VERO (ATCC®CRL-1586) and PK-15 cells (ATCC®CCL-33) were cultured in Dulbecco’s modified Eagle’s medium (CORNING, USA) supplemented with 10% fetal bovine serum (Gibco) at 37°C in a humidified atmosphere containing 5% CO2. The PRV strain ZJ 01 was isolated from a pig herd at China in 2012 (Gu et al., 2015), which was used for all experiments. The PRV were proliferated in PK-15 cells and stored at −80°C. β-Streptococcus equinus strain SheepZ001 were cultured in Tryptone soybean broth (TSB) + 0.05% fetal bovine serum (FBS) medium at 37°C, 220 rpm. Pasteurella multocida HB01 (Serotype A) were cultured in Brain-heart extract broth (BHI) medium at 37°C, 220 rpm. Bacillus subtilis 168, WB800 and other Bacillus subtilis group isolated from sheep nasal cavity were cultured in Luria-Bertani (LB) medium at 37°C, 220 rpm. Anti-PRV gB-protein monoclonal antibodies (1B1, prepared and stored in our laboratory), anti-GAPDH antibody (Proteintech), anti-cytokeratin18 (Abcam), anti-claudin 1(Abcam), anti-caspase3 (Proteintech); anti-caspase9 (Proteintech); anti-ISG15 antibody (Abcam), anti-PCNA antibody (Abcam), anti-CD3 antibody (Dako), anti-IgA antibody (Abcam) and anti-CD208 antibody (Dendritics) was used in the study.

The healthy 6-month-old lambs (n = 3) were euthanized by intravenous injection of pentobarbital sodium (100 mg/kg). After death, lambs were decapitated, the different part of nasal cavity (nasal concha and septum) was immersed into a PBS medium (5% double antibody, 1% amphotericin and gentamicin) (Figure 1A). Next, the different of nasal cavity were transferred into a 50 ml centrifuge tube filled with PBS and antibiotics. After shaking the tube, we washed it 3–4 times, with each wash lasting 5–10 min. Once complete, we transferred the washed nasal tissue onto a sterile plate. Using ophthalmic scissors and tweezers, we gently peeled off the nasal mucosa tissue. In addition, we extract a piece of nasal mucosa and place it on a new sterile plate with the surface facing upwards. Then, nasal mucosa is processed into uniform circular tissue blocks measuring 8 mm in diameter using a tissue sampler. The nasal cavity is a complex structure with varying thicknesses of mucosa and epithelial characteristics throughout its different parts. To ensure consistency, we chose to focus on the mucosa of the inferior nasal concha in the respiratory area of the nasal cavity. On average, about 40 pieces of mucosal tissue can be obtained from nasal cavity of 6-month-old sheep. The mucosal tissue was pretreated and placed onto a 12-hole transwell device with the mucosal surface facing upwards. The upper chamber filter membrane had a pore diameter of 0.4 μM. Approximately 600 μL of culture medium (Alfi et al., 2020) was added to the entire transwell device, which was only half the thickness of the mucosal tissue. The tissue was then cultured at 37°C and 5% CO2, with the medium being replaced daily.

The nasal mucosal explants were fold at different cultured times, make the cilia face outward. Then, the folded position was observed by optical microscope, and record by video.

The nasal mucosal explants were fixed in a phosphate-buffered 4% formaldehyde solution for 24 h. After fixation, the samples were embedded in paraffin. Sections (5 mm thick) were cut, deparaffinized in xylene, rehydrated in descending grades of alcohol. For immunohistochemistry analysis, antigen retrieval was performed for 30 min with citrate buffer at pH 6.0 in a Decloaking Chamber at 95°C. Slides were blocked with 5% bovine serum albumin (BSA) 1 h and then incubated with anti-cytokeratin18 (1: 200) overnight at 4°C in a humidified chamber. The SABC-POD Kit was used for the amplification and visualization of the signal.

Sections were rinsed and subjected to antigen demasking as described above. After were treated with 5% BSA for 1 h and incubated with mouse anti-Claudin1 (1: 200), anti-PCNA, anti-CD3 (1:200), anti-IgA (1:200) and anti-CD208 (1:100) overnight at 4°C, followed by incubation with fluorescent secondary antibodies for 1 h at 25°C. Cell nuclei were stained by incubation with diamidino-2-phenylindole (DAPI) for 5 min, and observed under a confocal laser microscope (LSM-710; Zeiss).

