Bacillus compound preparation containing grape seed extract and application thereof in preparation of medicine for treating or preventing PRRSV infection

Abstract

The present application belongs to the technical field of livestock and poultry anti-virus micro-ecological preparation, and particularly relates to a bacillus compound preparation containing grape seed extract and application of the bacillus compound preparation in preparation of a medicine for treating or preventing PRRSV infection. Two strains of bacillus with significant inhibition of PRRSV infection and synergistic effect after being combined with grape seed extract are screened, and the preservation numbers are CCTCC NO: M2026187 and CCTCC NO: M2026186 respectively. The test results show that the compound preparation can significantly reduce the infection activity and proliferation capacity of PRRSV in cells and pigs, effectively reduce the clinical symptoms of pigs caused by PRRSV infection, and relieve the damage degree of lung tissue organs of pigs. The potential synergistic effect between the bacillus compound bacteria and grape seed proanthocyanidin provides a new technical scheme for effective prevention and control of porcine reproductive and respiratory syndrome.

Description

Technical Field

[0001] This invention belongs to the field of antiviral microecological preparations for livestock and poultry, specifically relating to a compound preparation of Bacillus subtilis containing grape seed extract and its application in the preparation of drugs for the treatment or prevention of PRRSV infection. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as "blue ear disease," has long been a key challenge for the pig farming industry due to its high pathogenicity, high infectivity, and severe damage to the reproductive and growth performance of pigs. Currently, vaccination and antiviral therapy are commonly used disease control methods. However, PRRS attenuated and inactivated vaccines have poor immunogenicity and are difficult to effectively resist the invasion of heterologous strains. Furthermore, attenuated vaccines also have safety issues such as virus shedding and recombination, making the prevention and control of the disease difficult. The development of traditional PRRSV drugs also faces many bottlenecks: on the one hand, the virus is prone to mutation, leading to unstable drug targets and increasing the difficulty of development; on the other hand, the long development cycle and high cost of antiviral drugs severely restrict the industrialization process of anti-PRRSV drugs.

[0003] Against this backdrop, probiotics, with their advantages of being natural, safe, widely available, and relatively inexpensive, offer a novel approach to the development of anti-PRRSV agents. Studies have shown that specific strains of probiotics can not only competitively adhere to intestinal epithelial cells, preventing PRRSV invasion of host cells, but also inhibit viral replication by secreting antimicrobial peptides, organic acids, and other active substances. Furthermore, their metabolites can regulate the activity of immune cells, enhancing the host's resistance to PRRSV. In addition, probiotics can be further combined with traditional Chinese medicine, plant extracts, and other green ingredients to construct multi-target, multi-mechanism antiviral systems, further enhancing anti-PRRSV efficacy and providing new ideas and strategies for anti-PRRSV drug development. Summary of the Invention

[0004] In view of the difficulty in preventing and treating porcine reproductive and respiratory syndrome virus (PRRSV) infection, the present invention aims to provide a compound preparation of Bacillus subtilis containing grape seed extract. The compound preparation includes KY997 and KY2761, with accession numbers CCTCC NO: M2026187 and CCTCC NO: M2026186, respectively.

[0005] Another object of the present invention is to provide the use of the above-mentioned Bacillus compound preparation in the preparation of a medicament for treating or preventing porcine reproductive and respiratory syndrome virus infection.

[0006] To achieve the above objectives, the present invention employs the following technical measures:

[0007] This invention screened two strains, *Bacillus amyloliquefaciens* and *Bacillus subtilis*, which significantly inhibit porcine reproductive and respiratory syndrome virus (PRRSV) infection and exhibit synergistic effects when used in combination with grape seed extract. These strains were deposited at the China Center for Type Culture Collection (CCTCC) on January 22, 2026, at Wuhan University, Wuhan, China. Strain KY997 is classified as *Bacillus amyloliquefaciens* KY997, with accession number CCTCC NO: M2026187; strain KY2761 is classified as *Bacillus subtilis* KY2761, with accession number CCTCC NO: M 2026186.

