Patent ID: 12194072

DETAILED DESCRIPTION OF EMBODIMENTS

Technical solutions in embodiments of the disclosure are clearly and completely described in conjunction with the embodiments of the disclosure below.

The embodiments of the disclosure are described in detail in conjunction with actual situations.

Embodiment 1 Identification of the Anti-ASFV Effect of a Traditional Chinese Medicine Compound

The traditional Chinese medicine compound of the disclosure is made from the following raw materials in parts by weight: 20 g ofCodonopsis pilosula,20 g ofAtractylodes macrocephala,20 g ofWolfiporia cocos,9 g ofAuckiandialappa LappaDecne., 15 g ofTerminalia chebula,9 g ofAconitum carmichaeli,15 g ofMyristica fragrans,15 g ofCynanchum otophyllum,40 g ofDioscorea polystachyaand 5 g ofZiziphus jujuba. The raw materials are mixed and crushed to obtain mixed powder. The mixed powder is fermented withBacillus subtilis, Saccharomyces cerevisiae, BifidobacteriumandLactobacillus acidophilusin stages to obtain the traditional Chinese medicine compound, and contents of theBacillus subtilis, theSaccharomyces cerevisiae, theBifidobacteriumand theLactobacillus acidophilusare greater than or equal to 1.0×107CFU/L. The virus strain used in the experiment is the ASFV gene type II strain (ASFV CN/SC/2019), which is an isolated strain from the African swine fever regional laboratory of the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, a virus potency is 5×107TCID50/mL, a multiplicity of infection (MOI) of the ASFV CN/SC/2019 virus strain is 0.1.

1. Kidney cells of AfricanChlorocebus sabaeus(Vero cells) are cultivated as follows: 5 mL of serum (purchased from Biological industries) and 500 microliters (L) of double antibody (i.e., penicillin and streptomycin) are added into each 45 mL of a Dulbecco's modified Eagle's medium (DMEM), and the DMEM added with the serum and the double antibody is stored in a refrigerator at 4° C. after mixing evenly.

2. The Vero cells are divided into five groups that are a treatment group with a low dose of traditional Chinese medicine compound, a treatment group with a medium dose of traditional Chinese medicine compound (i.e., fermented liquid of Chinese medicine), a treatment group with a high dose of traditional Chinese medicine compound, a ribavirin group and a virus alone infection group (i.e., virus control), and each group has three replicates. In all groups, serum-free but 0.1% pancreatin-containing culture medium is used. The Vero cells with good growth status are inoculated at a 12-well plate, the serum-free but 0.1% pancreatin-containing culture medium is discarded when a cell confluence degree reaches 90%, and then the Vero cells are washed with phosphate-buffered saline (PBS) twice. The Vero cells are infected with the ASFV (MOI=0.1) in the treatment groups (i.e., the treatment group with the low dose of traditional Chinese medicine compound, the treatment group with the medium dose of traditional Chinese medicine compound, the treatment group with the high dose of traditional Chinese medicine compound) and the virus alone infection group, after 1 h of virus adsorption, the Vero cells are washed with PBS twice. The treatment groups are added with 1.5 mL of the traditional Chinese medicine compound (concentrations are 250 micrograms per milliliter abbreviated as g/mL, 500 μg/mL and 1000 g/mL, respectively) and continuously cultivated for 24 h. Pathological conditions of the Vero cells are observed and photographed under a microscope.

3. As samples, the 12-well plate with the Vero cells from step 2 is collected and placed at −80° C. for three freeze-thaw cycles. DMEM without serum is used to dilute the samples 10 times continuously to obtain samples with 10 dilutions; each dilution is repeated in 8 wells and inoculated until they are complete for culture, and the cell plates inoculated with the samples are cultured under 37° C. and 5% carbon dioxide (CO2) conditions. The pathological condition in each cell culture well is observed and recorded daily for 5 consecutive days. TCID50of the virus solution is calculated according to a Reed-Muench formula. TCID50=logarithm of reciprocal of high critical dilution for 50% infection+distance ratio×logarithm of dilution coefficient.

