Min pig source lactobacillus salivarius LSM231 for resisting PEDV infection and application of Min pig source lactobacillus salivarius LSM231

Abstract

The invention discloses a porcine epidemic diarrhea virus (PEDV) infection resistant Min pig source lactobacillus salivarius LSM231 and application thereof, and belongs to the technical field of microorganisms. The Min pig source lactobacillus salivarius is named as Ligilbacillus salivarius LSM231 and is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M 20242330, and the preservation time is October 25, 2024. The Min pig source lactobacillus salivarius has the advantages that the Min pig source lactobacillus salivarius is named as Ligilbacillus salivarius LSM231 and is preserved in the China Center for Type Culture Collection (CCTCC); the screened Min pig source lactobacillus salivarius is symbiotic probiotics in intestinal tracts and is high in safety performance, and experimental results show that the Min pig source lactobacillus salivarius LSM231 has relatively good antibacterial, acid-resistant and cholate-resistant characteristics, has a remarkable PEDV (porcine epidemic diarrhea virus) resisting effect, and can relieve clinical symptoms caused by PEDV infection and promote growth of piglets, so that the Min pig source lactobacillus salivarius LSM231 can be used for preventing and treating the porcine epidemic diarrhea. The compound can replace antibiotics to be used for preparing a micro-ecological preparation for resisting PEDV (porcine epidemic diarrhea virus) infection.

Description

Technical Field

[0001] The invention relates to a strain of Lactobacillus salivarius from pigs, and also relates to the use of the Lactobacillus salivarius from pigs in resisting bacterial or viral infection. The invention belongs to the technical field of microorganisms. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) belongs to the order Nidovirales, the family Coronaviridae, and the genus Alphacoronavirus. It is an enveloped RNA virus with a single positive-strand genome of about 28 kb. Its genome encodes four functional structural proteins near the 3' end: glycosylated spike protein (S protein), membrane protein (M protein), envelope protein (E protein), and nucleocapsid protein (N protein). Among them, the S protein is an important structural and functional protein on the surface of PEDV. It is located on the surface of the virus particle and has the function of recognizing target cells and promoting the fusion of virus and cell membranes. It also contains the targets recognized by most neutralizing antibodies. PEDV is infectious to pigs of all ages, but it is most infectious and highly contagious to suckling piglets, with a mortality rate of up to 100%. It mainly infects the small intestinal mucosa and causes symptoms such as anorexia, vomiting, diarrhea, dehydration, and weight loss in pigs. PED is still one of the major diseases that endanger my country's pig industry in my country's pig herds, causing huge economic losses to the pig industry.

[0003] The surface of the intestinal barrier of animals is full of symbiotic microorganisms, known as microbiota, which affect the physiological processes of the host, most of which are extracellular bacteria. These intestinal microorganisms can limit the expansion of pathogens on the surface of the host intestine by competing with bacterial and fungal pathogens for binding sites and producing antibacterial substances. In addition, the intestinal flora can also resist viral infection and maintain the health of the intestinal mucosa by secreting metabolites, maintaining the normal function of the intestinal barrier, and activating the innate immune system. Studies have shown that Lactobacillus fragilis can induce the expression of IFN-β through TLR4-TRIF signaling to resist infection with vesicular stomatitis virus or other influenza viruses. Recently, studies have found that Lactobacillus rhamnosus and its metabolites interact with type 3 innate lymphocytes (ILC3) in the jejunum of piglets through aromatic hydrocarbon receptors, which can promote ILC3 activation and IL-22 production to regulate the intestinal immune function of piglets and effectively prevent PEDV infection. Based on the close relationship between intestinal flora and body immunity, the use of probiotics to regulate intestinal flora has become an extremely effective way to improve body immunity.

[0004] Min pigs are a native Chinese breed, distributed in Northeast China, adapted to harsh climates and extensive breeding. Compared with ordinary three-way pigs, Min pigs have higher immunity, stronger disease resistance and adaptability, and have been included in the national list of livestock and poultry genetic germplasm resources for protection. Because the composition and structure of the intestinal flora of pigs are significantly different at different growth stages, especially the difference is the greatest during lactation, and the intestinal flora of different breeds of pigs is also obvious. The strong disease resistance of Min pigs may be the result of the interaction between the symbiotic microorganisms on the surface of the intestinal mucosa and its intestinal mucosal microecology. Therefore, studying the molecular mechanism of the interaction between specific symbiotic microorganisms and the intestinal mucosal microecology of the host itself is not only of biological significance, but also can provide new solutions for the treatment of enteroviral diseases.