[0008] The scope of protection of this invention includes:

[0009] A compound preparation of Bacillus containing grape seed extract, wherein the compound preparation comprises Bacillus amyloliquefaciens KY997, Bacillus subtilis KY2761 and grape seed extract, wherein the preservation number of Bacillus amyloliquefaciens KY997 is CCTCC NO: M 2026187 and the preservation number of Bacillus subtilis KY2761 is CCTCC NO: M2026186.

[0010] In the above-mentioned compound preparations, preferably, the effective bacterial concentrations of KY997 and KY2761 are the same;

[0011] In the above-mentioned compound preparation, preferably, the grape seed extract is a water extract of grape seeds.

[0012] The above-mentioned compound preparation, preferably, the preparation method of the grape seed extract includes: extracting crushed and sieved grape seeds multiple times at a material-to-water ratio of 1:5-20, combining the filtrates, heating and refluxing, ultrafiltration, concentrating, and then spray drying to obtain the final product.

[0013] In the above-described compound preparation, preferably, the mass ratio of *Bacillus amyloliquefaciens* KY997 and *Bacillus subtilis* KY2761 to grape seed extract is 2-8:0.2-0.5, and the effective bacterial concentration of *Bacillus amyloliquefaciens* KY997 and *Bacillus subtilis* KY2761 is 5.0 × 10⁻⁶. 9 CFU / g.

[0014] In the above-mentioned compound preparation, preferably, the mass ratio of Bacillus amyloliquefaciens KY997 and Bacillus subtilis KY2761 to grape seed extract is 4:0.4.

[0015] The above-mentioned compound preparations are used in the preparation of drugs for the treatment or prevention of porcine reproductive and respiratory syndrome virus infection.

[0016] The application of the above-mentioned compound preparation in the preparation of pig feed additives.

[0017] A therapeutic drug for porcine reproductive and respiratory syndrome virus infection, comprising the above-mentioned compound preparation.

[0018] The dosage forms of the drugs mentioned above are all pharmaceutically acceptable, including but not limited to tablets, capsules, granules, injections, powders, or drops.

[0019] Compared with the prior art, the advantages of the present invention are as follows:

[0020] This invention provides a compound preparation of Bacillus containing grape seed extract. This compound preparation contains two strains of Bacillus that were independently screened in this application and exhibit significant inhibitory effects against porcine reproductive and respiratory syndrome virus (PRRSV) infection. Furthermore, the combination of these Bacillus strains and grape seed extract demonstrates a synergistic effect. Results show that this compound preparation exhibits significant anti-PRRSV activity both in vivo and in vitro. The Bacillus strains contained within have advantages such as being non-toxic, residue-free, antiviral, growth-promoting, green and safe, and having low production costs and high economic benefits. Experiments have confirmed that the compound preparation of this invention achieves a 90%–100% efficacy in preventing and controlling porcine reproductive and respiratory syndrome virus (PRRSV), and also promotes growth and enhances immunity in animals, making it suitable for the prevention and control of PRRSV infection. Attached Figure Description

[0021] Figure 1 Gram staining microscopy and scanning electron microscopy results for strain KY997;

[0022] Wherein: A: Optical microscope examination results of strain KY997 (10×100); B: Electron microscope examination results of strain KY997.

[0023] Figure 2 Gram staining microscopy and scanning electron microscopy results for strain KY2761;

[0024] Wherein: A: Optical microscope examination results of strain KY2761 (10×100); B: Electron microscope examination results of strain KY2761.

[0025] Figure 3 Phylogenetic tree of 16S rDNA sequences of strains KY997 and KY2761.

[0026] Where: A: is the phylogenetic tree of the 16S rDNA sequence of KY997; B: is the phylogenetic tree of the 16S rDNA sequence of KY2761.

[0027] Figure 4 The results show the stress resistance of strains KY997 and KY2761;

[0028] Where: A: acid resistance results of KY997 and KY2761; B: bile salt resistance results of KY997 and KY2761.