As shown inFIG.1, the result shows that compared to the virus alone infection group, a virus titer (P<0.05) in the treatment group with low dose traditional Chinese medicine compound is significantly decreased, virus titers (P<0.01) in the treatment group with medium dose traditional Chinese medicine compound and the treatment group with high dose traditional Chinese medicine compound are extremely significantly decreased. Therefore, the traditional Chinese medicine compound's antiviral effect is dependent

Embodiment 2 Influence of Tetra-Strain Fermentation Product on the Activity of the ASFV

The tetra-strain fermentation product (i.e., traditional Chinese medicine compound fermented preparation) of the disclosure is made from the following raw materials in parts by weight: 10 g ofCodonopsis pilosula,10 g ofAtractylodes macrocephala,10 g ofWolfiporia cocos,3 g ofAuckiandialappa LappaDecne., 5 g ofTerminalia chebula,3 g ofAconitum carmichaeli,5 g ofMyristica fragrans,9 g ofCynanchum otophyllum,20 g ofDioscorea polystachyaand 2 g ofZiziphus jujuba. The raw materials are mixed and crushed to obtain mixed powder. The mixed powder is placed into a primary fermenter, and theBacillus subtilisand theSaccharomyces cerevisiaeare inoculated in the primary fermenter to obtain a mixture; the mixture is stirred continuously at 37° C. for 24 h. During stirring, air is introduced into the primary fermenter containing the mixture for aerobic fermentation to obtain an intermediate product. Half of the intermediate product is placed into a secondary fermenter, and anaerobic fermentation microorganismsLactobacillus acidophilusandBifidobacteriumare added into the secondary fermenter with half of the intermediate product. The secondary fermenter is placed into a fermentation bag with a one-way exhaust valve for anaerobic fermentation at 33° C. for 3 d to obtain a fermented mixture. Contents of theBacillus subtilis, theSaccharomyces cerevisiae, theBifidobacteriumand theLactobacillus acidophilusare greater than or equal to 1.0×107CFU/L. Fermentation effects of different combinations of theBacillus subtilis, Saccharomyces cerevisiae, BifidobacteriumandLactobacillus acidophilusare compared, and ratios of the different combinations are 1:1:1:1, 1:2:2:1, 1:1:2:2 and 2:1:1:1, respectively. The fermented mixture is directly aseptically filled in a fluid dosage form. A low-temperature spray drying treatment is performed on the fermented mixture to obtain the traditional Chinese medicine preparation fermented with probiotics. The virus strain used in the experiment is the ASFV gene type II strain (ASFV CN/SC/2019), which is an isolated strain from the African swine fever regional laboratory of the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, a virus potency is 5×107TCID50/mL, MOI of the ASFV CN/SC/2019 virus strain is 0.1.

1. Kidney cells of AfricanChlorocebus sabaeus(Vero cells) are cultivated as follows: 5 mL of serum (purchased from Biological industries) and 500 μL of double antibody (i.e., penicillin and streptomycin) are added into each 45 mL of DMEM, and the DMEM added with the serum and the double antibody is stored in a refrigerator at 4° C. after mixing evenly.

2. The Vero cells are divided into five groups that are fermentation groups with the different combinations of the four bacteria with ratios of 1:1:1:1, 1:2:2:1, 1:1:2:2 and 2:1:1:1, and a pure traditional Chinese medicine compound group, and each group has three replicates. In all groups, serum-free but 0.1% pancreatin-containing culture medium is used. The Vero cells with good growth status are inoculated at a 12-well plate, the serum-free but 0.1% pancreatin-containing culture medium is discarded when a cell confluence degree reaches 90%, and then the Vero cells are washed with PBS twice. The Vero cells are infected with the ASFV (MOI=0.1) in the fermentation groups and the pure traditional Chinese medicine compound group; after 1 h of virus adsorption, the Vero cells are washed with PBS twice. The fermentation and pure traditional Chinese medicine compound groups are added with 1.5 mL of the traditional Chinese medicine compound (concentrations are 500 μg/mL). They are continuously cultivated for 24 hours to collect samples.

3. DMEM without serum is used to dilute the samples 10 times continuously to obtain samples with 10 dilutions; each dilution is repeated in 8 wells and inoculated until they are complete for culture, and the cell plates inoculated with the samples are cultured under 37° C. and 5% CO2conditions. The pathological condition in each cell culture well is observed and recorded daily for 5 consecutive days. TCID50of the virus solution is calculated according to the Reed-Muench formula. TCID50=logarithm of reciprocal of high critical dilution for 50% infection+distance ratio×logarithm of dilution coefficient.

