups and 4 control groups. The experimental group sprayed with Bacillus subtilis NS15 (2X 10) at 14-day and 21-day ages of piglets⁹CFU/mL) 500 μ L/head, control PBS nasal spray. Injecting sodium pentobarbital into piglets aged for 28 days for euthanasia treatment, opening nasal cavity, fixing left nasal cavity in Boehn's solution, and preparing tissue slice; and (3) taking the right nasal cavity for preparing a nasal rinsing lotion, collecting nasal cavity mucous membrane tissues, and freezing the nasal cavity mucous membrane tissues to-80 ℃ for later use.

8.1 spraying nose with Bacillus subtilis NS15 does not affect the integrity of mucosal tissue structure of nasal cavity of pig

Fresh nasal tissue was immersed in boehringer's solution and fixed for 48 h. Samples of tissue were taken at the intersection of the median sagittal and transverse planes at nasal cavities 1/20, 2/5 and 4/5 (anterior: I; middle: II; posterior: III), respectively. Soaking and dehydrating the sample in gradient alcohol (75%, 85%, 95%, 100%, 100% ethanol) for 1h from low to high, and embedding the sample in paraffin for 2h, wherein the xylene is transparent. The wax block is continuously sliced with the thickness of 6 mu m and HE dyed after being prepared. As can be seen in figure 15, after the bacillus subtilis is sprayed to the nose, the tissue morphological structure of the nasal mucosa is not changed, and the epithelial integrity is kept unchanged, which indicates that the bacillus subtilis is sprayed to the nose to cause no pathological change of the tissue.

8.2 Bacillus subtilis NS15 promotes the secretion of innate immune cytokines in nasal mucosa of pigs

And detecting the contents of the innate immune cytokines IL-6, IFN-gamma, TNF-alpha and IL-11 in the nasal rinse solution and the nasal mucosa epithelium of the pigs in each treatment group. The nasal mucosa tissue sample is prepared as follows: homogenizing mucosa tissue 50 mg in 1mL PBS, repeatedly freezing and thawing the homogenate for three times (-20 ℃ C/37 ℃ C.), centrifuging the homogenate for 5min at 5000 g and 4 ℃ C, and collecting a supernatant sample. The ELISA kit is used for detecting the contents of IL-6, IFN-gamma, TNF-alpha and IL-11 proteins in the rinsing liquid of the nasal cavity and the mucous membrane tissues of the nasal cavity (Hangzhou Union Biotechnology Co., Ltd.), and the specific operation steps are carried out according to the instruction. The results show that the bacillus subtilis NS15 can remarkably promote the expression and secretion of innate immune cytokines such as IL-6, IFN-gamma, TNF-alpha, IL-11 and the like in nasal mucosa (figure 16 a).

8.3 Bacillus subtilis NS15 increases the number of innate immune cells in the nasal mucosa of pigs

Through immunohistochemistry and immunofluorescence detection respectively, the bacillus subtilis NS15 sprays the influence of nose on congenital immune cells such as T lymphocytes, IgA secretory cells and dendritic cells in nasal mucosa of pigs. T lymphocytes and IgA secretory cells in nasal mucosa of pigs are respectively displayed by using anti-pig CD3 and IgA antibodies, and 10 visual fields are randomly selected from 5 areas of the nasal mucosa of each pig for statistical analysis of data. As shown in the figure, after bacillus subtilis NS15 is sprayed into the nose, the number of T lymphocytes and IgA secretory cells in the inherent layer of the nasal mucosa of the pig are both increased remarkably (FIG. 16 b). Dendritic cells in nasal mucosa of pigs are displayed by using anti-pig CD11b (red fluorescence) and MHCII (green fluorescence) antibodies, 5 fields are randomly selected from 5 areas of the nasal mucosa of each pig after indirect immunofluorescence staining for statistical analysis of data, for example, the figure shows that after bacillus subtilis NS15 is sprayed to the nose, CD11b in the inherent layer of the nasal mucosa of pigs is⁺MHC II⁺Dendritic cells were significantly increased compared to the control group (fig. 16 c).

Claims

1. The Bacillus subtilis NS15 is characterized in that the Bacillus subtilis NS15 is preserved in China general microbiological culture Collection center (CGMCC) at 13.07.2020, and the preservation number is CGMCC No. 20344.

2. A bacterial agent produced by the Bacillus subtilis NS15 according to claim 1.

3. Use of the bacillus subtilis NS15 of claim 1 for the preparation of a nasally administrable formulation for the prevention of porcine epidemic diarrhea virus.

4. The use of claim 3, wherein the nasally administrable formulation is one of a spray or a powder.

5. The use of bacillus subtilis NS15 of claim 1 for the preparation of a nasally administrable formulation for adhering porcine epidemic diarrhea virus and preventing the virus from invading the epithelial cells of the nasal cavity.

6. The use of claim 5, wherein the nasally administrable formulation is one of a spray or a powder.

7. The use of bacillus subtilis NS15 of claim 1 in the preparation of a nasal administration preparation for enhancing innate immunity function of nasal mucosa of pigs.

8. The use of claim 7, wherein the nasally administrable formulation is one of a spray or a powder.