[0029] Figure 5 The effect of the compound preparation on the lesions of tissues and organs of PRRSV-infected pigs. Detailed Implementation

[0030] To better understand the content of this invention, the following detailed embodiments further illustrate the invention. It should be understood that the embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0031] Example 1:

[0032] Screening and identification of strains resistant to PRRSV infection

[0033] 1.1 Culture of Marc-145 cells

[0034] Frozen Marc145 cells were removed from the liquid nitrogen tank and rapidly thawed in a 37°C water bath. The cell suspension was then centrifuged in centrifuge tubes, and the supernatant was discarded. The cells were resuspended in DMEM complete medium containing 10% serum and transferred to T25 cell culture flasks. The flasks were placed in a 37°C incubator containing 5% CO2. Once a uniform monolayer of cells had formed, the supernatant was discarded. The cells were washed three times with PBS, and then digested with trypsin for 3-5 minutes. Digestion was stopped with DMEM complete medium, and the cells were centrifuged, the supernatant was discarded, and the cells were resuspended in fresh complete medium. The cells were then inoculated into new cell culture flasks or plates for later use.

[0035] 1.2 Preparation of Fermentation Broth for Strains

[0036] Wuhan Keqian Biotechnology Co., Ltd. preserved 100 strains of Bacillus glycerol for streak culture. Then, a single colony was picked and inoculated into 50ml of NB liquid medium. After incubation at 37℃ for 24h on a shaker, the culture was centrifuged at 3500r / min for 10min. The supernatant was collected and stored at -80℃ for later use.

[0037] 1.3 Treatment of Marc 145 cells with anti-PRRSV fermentation broth of the strain

[0038] Marc145 cells were evenly seeded into 12-well cell culture plates and cultured in a constant-temperature cell culture incubator. Once a uniform monolayer of cells was formed, the supernatant was discarded, and the cells were washed three times with sterile PBS before being inoculated into DMEM medium containing different Bacillus fermentation broths at a ratio of 1:100. A blank control group without fermentation broth was also included. After 6 hours, the supernatant was discarded, and the cells were washed three times with PBS before being inoculated into a mixture of DMEM medium containing different Bacillus fermentation broths and PRRSV HP-WH3 virus (MOI=0.1). After 2 hours of incubation, the supernatant was discarded, the cells were washed three times with PBS, and then inoculated into fresh medium containing different Bacillus fermentation broths. The 12-well cell culture plates were then incubated in a constant-temperature cell culture incubator for 48 hours.

[0039] 1.4 Determination of viral load

[0040] The 12-well cell plates cultured for 48 hours were repeatedly frozen and thawed three times at -80°C to ensure complete release of viral particles from the cells. The supernatant was then collected by centrifugation for viral load detection. The detection method followed the People's Republic of China National Standard for Porcine Reproductive and Respiratory Syndrome (PRRS) detection. Primer sequence:

[0041] F: 5'-TTGCTAGGCCGCAAGTAC-3';

[0042] R: 5'-ACGCCGGACGACAAATGC-3';

[0043] P: 5'-(FAM)CTGGCCCCTGCCCACCAC(BHQ1)-3'.

[0044] According to the standard curve: y = -3.316X + 37.771, R 2 =1; Inhibition rate = (copy number) 对照组 - Copy number 芽孢杆菌组 ) / number of copies 对照组 *100% is used to calculate the inhibition rate.

[0045] The results are shown in Table 1. Using PRRSV HP-WH3 (GenBank: HM853673.2) as an indicator strain, 100 Bacillus strains were initially screened. The results showed that compared with the control group, 12 Bacillus strains had significant anti-PRRSV activity in their fermentation broth, with inhibition rates greater than 90%. Further screening of the 12 Bacillus strains revealed that two strains not only significantly inhibited the highly pathogenic strain WH3, but also significantly inhibited the proliferation of the attenuated PRRSV strain NAD30 (CN119899783B) in Marc145 cells, with inhibition rates greater than 95%, exhibiting broad-spectrum antiviral activity. These strains were named KY997 and KY2761.

[0046] Table 1. Screening for PRRSV (HP-WH3) spore-forming bacteria

[0047] .

[0048] 1.5 Biological characteristics and identification of strains KY997 and KY2761

[0049] Based on comprehensive colony characteristics, physiological and biochemical properties, and molecular biological detection results, KY997 was identified as *Bacillus amyloliquefaciens*, and KY2761 as *Bacillus subtilis*. Their characteristics are as follows:

[0050] (1) Colony characteristics and bacterial cell morphology

[0051] Tests revealed that strains KY997 and KY2761 are both Gram-positive bacteria with short rod-shaped cells. KY997 is encapsulated in mucus, while KY2761 is not. Both strains can produce spores, with plump spores at the midpoint. On NB solid medium, the colonies are milky white or slightly yellowish-white. In the early stages, they are concave or round, with a moist, opaque surface and neat edges. Later, they grow into non-transparent colonies with wrinkles in the middle and irregular edges.