For ISG15 and PRV observation, PK15 cells were inoculated on the climbing plate. After treatment, 4% paraformaldehyde was fixed for 1 h, PBS was washed three times, and then directly incubated with anti-ISG15 (1: 200) and anti-PRV-gB (1: 500) antibody overnight at 4°C, followed by incubation with fluorescent secondary antibodies for 1 h at 25°C. Cell nuclei were stained by incubation with diamidino-2-phenylindole (DAPI) for 5 min, and observed under a confocal laser microscope (LSM-710; Zeiss).

For PAS stain, the sections are hydrated as previously mentioned and then treated with periodic acid for 7 min. They are rinsed under running water and soaked for 5 min. Next, the sections was soaked in Scheff’s reagent for 15 min in the dark and rinsed under running water. Then, the sections were counterstained with hematoxylin for 2 min and treated with acid differentiation solution for 3 s. Finally, the sections are subjected to conventional dehydration and transparency before being sealed.

After hydrating the sections as previously mentioned, apply a diluted Giemsa dye solution for 20 s (adjusting the dyeing time as needed), and rinse in water three times, for 2 minutes each time. Finally, the sections are subjected to conventional dehydration and transparency before being sealed.

Apoptosis was analyzed by TUNEL Cell Apoptosis Detection Kit (Alexa Fluor 488, Yisheng) which determines DNA fragmentation. The main steps are as follows: After sections hydration, incubate at room temperature for 20 min with Protease K, and moisten the sample with PBS solution for 2–3 times (positive control: treat with DNase I; negative control: replace TdT enzyme with ddH₂O). Add 10 μL 1 × The equilibration buffer 25°C 20 min. Remove most of 1× Equilibration Buffer, then add 50 μL TdT incubation buffer. Keep out of light and incubate at 37°C for 60 min. The sections were placed in PBS solution and washed repeatedly for 3 times. Cell nuclei were stained by incubation with DAPI for 5 min, and observed under a confocal laser microscope (LSM-710; Zeiss).

Total RNA from nasal cavity mucosa and PK15 cells was extracted using a TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was generated by reverse transcription using HiScript™ Q RT SuperMix for qPCR (Vazyme, China) according to the manufacturer’s instructions. The target gene transcription was determined using quantitative RT-PCR (RT-qPCR) with the SYBR Green qPCR Kit (Takara, Beijing, China) and analyzed using the double standard curve method. All primers used for RT-qPCR are presented in Supplementary Table S1. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was quantified as the internal control. The relative levels of cytokine RNA were calculated using the 2^-ΔΔ CT method.

Tissue and cell samples were assessed by western blotting with specific antibodies. At indicated times post-infection, the collected samples were lysed in RIPA buffer containing a protease inhibitor cocktail (Thermo Scientific). The concentration of the lysates was determined using a BCA protein quantification kit (Thermo Scientific). Equal amounts of protein were separated via SDS-PAGE and electrophoretically transferred onto a PVDF membrane (Millipore, Shanghai, China). The membranes were blocked with 5% skim milk for 1.5 h and incubated with anti-GAPDH (1:1,000), anti-caspase3 (1:1,000), anti-caspase9 (1:1,000), anti-PCNA (1:1,000) and anti-PRV-gB (1:3,000) overnight at 4°C, and followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies. The intensity of the bands in terms of density was measured and normalized against GAPDH expression.

Confluent monolayers of Vero E6 cells grown in 12-well tissue culture plates were infected with 500 μL of serial tenfold dilutions of the supernatant samples. After being incubated for 1 h at 37°C, cells were overlaid with 0.7% Sea-Plague agarose in DMEM containing 2% FBS and incubated at 37°C. Three days post-infection, the plaques were visualized via crystal violet staining.

The production of IFN-α, IFN-β and IFN-γ were detected using an ELISA kit (MEIMIAN), according to the manufacturer’s instructions.

The nasal mucosa explant was placed in 24-hole plate and inoculated with PRV (5 × 10⁵, 400 μL) to immerse the mucosal tissue. After 1 h, the nasal mucosal tissue was washed for 3 times and transferred to 12-hole transwell devices. The nasal mucosal was cultured at 37°C with 5% CO2. Infected nasal mucosa was collected at different time points (12, 24, 36, 48, 60 and 72 h) for subsequent analysis.

Th Sheep-derived Bacillus subtilis, Streptococcus and Pasteurella multocida were harvested by centrifugation at 300 g for 5 min, washed 3 times in PBS, and suspended to an OD600 of 0.6 (approximately 10⁶ CFU/ml) in PBS. Explants were washed with the cultivation medium without antibiotics and kept without antibiotics for at least 12 h prior inoculation. Next, explants were inoculated with 250 μL of bacterial suspension and allowed to adhere to the explants for 12 h at 37°C and 5% CO2. Drop the prepared bacterial solution with 250 μL onto the nasal mucosal explant cultured in the transwell devices, and allowed co-culture for 24 h. Then, the sheep nasal mucosa was collected for subsequent detection.