[0005] The present invention separates a strain of Lactobacillus salivarius from the intestinal contents of pigs, and finds through antiviral experiments that it has a significant antiviral effect, which can provide a technical means for the research and development of microecological live bacteria preparations. Summary of the invention

[0006] The purpose of the present invention is to provide a strain of Lactobacillus salivarius from pigs and use of the Lactobacillus salivarius from pigs in resisting bacterial or viral infection.

[0007] In order to achieve the above object, the present invention adopts the following technical means:

[0008] The inventors of the present invention isolated and purified a strain of Lactobacillus salivarius from the colon contents of pigs, named it Ligilactobacillus salivarius LSM231, classified it and named it Ligilactobacillus salivariusLSM231, which was deposited in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with a deposit number of CCTCCNO: M 20242330 and a deposit date of October 25, 2024.

[0009] Furthermore, the present invention also proposes the use of the pig-derived Lactobacillus salivarius in the preparation of a medicine or a microecological preparation for inhibiting Serratia marcescens, Salmonella enteritidis, Escherichia coli, Staphylococcus aureus (S.aureus) and / or Salmonella typhimurium.

[0010] Furthermore, the present invention also proposes the use of the pig-derived Lactobacillus salivarius in the preparation of drugs or microecological preparations for resisting porcine epidemic diarrhea virus (PEDV) infection.

[0011] Among them, preferably, the pig-derived Lactobacillus salivarius can effectively block porcine epidemic diarrhea virus from infecting cells before virus infection and during virus adsorption stages, thereby achieving an antiviral effect.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1. At present, antibiotics are still used in the treatment of viral diseases, but antibiotics will seriously destroy the balance of intestinal microbial flora and reduce the content of beneficial intestinal bacteria, which will be counterproductive and aggravate the viral infection of animals. Lactobacillus not only has the advantages of good safety and low cost, but also as an intestinal symbiotic bacteria, it can regulate the balance of intestinal flora and intestinal mucosal immunity and resist viral infection; it can be used as an important alternative to antibiotic treatment.

[0014] 2. The pig-derived Lactobacillus salivarius screened by the present invention has high safety performance because it is a symbiotic probiotic in the intestine. The above experimental results show that the probiotic LSM231 has good antibacterial and acid- and bile-resistant properties, and has a significant anti-PEDV effect, can alleviate the clinical symptoms caused by PEDV infection, and promote the growth of piglets. Therefore, it can replace antibiotics for the preparation of microecological preparations against PEDV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Morphology (A) and Gram staining observation (B) of Lactobacillus salivarius LSM231;

[0016] Figure 2 The growth curve (A) and acid production trend curve (B) of LSM231;

[0017] Figure 3 To evaluate the antibacterial effect of LSM231;

[0018] ****P<0.0001vs MRS;

[0019] Note: Wells A, B and C are all probiotic supernatants; Well D is the MRS culture medium control well;

[0020] Figure 4 Results of acid resistance (A) and bile resistance (B) of LSM231;

[0021] Figure 5 The infection status of IPI-2I cells was detected by IFA 24 h after PEDV infection;

[0022] Figure 6 This is the one-step growth curve of PEDV proliferation on IPI-2I cells;

[0023] Figure 7 This is the test result of the optimal concentration of LSM231;

[0024] ****P<0.0001vs CON; *P<0.05vs CON;

[0025] Note: A: optimal dosage of bacterial suspension; B: optimal dosage of cell-free supernatant;

[0026] Figure 8 To evaluate the inhibitory effect of LSM231 on PEDV proliferation;

[0027] ****P<0.0001vs PEDV; ***P<0.001vs PEDV; *P<0.05vs PEDV;

[0028] A: Before virus infection; B: Virus adsorption stage; C: Virus invasion stage;

[0029] Fig. 9 The PEDV titer was determined by plaque method;

[0030] ****P<0.0001vs PEDV; ***P<0.001vs PEDV;

[0031] Fig.10 For indirect immunofluorescence intensity analysis;

[0032] ****P<0.0001vs PEDV;

[0033] Fig.11 is the mRNA expression level of cell barrier-related factors;