[0052] (2) Identification of physiological and biochemical characteristics

[0053] Single colonies were picked and inoculated into microbial assay tubes for the determination of physiological and biochemical parameters. Classification and determination were performed according to Bergey's Manual of Bacteriological Identification and the Manual of Systematic Identification of Common Bacteria. The results are shown in Tables 2 and 3. Strains KY997 and KY2761 were preliminarily identified as belonging to the genus *Bacillus*.

[0054] Table 2. Physiological and biochemical test results of strain KY997

[0055] .

[0056] Note: + indicates positive, – indicates negative.

[0057] Table 3. Physiological and biochemical test results of strain KY2761

[0058] .

[0059] Note: + indicates positive, – indicates negative.

[0060] (4) Phylogenetic tree of strains KY997 and KY2761

[0061] BLAST analysis was performed on 16S rDNA samples from strains KY997 and KY2761, and phylogenetic trees were constructed using MEGA 7.0 with the Neighbor-Joining method, selecting standard strains with high homology (e.g., [example missing]). Figure 3 ).

[0062] Based on the phylogenetic tree and physiological and biochemical characteristics analysis of 16S rDNA, and combined with the genomic species information comparison results of strain KY997, strain KY997 was identified as Bacillus amyloliquefaciens; strain KY2761 was identified as Bacillus subtilis.

[0063] These two strains were deposited at the China Center for Type Culture Collection (CCTCC) on January 22, 2026, at Wuhan University, Wuhan, China. Strain KY997 is classified as *Bacillus amyloliquefaciens* KY997, with accession number CCTCC NO: M 2026187; strain KY2761 is classified as *Bacillus subtilis* KY2761, with accession number CCTCC NO: M 2026186.

[0064] 1.3 Enzyme production capacity of strains KY997 and KY2761

[0065] (1) Protease activity

[0066] Two μL of KY997 and KY2761 bacterial cultures were vertically dropped onto NB solid medium containing 10% skim milk powder, respectively. After 10 min, the cultures were incubated at 37℃ for 24 h and 5 d, respectively. The presence of a distinct proteolytic zone around the strains was observed, and the diameter of the zone was measured to assess the protease production capacity of the strains. The results showed that the diameters (D) of the proteolytic zones for KY997 were 14.10±0.66 mm and 34.01±0.85 mm, respectively, while those for KY2761 were 13.81±0.40 mm and 33.11±0.57 mm, respectively. This indicates that both Bacillus KY997 and KY2761 can produce proteases, with a more prominent protease production characteristic. As feed additives, they can significantly improve the utilization rate of feed protein.

[0067] (2) Amylase activity

[0068] Two μL of KY997 and KY2761 bacterial cultures were vertically dropped onto NB solid medium containing 1% soluble starch. After incubation at 37°C for 24 hours, 1 mL of iodine solution was added. The presence of a clear starch-dissolving zone around the strain was observed within 1 minute, and the diameter of the dissolving zone was measured to assess the strain's amylase production capacity. The measured diameters (D) of the dissolving zones for KY997 and KY2761 were 23.13 ± 0.45 mm and 24.11 ± 0.36 mm, respectively, indicating that both Bacillus KY997 and KY2761 can produce amylase.

[0069] 1.4 Stress resistance characteristics of Bacillus KY997 and KY2761

[0070] (1) Acid resistance test

[0071] Strains KY997 and KY2761 were cultured on NB solid medium for 3 days. Colonies were washed off with 6 mL of physiological saline and thoroughly mixed. The bacterial suspension was then inoculated at a 10% inoculum into NB liquid medium at pH values ​​of 2, 3, 4, 5, 6, and 7 (initial bacterial concentration was 1.4 × 10⁻⁶). 8 (CFU / mL), with 3 replicates per group. After incubation at 37℃ with shaking for 2 hours, observe the growth and count the colonies.