The LPS suspended to 0.01, 0.1, 1, 10 and 100 μg/ml in PBS. Explants were washed with the cultivation medium without antibiotics and kept without antibiotics for at least 12 h prior inoculation. Next, explants were inoculated with 250 μL of LPS of different concentrations and culture for 12 h at 37°C and 5% CO2, and wash the nasal mucosal tissue for 3 times. Then, the nasal mucosal culture it at 37°C with 5% CO2. The infected nasal mucosa was collected at 24 h for subsequent analysis.

Prior to the PRV infection, the sheep nasal mucosal explant model was pretreated with Sheep-derived Bacillus subtilis from the sheep nasal cavity, as outlined in Methods 2.11. The sheep nasal mucosa was moistened and washed three times with PBS. Following this, the PRV virus was inoculated according to Methods 2.11. After 24 h of culture, the collected samples were tested for viral load and distribution characteristics.

A total of eight newborn lambs were randomly divided into two groups of four each. The control group received an intranasal administration of PBS (500 μL per nostril), while the NSV2 group received intranasal administration of 109 CFU/ml at 12, 21, and 26 days after birth. The dosage for each administration was 0.5 ml, 1 ml, and 1 ml, respectively. At 30 days, lambs were euthanized using intravenous injection of sodium pentobarbital (100 mg/kg). The nasal cavity was then opened and 5 ml of PBS was added to rinse the cavity. The rinse solution was collected from the nostrils and the content of IFN-α/β/γ was detected using ELISA. Additionally, fixed and molecular samples of the nasal mucosa were collected to detect the expression of ISG15.

The nasal swabs collected from the stocking (n = 4) and feeding (n = 4) sheep, respectively. After the sheep are pinioned, the sterile swab inserts the nasal cavity for more than 10 cm and rotate it for 3 times. DNA was extracted from nasal swabs using HiPure soil DNA kit (Magen). 16S ribosomal RNA (rRNA) V3-V4 was amplified with specific primers (F: 5’-GTGCCAGCCGGGTAA-3′; R: 5′- GGACTACHVGGGTWTCTAAT-3′). The HiSeq 2500 system (Illumina Inc) was used used for sequencing and the USEARCH (v7.0.1090) is used to cluster the sequences into operation classification units (OTUs), which have 97% identity. The relative abundance of OTUs and classify was calculate at different taxonomic levels (phyla, order, class, family, genus and species). Based on OTU and species annotation, the complexity of sample species and species differences among populations were analyzed.

In the meantime, the centrifuge tube containing the nasal swab was open in the ultra-clean table aseptically, and add 500 μL of PBS solution, and then shake repeatedly three times. The leachate was collected and heat in 80°C water bath for 15 min, and then transfer the 100 μL leaching solution is transferred to LB (Invitrogen) and cultured overnight at 37°C and 220 rpm. According to the colony morphology and Gram staining results select single colony. After colony suspension was prepared, 16S rDNA universal primers 27F and 1492R were used primers for PCR amplification. Primer sequence 27F: AGAGTTTGATGCTCAG, 1492R: TACCTTTGTTACGACTT. The PCR product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing, and was compared by NCBI blast. According to the sequence comparison results, all strains of Bacillus subtilis group were stored for the next test.

The PK15 were transfected with 100 nM ISG15-specific or scrambled control siRNA duplexes (Supplementary Table S2) using the X-tremeGENE HP DNA transfection reagent (Roche) according to the manufacturer’s instructions. After 36 h of transfection, the cells were inoculated with 250 μL of bacterial suspension for 12 h, infected with PRV (MOI = 0.1) 12 h for subsequent analysis.

Results are presented as means ± SD and analyzed with SPSS 17.0. One-way ANOVA was employed to determine statistical differences among multiple groups, and T-test was employed to determine differences between the two groups. *: p < 0.05. Different letters also indicate significant differences.

The integrity and vitality of the NMEM are crucial for further experimental research and practical applications. To ensure this, we have evaluated the activity of the NMEM through multiple indicators. Firstly, we observed that the cilia of sheep nasal mucosa explants vibrated at a high speed for about 11 days (Figure 1B; Supplementary Video). Furthermore, the integrity of the nasal mucosal explant was good, and the expression of cytokeratin 18 and the tight junction protein claudin-1 in the epithelial layer of the explant was high. PAS staining was used to observe the secretory characteristics of the sheep nasal mucosa explants, and the secretory capacity of the gland decreased with longer culture time (Figure 1C). At last, CD3⁺ T-, IgA⁺ B-, and CD208⁺-positive cells (antigen markers of sheep dendritic cells) (Salaun et al., 2004) were distributed in the sheep nasal mucosa explant of 6-month-old sheep (Supplementary Figure S1).