[0034] Note: ****P<0.0001vs CON; ***P<0.001vs CON; **P<0.01vs CON; *P<0.05vs CON; ####P<0.0001vs PEDV; ###P<0.001vs PEDV; ##P<0.01vs PEDV; #P<0.05vs PEDV; ns: no significant difference;

[0035] Fig.12 is the mRNA expression level of genes related to inflammatory factors;

[0036] ***P<0.001vs CON; **P<0.01vs CON; ###P<0.001vs PEDV; ##P<0.01vs PEDV; ns: no significant difference;

[0037] Fig.13 is the antiviral protein mRNA expression level;

[0038] ***P<0.001vs CON; **P<0.01vs CON; *P<0.05vs CON; ##P<0.01vs PEDV; #P<0.05vs PEDV;

[0039] Fig.14 For clinical symptoms and autopsy changes;

[0040] Fig.15 Daily weight gain changes;

[0041] Fig.16 Regularity of fecal detoxification;

[0042] ***P<0.001vs PEDV; **P<0.01vs PEDV; *P<0.05vs PEDV;

[0043] Fig.17 The viral loads in the duodenum, jejunum and ileum of piglets;

[0044] ***P<0.001vs PEDV; *P<0.05vs PEDV;

[0045] Fig.18 is the positive rate of LSM231 labeled with CFDA-SE;

[0046] Fig.19 To detect the colonization of LSM231 in the intestine of piglets;

[0047] Fig. 20 This is a HE-stained section of piglet small intestine;

[0048] Fig.21 The changes of villus length, crypt depth and villus / crypt ratio in the small intestine of piglets;

[0049] ****P<0.0001vs CON; ***P<0.001vs CON; **P<0.01vs CON;

[0050] ####P<0.0001vs PEDV; ###P<0.001vs PEDV; ##P<0.01vs PEDV; ns: no significant difference;

[0051] Fig. 22This is the AB-PAS stained section of piglet small intestine;

[0052] Fig.23 The changes of goblet cell density and mucus layer thickness in the small intestine of piglets;

[0053] ****P<0.0001vs CON; ***P<0.001vs CON; **P<0.01vs CON; *P<0.05vs CON; ####P<0.0001vs PEDV; ###P<0.001vs PEDV; ##P<0.01vs PEDV; ns: no significant difference;

[0054] Fig.24 is the mRNA expression level of intestinal barrier-related genes;

[0055] ****P<0.0001vs CON; ***P<0.001vs CON; **P<0.01vs CON; *P<0.05vs CON; ####P<0.0001vs PEDV; ###P<0.001vs PEDV; ##P<0.01vs PEDV; ns: no significant difference;

[0056] Fig.25 is the mRNA expression level related to inflammatory factors;

[0057] ****P<0.0001vs CON; ***P<0.001vs CON; ###P<0.001vs PEDV; ##P<0.01vs PEDV; ns: no significant difference;

[0058] Fig.26 is the mRNA expression level of antiviral-related genes;

[0059] ****P<0.0001vs CON; ***P<0.001vs CON; **P<0.01vs CON; *P<0.05vs CON; ###P<0.001vs PEDV; ##P<0.01vs PEDV; #P<0.05vs PEDV; ns: no significant difference. DETAILED DESCRIPTION

[0060] The embodiments of the present invention are described in detail below. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0061] Example 1 Isolation and biological characteristics of Lactobacillus salivarius LSM231 from pigs

[0062] 1. Isolation and purification of LSM231

[0063] A strain of Lactobacillus salivarius was isolated and purified from the colon contents of pigs and named Ligilactobacillus salivarius LSM231. After culturing it in MRS solid medium for 24 hours, it formed milky white colonies with smooth surface and slight protrusions, such as Figure 1 A. The morphology of the strains was observed by Gram staining, and the results showed that all strains were blue-purple and were Gram-positive bacteria (G + ), the shape is short rod, such as Figure 1 As shown in B.

[0064] 2. Determination of LSM231 growth curve and acid production trend

[0065] The isolated Lactobacillus salivarius LSM231 was streaked onto MRS solid medium, incubated at 37°C for 24 h, and a single colony was picked and inoculated into MRS liquid medium; incubated at 37°C for 24 h; inoculated into MRS liquid medium at a ratio of 1:100, incubated at 37°C for 24 h, and the bacterial suspension was taken every 2 h to measure the OD 600 The plate count method was used to calculate the number of viable bacteria and draw a bacterial growth curve. Figure 2 As shown in A, 0-2h is the slow growth period, 4-8h is the logarithmic growth period, 10-18h is the plateau period, and after 20h it enters the decline period. Figure 2 B) It can be seen that the acid production capacity showed a linear trend during the logarithmic growth phase, indicating that the acid production capacity was the strongest. Lactobacillus was able to reduce the initial pH value of the culture solution from 5.7 to about 4.0 after 24 hours of culture.