[0072] The results show that ( Figure 4 In section A), within the pH range of 2–7, the viable count of strain KY997 was consistently 2 × 10⁻⁶. 9 CFU / mL or higher, viable count of strain KY2761 was 1×10⁻⁶. 9 The presence of CFU / mL or higher indicates no significant difference, suggesting that Bacillus KY997 and KY2761 in this invention have a high survival rate in a low-pH gastric acid environment, allowing a sufficient number of live bacteria to pass through the stomach and reach the intestines to exert their effects.

[0073] (2) Bile salt tolerance test

[0074] Strains KY997 and KY2761 were cultured on NB solid medium for 3 days. Colonies were washed off with 6 mL of physiological saline and thoroughly mixed. The bacterial suspensions were then inoculated at 10% inoculum into NB liquid medium at concentrations of 0.0%, 0.1%, 0.2%, 0.3%, and 0.4%, respectively (initial bacterial concentration was 1.4 × 10⁻⁶). 8 (CFU / mL), with 3 replicates per group. After incubation at 37℃ with shaking for 2 hours, observe the growth and count the colonies.

[0075] The results show that ( Figure 4 In the B group, after 2 hours of tolerance in a liquid with a bile salt concentration of 0.1%-0.4% (mass percentage), the viable count of strain KY997 and the zero-bile-salt group (bile salt concentration of 0.0%) were both above 2×10⁻⁶.9 CFU / mL or higher, the viable count of strain KY2761 and the zero bile salt group (bile salt concentration of 0.0%) were both above 1×10⁻⁶. 9 With CFU / mL and no significant difference, it is indicated that both Bacillus KY997 and KY2761 can tolerate high bile salt environments.

[0076] Example 2:

[0077] Fermentation process of strains KY997 and KY2761

[0078] After activating Bacillus KY997 or KY2761, a single colony was picked and inoculated into NB liquid medium and cultured at 37℃ and 220r / min for 12h to obtain seed culture. The seed culture was then inoculated into a 30 L small industrial fermenter at a ratio of 1:1000. The fermentation medium consisted of: 25g corn starch, 10g peptone, 2g soybean meal powder, 3g calcium chloride, 2g sodium chloride, 2g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.3g manganese sulfate, 0.002g defoamer, and 1000ml distilled water. The pH was adjusted to 7.0 with sodium hydroxide. The fermentation was autoclaved at 121℃ for 15 minutes and incubated at 37℃ with a pH of 6.5±0.2. The initial stirring speed was 150 rpm, and the stirring speed was gradually increased as the dissolved oxygen level decreased, eventually reaching 700 rpm. The initial aeration rate was 1 m³ / h, and the aeration rate was gradually increased as the fermentation time increased, the fermentation density increased, and the dissolved oxygen level decreased, controlling the dissolved oxygen (DO) at 5%-10%, until the aeration rate reached 8 m³ / h. 3 / h, under these conditions, culture for 24 hours and then place in a tank. Once the transspore rate reaches 90%, the fermentation broth of Bacillus amyloliquefaciens KY997 or KY2761 is obtained.

[0079] The fermentation broth obtained using the above fermentation method can achieve an effective bacterial count of 80-100 × 10⁻⁶ for either KY997 or KY2761. 9 The concentration of CFU / mL was then measured, followed by spray drying of the fermentation broth. After spray drying, the effective bacterial concentration of strains KY997 or KY2761 was 80-100 × 10⁻⁶ CFU / mL. 9 CFU / g, store at 4℃ for later use.

[0080] Example 3:

[0081] Preparation of Bacillus compound preparation containing grape seed extract and other control groups:

[0082] Compound preparation group:

[0083] (1) The bacterial powders of strains KY997 and KY2761 prepared in Example 2 were mixed at a mass ratio of 1:1 to obtain a Bacillus complex. The total bacterial concentration of the complex was 5.0 × 10⁻⁶. 9 CFU / g.

[0084] (2) The Bacillus complex prepared in step (1) and grape seed extract are mixed in a mass ratio of 2:0.5, 4:0.4, 6:0.3 or 8:0.2 to obtain compound preparations, which are named compound preparation group 1, compound preparation group 2, compound preparation group 3 and compound preparation group 4 respectively.