To verify the reliability of the culture model, we detected apoptosis and proliferation in the cultured sheep nasal mucosa explants. The results indicated no significant differences between the ratios of cleaved-caspase-3/procapase-3 and cleaved-caspase-9/procapase-9 with increased incubation time (Figures 2A,B). TUNEL assay revealed no apoptosis after 1–11 days compared with the positive control group (Figure 2C). Few apoptotic cells were observed under the epithelial layer of the nasal mucosa (Supplementary Figure S3). The nasal mucosa explants exhibited good proliferative ability within the first 3 days, which decreased gradually thereafter (Figures 2C,D). Furthermore, the expression levels of innate immunity factors decreased as the culture time of nasal mucosal explants increased (Supplementary Figure S2A). The expression pattern of innate immune factors at different times closely resembled that observed in vivo (Supplementary Figure S2B).

PRV can cause fatal infection in sheep, and even attenuated vaccines may cause an outbreak of Pseudorabies in sheep (Kong et al., 2013). Therefore, we constructed a model of PRV-infected sheep nasal mucosa explants to explore the infection and characteristics of PRV. The results show that the endoturbinates explants, located in the olfactory region of the sheep’s nasal cavity, had a significantly higher PRV infection level at 24 and 36 h compared to other tissues such as the nasal vestibule, inferior and superior nasal concha (Figures 3A,B). As the infection time increased, the nuclei of the mucosal epithelium became vacuolated after 24 h, the upper cortex detached after 48 h, and almost all epithelial layers detached at 72 h (Figure 3C). The results of immunofluorescence and western blot assays were consistent, and the PRV viral load increased significantly over time (Figures 3D,E). Additionally, the expression of the inflammatory factors interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-c in the PRV infected group was higher than in the control. However, the expression of inflammatory factors increased at other time points, although not significantly, and some inflammatory factors were downregulated (Figure 3F).

Streptococcus and Pasteurella multocida are important pathogenic microorganisms colonized in sheep nasal cavities (Scott, 2011; Kumar et al., 2014). In this study, HE staining results showed that the cilia integrity of mucosal explants treated with Streptococcus and Pasteurella multocida became worse compared with the control group (Supplementary Figure S4B). Besides, we observed the presence of Pasteurella-like bacteria in the sheep nasal mucosa through Giemsa staining (Supplementary Figure S4B). When the nasal mucosa explants of sheep were pretreated with Streptococcus and Pasteurella multocida, the expression levels of inflammatory factors such as IL-1β, IL-6, IL-8, and TNF-α were significantly increased (Supplementary Figure S4C). Lipopolysaccharide (LPS) is a component of the outer wall of the cell wall of gram-negative bacteria, which can effectively induce host inflammation. After being treated with LPS, the expression levels of inflammation-related factors, including IL-1, IL-6, and TNF-α were significantly increased in sheep nasal mucosa explants. These results were consistent with those of pathogen treatment, and have a significant concentration dependent (Supplementary Figures S5A,B).

In addition to pathogenic bacteria and viruses, there are also a large number of commensal microorganisms in the nasal cavity, which is essential to maintain the health and stability of the respiratory tract (Bosch et al., 2013). A 16sRNA sequencing system was used to compare the structural characteristics and differences in nasal flora between stocking and feeding sheep. There is no significant difference in diversity and characteristics of flora between Stocking and Feeding (Supplementary Figure S4A). Based on the permutational multivariate analysis of variance and the principal coordinates analysis (PCoA), there was significant difference in nasal microbes between Stocking and Feeding (Supplementary Figure S4B). There are 1,386 unique OTUs in Stocking, 1,455 unique OTUs in Feeding group (Supplementary Figure S4C). Stocking and feeding sheep are mainly enriched in Firmicutes (17.7%/38.0%), Proteobacteria (17.2%/28.3%), Tenericutes (14.2%/10.5%), Actinobacteria (20.1%/14.5%), and Bacteroidetes (12.5%/4.0%) (Supplementary Figure S4D). Statistics were made on the top 35 genera with the highest enrichment, mainly including Staphylococcus (15.7%/78%), Mycoplasma (1.5%/12.0%), Moraxella (3.0%/11.9%), Filamentous bacteria (12.4%/1.5%) and Acinetobacter (1.1%/6.0%) (Supplementary Figure S4E). The abundance of Bacillus spp. in the stocking group was significantly higher than in the Feeding group (p < 0.05, Supplementary Figure S4F).