[0066] 3. Antibacterial effect of LSM231

[0067] The inhibition zone size of LSM231 against Serratia marcescens, Salmonella enteritidis, Escherichia coli, Staphylococcus aureus (S. aureus) and Salmonella typhimurium is as follows: Figure 3 As shown, the results showed that LSM231 had a good antibacterial effect on the five pathogenic bacteria.

[0068] 4. Acid and bile salt resistance test

[0069] After Lactobacillus salivarius LSM231 was incubated in MRS (PH4, PH5, PH6, PH7 and PH8) and MRS (0.01%, 0.02%, 0.04%, 0.06%, 0.08% and 0.10% pig bile salt) liquid culture medium for 3 hours, the viable bacteria counting method was used to detect the tolerance of LSM231 to different pH conditions and different pig bile salt concentrations. Figure 4 As shown in A, under different acid and alkaline conditions, LSM231 has a good survival rate; the bile salt resistance results are as follows Figure 4 As shown in B, LSM231 can tolerate 0.06% concentration of porcine bile salts.

[0070] Example 2 Exploring PEDV infection and LSM231 anti-infection effects at the IPI-2I cell level

[0071] 1. Indirect immunofluorescence identification of PEDV-infected IPI-2I cells

[0072] The PEDV-SX strain was used to infect the IPI-2I cell culture plate with a monolayer. After 2 hours of adsorption, the maintenance medium was replaced and cultured until obvious syncytial lesions appeared. Indirect immunofluorescence assay (IFA) was performed using a monoclonal antibody against the PEDV S protein as the primary antibody and goat anti-mouse IgG-FITC as the secondary antibody. Figure 5 The IPI-2I cells infected with PEDV showed obvious green fluorescence, while the control group had no fluorescence, indicating that IPI-2I cells were successfully infected with PEDV.

[0073] 2. Determination of the growth curve of IPI-2I cells infected with PEDV

[0074] According to the measured PEDV TCID 50 , at 100TCID 50 Infect IPI-2I cells, change the maintenance medium, measure the PEDV virus copy number every 6 hours, and obtain the one-step growth curve of PEDV virus on IPI-2I cells. Figure 6 It can be seen that the incubation period of PEDV adsorption and infection of IPI-2I cells is about 6 hours, 12-18 hours is the period of rapid virus proliferation and lysis, after which the virus replication shows a downward trend.

[0075] 3. Detection of the optimal concentration of LSM231 on IPI-2I cells

[0076] Lactobacillus salivarius LSM231 was cultured to the optimal harvest time, and the bacterial solution concentration was 10 8 -10 9 CFU / mL. Collect the bacterial suspension (LSM231) and filter the cell-free supernatant (CFS) using a 0.22 μm filter. Dilute the bacterial suspension 10 -1, 10 -2 , 10 -3 , 10 -4 The cell-free supernatant was diluted to 2 times the original concentration. -1 , 2 -2 , 2 -3 , 2 -4 times; added to a 96-well plate filled with a monolayer of IPI-2I cells, and after 2 h of co-culture, CCK8 reagent was added to evaluate the effect on the cells, such as Figure 7 As shown in Figure 2, the optimal concentration of LSM231 bacterial suspension is 10% of the original concentration. -3 The cell-free supernatant did not cause damage to the cells after dilution, so the optimal concentration was selected as 2 times the original concentration. -1 times.

[0077] 4. Effect of LSM231 on the proliferation of PEDV in IPI-2I cells

[0078] In order to detect the best mode of action of LSM231 in inhibiting the proliferation of PEDV, LSM231 bacterial suspension or cell-free supernatant was added to cells before infection, at the virus adsorption stage, and at the virus invasion stage, and the effect of different infection stages on the proliferation of PEDV was detected. Figure 8 As shown in the figure, among the three modes of action, the use of LSM231 bacterial suspension or cell-free supernatant before virus infection can significantly reduce (P<0.05) the level of PEDV RNA replication; the use of LSM231 bacterial suspension or cell-free supernatant during the virus adsorption stage can extremely significantly reduce (P<0.0001) the level of PEDV RNA replication. When the bacterial suspension is added during the virus invasion stage, it will multiply in large quantities during the culture process, so it is impossible to extract cell RNA. In summary, LSM231 can effectively block virus-infected cells before virus infection and during the virus adsorption stage, achieving an antiviral effect.