[0085] Grape seed extract is an aqueous extract of grape seeds, which is obtained by drying, pulverizing and sieving grape seeds, extracting twice at a ratio of 1:10 between the material and the liquid (i.e., pure water), combining the filtrates, heating and refluxing, ultrafiltration to obtain the extract, concentrating the obtained extract and spray drying it, and is also used in the following examples.

[0086] Comparative Example 1:

[0087] Bacillus complex: This comparative example contains only the Bacillus complex prepared in step (1) above, and the total bacterial concentration of the complex is 5.0 × 10⁻⁶. 9 CFU / g.

[0088] Comparative Example 2:

[0089] Grape seed extract group: The grape seed extract group in this comparative example contains only grape seed extract.

[0090] Comparative Example 3:

[0091] Group KY997: Contains only the Bacillus amyloliquefaciens KY997 spray-dried bacterial agent prepared in step (1) above, with a bacterial concentration of KY997 of 5.0 × 10⁻⁶. 9 CFU / g.

[0092] Comparative Example 4:

[0093] Group KY2761: Contains only the Bacillus amyloliquefaciens KY2761 spray-dried bacterial agent prepared in step (1) above, with a bacterial concentration of KY2761 of 5.0 × 10⁻⁶. 9 CFU / g.

[0094] Example 4:

[0095] Preparation of animal models and animal experiments

[0096] 1.1 Experimental Animals

[0097] Fifty PRRSV-negative piglets (weaned piglets aged 30-42 days) were randomly divided into 10 groups:

[0098] The study included a blank control group, a challenge control group, compound preparation group 1, compound preparation group 2, compound preparation group 3, compound preparation group 4, Bacillus complex group, KY997 group, KY2761 group, and grape seed extract group.

[0099] All animals were housed in animal facilities that met GMP animal testing standards. After 3 days of acclimatization, they were infected with the highly pathogenic PRRSV strain WH3 (CN119899783B) via nasal drops and intramuscular injection. The infectious dose was 10. 4.5 TCID 50 During the experiment, the mental state of the experimental animals was constantly observed, and cases of illness and death were recorded.

[0100] The blank control group and the challenge control group were fed with basic piglet feed; Compound preparation group 1 was fed with Compound preparation group 1 of Example 3, with the amount of compound preparation added at 2000g / T feed; Compound preparation group 2 was fed with Compound preparation group 2 of Example 3. The compound preparation was added at a rate of 2000 g / T of feed; Compound preparation group 3 was fed with the compound preparation group 3 of Example 3, and the compound preparation was added at a rate of 2000 g / T of feed; Compound preparation group 4 was fed with the compound preparation group 4 of Example 3, and the compound preparation was added at a rate of 2000 g / T of feed; Grape seed extract group was fed with grape seed extract from Comparative Example 2 of Example 3, and the grape seed extract preparation was added at a rate of 1000 g / T of feed; Bacillus complex group was fed with Comparative Example 1 of Example 3, and the Bacillus complex was added at a rate of 2000 g / T of feed; KY997 group was fed with Comparative Example 3 of Example 3, and the KY997 was added at a rate of 2000 g / T of feed; KY2761 group was fed with Comparative Example 4 of Example 3, and the KY2761 was added at a rate of 2000 g / T of feed.

[0101] 1.2 Measurement Indicators

[0102] 1.2.1 Impact of clinical incidence

[0103] Following PRRSV infection, the body temperature (rectal temperature) and weight of piglets were measured. Clinical characteristics of infected piglets were observed and recorded, including appetite, mental state, body surface condition, respiratory symptoms, and neurological symptoms. Clinical indicators were scored according to Table 4, where the score for deaths was calculated as total clinical score + 5 points = 20 points. The clinical assessment criteria are shown in Table 4. The results are shown in Table 5. Compared with the blank control group, the challenged control group showed a significantly higher incidence of clinical symptoms on day 3 after PRRSV challenge (P<0.05), exhibiting typical PRRSV infection symptoms such as depression, decreased appetite, elevated body temperature, and cyanosis of the ears in some pigs. Deaths began to occur after 14 days. The compound bacteria group, grape seed extract group, and single bacteria group showed some relief of clinical symptoms compared to the challenged control group, but deaths still occurred. No deaths occurred in compound preparation groups 1, 2, 3, and 4, and the blank control group after PRRSV infection, and clinical symptoms were significantly reduced (P<0.05).