Accordingly, we isolated Bacillus strains from the nasal cavities of sheep and observed that their colony morphology matched the Gram staining characteristics of Bacillus subtilis group (Supplementary Figures S4G,H). Furthermore, we sequenced the Bacillus in the sheep nasal cavity and obtained six Bacillus subtilis strains (NSV1, NSV2, NSV3, NSS1, NSS2, and NSP1) and three unclassified Bacillus strains (NSS3, NSS4, and NSS5) (Supplementary Figure S4F).

The results showed that treating sheep nasal mucosa explants with sheep-derived Bacillus subtilis strains NSV2 and NSV3 significantly increases the expression of IFN-α, IFN-β, and IFN-γ (p < 0.05). NSV1, NSS2, and NSS4 tended to upregulate IFNs expression (Figures 4A,B). Then, we detected the ISGs downstream of interferon. NSV2 and NSV3 can significantly induce the expression of ISG15, and significantly upregulate IFITM1 and OAS2, respectively (Figure 4C). Furthermore, NSV2 nasal spray lamb significantly induced IFN-α and IFN-β (Figures 4D,E). The immunofluorescence and Western blot results showed that the expression of ISG15 increased significantly in the nasal mucosa (Figures 4F,G).

In order to further explore the possible interaction of sheep nasal microorganisms. We used the nasal mucosal explants model pretreated with sheep-derived Bacillus subtilis of different strains (Figures 5A,B). The result showed that NSV2 and NSV3 significantly inhibited PRV infection of the sheep nasal mucosal explants (Figures 5C,D). Hematoxylin and eosin (HE) staining and immunofluorescence observations revealed that the degree of pathological change and fluorescence intensity of PRV in the NSV2 and NSV3 treatment groups were significantly lower than those in the control group (Figure 5E).

To further explore the role of Sheep-derived Bacillus subtilis in resisting PRV infection, we selected PK15 as a cell model to explore its specific mechanism. qPCR results showed that the level of viral mRNA in cells treated with Bacillus subtilis was significantly lower than that in control cells (Figures 6A,B). The results of immunofluorescence assay showed that the PRV fluorescence intensity at 6, 12, and 24 h in the Bacillus subtilis treatment group was significantly lower than that of the control group (Figure 6D). ISG15 expression was high within the first 24 h after Bacillus subtilis treatment (Figure 6C). In order to further explore the role of ISG15 in the inhibition of pseudorabies virus by Bacillus subtilis, we designed the interfering RNA of ISG15 for further exploration (Figure 6E). The results showed that after using siISG15, the ability of Bacillus subtilis to inhibit PRV infection decreased significantly compared with Scrambled RNA (Figures 6F–H), and the content of upstream IFN-α/β/γ significantly increased (Figure 6I).

ISG15 is an important ubiquitin-like protein that significantly inhibits viral replication by binding to host or viral proteins. Samples with a parallel infection in the B. subtilis treatment group were analyzed by western blot, to confirm the results and determine the ISG15 ubiquitination level after treatment with B. subtilis. The PRV gB protein was first detected at 0 h (Figure 7A) and continued to increase until 48 h. The results showed that the accumulation of free ISG15 was time-dependent and became obvious at 36 h, while the increase of ISG15 conjugate was not obvious until 36 h. Compared with the accumulation of PRV-gB protein, the increase of ISG15 protein has a 24-h delay (Figure 7A). However, significant differences can be seen in the treatment group of Bacillus subtilis NSV2. After NSV2 treatment, a large amount of free ISG15 was accumulated in the early stage of PRV infection, while a large number of ISG15 conjugates appeared in the early stage of infection (Figure 7A). At the same time, the expression of PRV-gB protein also appeared in large numbers after 24 h (Figure 7B). To further compare the presence of free and bound ISG15 after treatment with Bacillus subtilis, we detected the blank group, PRV group, Bacillus subtilis group and Bacillus subtilis + PRV group 12 h after virus infection. The results showed that NSV2 could significantly enhance the expression of free ISG15 and the accumulation of bound ISG15 (Figure 7B). In addition, the results were consistent with that of ISG15 induced by Bacillus subtilis in explants, the NSS2 could not induce its expression (Figure 7C) and NSV3 could significantly enhance the expression of ISG15 (Figure 7D). Morever, immunofluorescence results indicate a significant co-localization between ISG15 and PRV-gB (Figure 7E).