[0079] 5. Plaque assay to determine the effect of LSM231 on PEDV titer

[0080] The LSM231 bacterial suspension and cell-free supernatant were diluted to the optimal concentration and then mixed with PEDV (100 TCID 50 ) acted together with IPI-2I cells, and the inhibitory effect of LSM231 on the reproduction and replication of PEDV in IPI-2I cells was further determined by virus plaque reduction experiment. Fig. 9 As shown in the figure, compared with the PEDV-infected Control group, the virus reproduction titer was significantly inhibited after treatment with LSM231 bacterial suspension and supernatant (P<0.001). This indicates that the use of LSM231 bacterial suspension or cell-free supernatant to treat IPI-2I cells during the virus adsorption stage can inhibit the proliferation of PEDV.

[0081] 6. LSM231 inhibits PEDV infection of IPI-2I cells

[0082] After PEDV infects IPI-2I cells, it promotes cell-cell membrane fusion, resulting in syncytial pathology. In order to further verify the inhibitory effect of LSM231 on PEDV infection of IPI-2I cells, the IFA test was used to detect the changes in the fluorescence intensity of cells infected with the virus after the use of LSM231 in the virus adsorption stage. The results are as follows Fig.10 As shown in the figure, cells infected with PEDV all showed typical syncytial lesions, but compared with the PEDV-infected Control group, the cell lesions in the LSM231 group were significantly reduced and the fluorescence intensity of the cells was relatively lower (P<0.0001). Therefore, LSM231 treatment can inhibit the replication of PEDV in IPI-2I cells, reduce cell syncytial lesions, and thus play a role in protecting cells.

[0083] 7. LSM231 regulates the expression of junction proteins in PEDV-infected IPI-2I cells

[0084] After PEDV and LSM231 were used together to act on IPI-2I cells, total RNA was extracted from the cells after 24 h of culture, and the transcription levels of tight junction proteins (Claudin-1, Occludin, and ZO-1) and adherens junction proteins (integrin, E-Cadherin, and β-Catenin) were detected by RT-qPCR. The results are shown in Fig.11 As shown in the figure, compared with the CON group, the mRNA levels of tight junction proteins (Claudin-1, Occludin and ZO-1) and adhesion junction proteins (integrin, E-Cadherin and β-Catenin) were significantly decreased after PEDV infection (P<0.01); compared with the PEDV group, the transcription levels of Claudin-1, Occludin and ZO-1, as well as E-Cadherin and β-Catenin in the LSM231+PEDV group were significantly increased (P<0.05). This indicates that LSM231 can enhance the intestinal epithelial barrier function by increasing the expression of tight junction proteins and adhesion junction proteins.

[0085] 8. LSM231 regulates the expression of inflammatory factors in PEDV-infected IPI-2I cells

[0086] The effect of TP1 on cellular inflammatory factors was analyzed, and RT-qPCR was used to detect the expression of IL-6, IL-18 and TNF-α mRNA. Fig.12As shown in the results, after PEDV infected IPI-2I cells, the mRNA expression levels of inflammatory factors IL-6, IL-18, and TNF-α were significantly increased (P<0.01). Compared with the PEDV group, the mRNA expression levels of inflammatory factors in the LSM231 group were significantly decreased (P<0.01). This indicates that LSM231 alleviates the inflammatory response caused by PEDV by reducing the expression of inflammatory factors.

[0087] 9. LSM231 regulates the expression of antiviral proteins in PEDV-infected IPI-2I cells

[0088] The effect of LSM231 on the expression of antiviral proteins in IPI-2I cells was analyzed. Fig.13 As shown in the figure, RT-qPCR detection found that after PEDV infected IPI-2I cells, the mRNA expression levels of RIG-I, MDA5 and its downstream related genes IRF7, IRF3 and interferon IFN-λ3 in the antiviral signaling pathway were significantly increased (P<0.05). Compared with the PEDV group, the mRNA expression levels of RIG-I, MDA5, IRF7, IRF3 and interferon IFN-λ3 in the LSM231 treatment group were significantly increased (P<0.05). This shows that LSM231 can resist viral infection by increasing the expression of related antiviral genes.