[0104] Table 4. Assessment criteria for overall clinical symptoms in PRRSV-infected piglets

[0105] .

[0106] Table 5 Clinical symptom scores of PRRSV-infected piglets

[0107] .

[0108] Note: * indicates a significant difference compared to the challenge control group (p < 0.05), and no indication indicates no significant difference (p > 0.05). The same applies below.

[0109] 1.2.2 Effect of compound preparation on mortality rate of PRRSV-infected pigs

[0110] Following PRRSV infection, the health status of pigs was observed daily, and their clinical condition was recorded and clinically assessed. The results are shown in Table 6: the survival rate of the challenge control group was 20% after challenge; the survival rates of the single-bacterial group (KY2761), the Bacillus complex group, and the grape seed extract group were 80%; the survival rate of the single-bacterial group (KY997) was 60%; and the survival rates of compound preparation groups 1, 2, 3, and 4, and the blank control group all reached 100%. The experimental results indicate that the combination of compound Bacillus and grape seed extract is more effective than intervention by a single substance, and exhibits a synergistic effect.

[0111] Table 6. Effects of Bacillus preparations on mortality rates in PRRSV-infected pigs.

[0112] .

[0113] 1.2.3 Effect of the compound preparation on body temperature in PRRSV-infected pigs

[0114] Starting from day 0 of PRRSV infection, the pigs' body temperature was measured on day 3, and then every week until day 42. The results are shown in Table 7. In the challenge control group, the overall body temperature of the pigs rose on day 3 of infection, reaching 41℃ on day 7, and remained above 41℃ until day 14. Three pigs died between day 7 and day 14 of infection. In the treatment groups, the pigs in each group began to show body temperatures above 40℃ on day 3 of infection, which remained above 40℃ until day 14, after which fluctuated between 40℃ and 39℃, and finally stabilized below 40℃. No deaths occurred in the compound preparation groups 1, 2, 3, and 4.

[0115] Table 7. Average body temperature of pigs in each group after PRRSV infection.

[0116] .

[0117] 1.2.4 Effect of the compound preparation on the body weight of PRRSV-infected pigs

[0118] The experiment was conducted on day 0 of PRRSV infection. All pigs were weighed on an empty stomach on day 42, and the weight was calculated until day 42. Mean body weight (BW) and mean daily gain (ADG) were calculated.

[0119] Average body weight (BW) = Body weight at the end of the experiment / Number of pigs in each group;

[0120] Average daily gain (ADG) = (body weight at the end of the experiment - body weight at the beginning of the experiment) / (number of days in the experiment × number of pigs in each group);

[0121] The results are shown in Table 8. Pigs in the challenge control group showed slow weight gain after PRRSV infection. The average weight of the compound preparation groups 2, 3 and 4 increased significantly (P<0.05), with compound preparation group 2 showing the best weight gain.

[0122] Table 8. Average body weight of pigs in each group after PRRSV infection.

[0123] .

[0124] 1.2.5 Determination of serum viral load in PRRSV-infected pigs by the compound preparation

[0125] Starting from day 0 of PRRSV infection, blood was collected from the anterior vena cava of infected pigs on day 3 and every 7 days. Serum was incubated at 37℃ for 2 hours, then transferred to 4℃ for 1-2 hours, and centrifuged at 3000 rpm for 10 minutes at 4℃. The supernatant was collected, aliquoted, and stored at -80℃ for subsequent serum viral load testing. The results are shown in Table 9. In the challenge control group, serum viral load significantly increased after PRRSV infection (P<0.05), with serum CT values ​​remaining around 20 from day 3 to day 28. Four pigs died during this period. In the treatment groups, serum viral load remained high from day 7 to 14 post-infection, gradually decreasing after day 21. No deaths occurred in the compound preparation groups 1, 2, 3, and 4 during the infection period, and the compound preparation effectively reduced serum PRRSV viral load, demonstrating a good antiviral effect.

[0126] Table 9. Effects of the compound preparation on viral load in PRRSV-infected porcine serum.

[0127] .