[0089] Example 3 Study on the protection of piglets against PEDV infection by LSM231 strain

[0090] 1. Clinical symptoms and autopsy results

[0091] Nine newborn piglets were randomly divided into three groups, each with 3 piglets, namely, CON group, PEDV group, and LSM231+PEDV group. The LSM231+PEDV group was given 2 mL (10 9 The CON group and the PEDV group were gavaged with an equal amount of PBS once a day for 3 consecutive days. On the fourth day, the PEDV group and the LSM231+PEDV group were gavaged with 1.5×10 6 TCID 50 The CON group was given an equal amount of PBS. The experimental observation, sampling and weight changes were performed during the 72-hour period. The clinical and autopsy characteristics and daily weight gain changes were as follows: Fig.14 and 15. The PEDV group began to have diarrhea 16 hours after infection, accompanied by vomiting, loss of appetite and other symptoms, and their weight gradually decreased; the LSM231+PEDV group began to have diarrhea 24 hours after infection with PEDV, but the degree of diarrhea was lower than that of the PEDV group, and the daily weight gain was higher than that of the PEDV group but lower than that of the CON group. After 72 hours, the piglets in each group were autopsied to observe the pathological changes in the small intestine. The results are shown in the figure. The autopsy of the piglets in the PEDV group showed that the intestinal wall was significantly thinned and almost transparent, with obvious yellow watery contents and obvious bloating; the piglets in the LSM231+PEDV group showed mild thinning of the intestinal wall, a small amount of yellow contents, and a lower degree of bloating than the PEDV group.

[0092] 2. Effect of oral administration of LSM231 on fecal excretion of PEDV in piglets

[0093] After the piglets were infected with PEDV, anal swab samples were collected from each group of piglets every 6 hours, and the fecal virus excretion was detected using the RT-qPCR method. The results are as follows: Fig.16 As shown in the data, at 12h, 24h, 30h, 36h, 48h, 54h, 66h, and 72h after PEDV infection, the fecal virus content in the PEDV group was significantly higher than that in the LSM231+PEDV group (P<0.05), indicating that oral LSM231 intervention can effectively reduce the replication of PEDV in the body.

[0094] 3. Effect of oral administration of LSM231 on PEDV viral load in the intestine of piglets

[0095] The effect of LSM231 on viral load in each intestinal segment was tested. The PEDV viral load in the duodenum, jejunum, and ileum tissues was tested using RT-qPCR. The results are as follows: Fig.17 As shown in the results, after intervention with LSM231, the viral load in the duodenum of the LSM231+PEDV group was significantly lower than that in the PEDV group (P<0.05); the viral load in the jejunum and ileum was extremely significantly lower than that in the PEDV group (P<0.001). The above results indicate that oral administration of LSM231 can inhibit the replication of PEDV in vivo.

[0096] 4. Detection of colonization of LSM231 in the intestine of piglets after oral administration

[0097] The fluorescent dye CFDA-SE was used to label Lactobacillus salivarius LSM231, and the labeling positive rate was detected by flow cytometry. The results are as follows Fig.18 Compared with the unlabeled control in the left figure, the labeling rate of positive labeled strains in the right figure reached 99%. After three consecutive days of oral administration, the LSM231+PEDV group piglets were respectively taken from the duodenum, jejunum, and ileum to detect the colonization rate of labeled bacteria in each small intestine segment of the piglets. The results are shown in Fig.19The results are summarized in Table 1. The flow cytometry results showed that LSM231 had the highest colonization rate in the ileum, reaching 60.1%.

[0098] Table 1 Determination of the colonization ability of LSM231 in piglets

[0099]

[0100] Note: Different superscript letters in the same row indicate significant differences among groups (P<0.05).

[0101] 5. Effect of LSM231 on the morphology of small intestinal mucosal tissue in piglets infected with PEDV

[0102] The duodenum, jejunum and ileum tissues were obtained by autopsy and pathological histological sections were made. The morphology and pathological damage of the small intestinal epithelial tissue were observed after HE staining. Fig. 20 As shown in the figure, the small intestine of piglets in the PEDV group showed severe pathological damage, the intestinal villi were atrophied, broken, and irregularly arranged, and the intestinal mucosal structure was incomplete. The intestinal structure and intestinal villi of piglets in the CON group were intact. The villi in the ileum and jejunum of piglets in the oral LSM231 group showed slight atrophy and shedding, the intestinal villus structure was clearly visible, and the pathological changes were not obvious. Compared with the PEDV group, the LSM231+PEDV group significantly increased the villus length (P<0.001) and significantly increased the villus / crypt ratio of the duodenum and ileum (P<0.01). The results are shown in the figure. Fig.21 In summary, Lactobacillus salivarius LSM231 can alleviate the damage of PEDV to the small intestine of piglets.