[0128] 1.2.6 Effect of the compound preparation on viral load in PRRSV-infected porcine anal swabs

[0129] Anal swabs were collected from infected pigs starting on day 0 of PRRSV infection, and then every 7 days thereafter for a total of 42 days. The results are shown in Table 10. The viral load in the anal swabs of pigs in the challenge control group increased significantly from day 3 to day 14 after PRRSV infection (P<0.05). The viral load in the anal swabs of the treatment group was significantly lower than that in the challenge control group, and the viral load gradually decreased after day 21.

[0130] Table 10 Effect of the compound preparation on viral load in PRRSV-infected pig anal swabs

[0131] .

[0132] 1.2.7 Effect of the compound preparation on viral load in pharyngeal and nasal swabs of PRRSV-infected pigs

[0133] Pharyngeal and nasal swabs were collected from infected pigs starting on day 0 of PRRSV infection, and then every 7 days thereafter, for a total of 42 days. The results are shown in Table 11. The viral load in the pharyngeal and nasal swabs of the challenged control group was significantly higher (P<0.05), and the CT value was below 30 from day 3 to day 35 after infection. The CT value of the treatment group started to decrease on day 3 after infection and then gradually increased. During this period, no deaths occurred in groups 1, 2, 3, and 4 of the compound preparation.

[0134] Table 11 Effect of the compound preparation on viral load in pharyngeal and nasal swabs of PRRSV-infected pigs

[0135] .

[0136] 1.2.8 Effects of the compound preparation on lung tissue lesions in PRRSV-infected pigs

[0137] Autopsy results (such as) Figure 5 The results showed that the challenge control group had obvious solid lesions in the lungs of pigs after PRRSV infection (dark red consolidation area in the lower right corner). The degree of tissue damage in the lungs of piglets in each treatment group was significantly reduced compared with the challenge control group. Among them, the lung tissue symptoms of compound preparation groups 1, 2, 3 and 4 basically reached the anatomical level of the blank control group.

[0138] In summary, the compound preparation of the present invention can effectively treat or prevent porcine reproductive and respiratory syndrome, while also enhancing the body's immunity and promoting growth. Furthermore, the combination of Bacillus amyloliquefaciens, Bacillus subtilis, and grape seed extract is more effective than intervention by any single substance, and exhibits a synergistic effect.

[0139] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A compound preparation of Bacillus containing grape seed extract, wherein the compound preparation comprises Bacillus amyloliquefaciens KY997, Bacillus subtilis KY2761 and grape seed extract, wherein the preservation number of Bacillus amyloliquefaciens KY997 is CCTCC NO: M2026187 and the preservation number of Bacillus subtilis KY2761 is CCTCC NO: M2026186.

2. The compound preparation according to claim 1, characterized in that, The effective bacterial concentrations of Bacillus amyloliquefaciens KY997 and Bacillus subtilis KY2761 are the same.

3. The compound preparation according to claim 1, characterized in that, The grape seed extract mentioned above is a water extract of grape seeds.

4. The compound preparation according to claim 3, characterized in that, The method for preparing the grape seed extract includes: extracting crushed and sieved grape seeds multiple times at a material-to-water ratio of 1:5-20, combining the filtrates, heating and refluxing, ultrafiltration, concentrating, and then spray drying to obtain the extract.

5. The compound preparation according to claim 1, characterized in that, The mass ratio of *Bacillus amyloliquefaciens* KY997 and *Bacillus subtilis* KY2761 to grape seed extract is 2-8:0.2-0.5, and the effective bacterial concentration of *Bacillus amyloliquefaciens* KY997 and *Bacillus subtilis* KY2761 is 5.0 × 10⁻⁶. 9 CFU / g.

6. The compound preparation according to claim 5, characterized in that, The mass ratio of Bacillus amyloliquefaciens KY997 and Bacillus subtilis KY2761 to grape seed extract is 4:0.

4.

7. The use of the compound preparation according to claim 1 in the preparation of a medicament for treating or preventing porcine reproductive and respiratory syndrome virus infection.

8. The application of the method described in claim 1 in the preparation of pig feed additives.

9. A therapeutic drug for porcine reproductive and respiratory syndrome virus infection, comprising the above-mentioned compound preparation.

10. The application according to claim 8 or the medicament according to claim 9, wherein the dosage form of the medicament is any pharmaceutically acceptable dosage form.

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