[0103] 6. Effect of LSM231 on intestinal goblet cells and mucus layer in piglets infected with PEDV

[0104] After piglets were orally administered Lactobacillus salivarius LSM231, the small intestinal tissue was stained using the AB-PAS staining method. Figure 22-23 As shown in the figure, compared with the CON group, the goblet cell density and mucus layer thickness in the PEDV group were significantly reduced (P<0.05). Compared with the PEDV group, the goblet cell density in the LSM231+PEDV group was significantly increased (P<0.001), and the mucus layer thickness in the duodenum and jejunum was significantly increased (P<0.001).

[0105] 7. Effect of LSM231 on the expression of intestinal-related junctional proteins in PEDV-infected piglets

[0106] The RT-qPCR method was used to detect the effect of LSM231 intervention on intestinal barrier junction proteins in piglets infected with PEDV. Fig.24The figure shows the expression level of intestinal barrier genes in the ileum of piglets. Compared with the CON group, the mRNA transcription levels of tight junction proteins (Claudin-1, Occludin and ZO-1) and adhesion junction proteins (integrin, E-Cadherin and β-Catenin) in the PEDV group were significantly decreased (P<0.05); compared with the PEDV group, the tight junction proteins Claudin-1, Occludin and ZO-1 and adhesion junction proteins E-Cadherin and β-Catenin in the LSM231+PEDV group were significantly increased (P<0.01). The above results indicate that PEDV infection affects the expression of tight junction and adhesion junction proteins, and LSM231 can increase the expression of tight junction and adhesion junction proteins and improve the intestinal barrier function of piglets.

[0107] 8. Effect of LSM231 on the expression of intestinal inflammation-related factors in PEDV-infected piglets

[0108] In order to detect the effect of LSM231 on the expression of intestinal inflammatory factors in piglets infected with PEDV, the expression levels of inflammatory factors IL-6, IL-18 and TNF-α were detected by RT-qPCR. Fig.25 As shown in the results, compared with the CON group, the expression levels of the three inflammatory factors in piglets infected with PEDV were significantly increased (P<0.001); compared with the PEDV group, the LSM231+PEDV group significantly reduced the expression levels of IL-6 and IL-18 (P<0.001). The above results show that intervention with LSM231 can significantly reduce the increase in the expression levels of inflammatory factors caused by PEDV infection, thereby alleviating intestinal damage.

[0109] 9. Effect of LSM231 on the expression of genes related to intestinal antiviral pathways in PEDV-infected piglets

[0110] To evaluate the effect of LSM231 intervention on the intestinal mucosal immune antiviral signaling pathway in piglets. Fig.26 As shown in the figure, RT-qPCR detection showed that compared with the CON group, the antiviral proteins RIG, MDA5 and their downstream factors IRF7 and IFN-λ3 in piglets infected with PEDV were significantly increased (P<0.05). Compared with the PEDV group, the expression levels of RIG, MDA5 and IRF3 in the LSM231+PEDV group were higher than those in the PEDV group (P<0.05). The above results show that the use of LSM231 can alleviate PEDV infection by increasing the expression of antiviral proteins.

Claims

1. A strain of Lactobacillus salivarius from Min pigs, named Ligilactobacillus salivarius LSM231, deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20242330 and the deposit date of October 25, 2024.

2. Use of the pig-derived Lactobacillus salivarius according to claim 1 in the preparation of a medicine or microecological preparation for inhibiting Serratia marcescens, Salmonella enteritidis, Escherichia coli, Staphylococcus aureus (S. aureus) and / or Salmonella typhimurium.

3. Use of the pig-derived Lactobacillus salivarius according to claim 1 in the preparation of drugs or microecological preparations for resisting porcine epidemic diarrhea virus (PEDV) infection.

4. The use according to claim 3, characterized in that The swine-derived Lactobacillus salivarius can effectively block porcine epidemic diarrhea virus infection of cells before virus infection and in the virus adsorption stage, thereby achieving an antiviral effect.

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