As shown inFIG.2, the fermentation effects of the different combinations ofBacillus subtilis, Saccharomyces cerevisiae, BifidobacteriumandLactobacillus acidophilusare compared, the ratios of the different combinations are 1:1:1:1, 1:2:2:1, 1:1:2:2 and 2:1:1:1, respectively, and the different combinations are labelled as ASFV+1, ASFV+2, ASFV+3 and ASFV+4. It can be seen from the result that the antiviral effects of the fermentation groups are more significant than the pure traditional Chinese medicine compound group, and the ASFV+4 group has the optimal antiviral effect. That is, when the ratio of the combination is 2:1:1:1, the virus titer is the lowest, and the antiviral effect is optimal.

Embodiment 3 Actual Prevention Effect of the Tetra-Strain Fermentation Product on the ASFV

A traditional Chinese medicine compound fermented preparation for treating ASFV is provided. The traditional Chinese medicine compound fermented preparation is made from the following raw materials in parts by weight: 15 g ofCodonopsis pilosula,15 g ofAtractylodes macrocephala,15 g ofWolfiporia cocos,6 g ofAuckiandialappa LappaDecne., 10 g ofTerminalia chebula,6 g ofAconitum carmichaeli,10 g ofMyristica fragrans,12 g ofCynanchum otophyllum,30 g ofDioscorea polystachyaand 3 g ofZiziphus jujuba. The raw materials are mixed and crushed to obtain mixed powder. The mixed powder is placed into a primary fermenter, and theBacillus subtilisand theSaccharomyces cerevisiaeare inoculated in the primary fermenter to obtain a mixture; the mixture is stirred continuously at 38° C. for 48 h. During stirring, air is introduced into the primary fermenter containing the mixture for aerobic fermentation to obtain an intermediate product. Half of the intermediate product is placed into a secondary fermenter, anaerobic fermentation microorganismsLactobacillus acidophilusandBifidobacteriumare added into the secondary fermenter with half of the intermediate product, and the secondary fermenter is placed into a fermentation bag with one-way exhaust valve for anaerobic fermentation at 37° C. for 2 d to obtain a fermented mixture. Contents of theBacillus subtilis, theSaccharomyces cerevisiae, theBifidobacteriumand theLactobacillus acidophilusare greater than or equal to 1.0×107CFU/L. The fermented mixture is directly aseptically filled in a fluid dosage form. A low-temperature spray drying treatment is performed on the fermented mixture to obtain the traditional Chinese medicine preparation fermented with probiotics.

A pig farm in Nanyang, Henan, has over 3000 sows. From April to June 2021, an ASF epidemic broke out in the surrounding pig farms of this pig farm, which once caused more than 90% of the pig farms to close down and clear the fields. The site selection of the pig farm has a reasonable layout, scientific pig farm management, and strict disinfection and prevention measures for people, cars and animals entering the farm. The pig farm controls biological safety well and focuses on protecting susceptible pig populations. The traditional Chinese medicine preparations were mixed into pig feed for feeding the pig based on the weight of freeze-dried traditional Chinese medicine compound once a day. The feeding ratio of the frozen-dried traditional Chinese medicine compound and the pig weights is 0.1 g: 1 kg, and the traditional Chinese medicine compound is used for a long time during the ASFV. At the beginning of this study, 600 sows were selected for experimental verification. The 600 sows were divided into 3 groups: the first group was blank, the second group was a traditional Chinese medicine compound fermented preparation control group, and the third group was a traditional Chinese medicine compound group, which lasted 7 days and was observed and recorded at any time.

TABLE 1Prevention effect of traditional Chinese medicinecompound and fermentation preparation thereof on sows in a pig farmNumberStartingEndDailyDeathof testingSurvivalaverageaverageweighttollGroupanimalsMortalityrateweight (kg)weight (kg)gain (g)(head)Blank group2001.5%98.5%80.887.5956.673Traditional Chinese2000.5%99.5%81.290.8613801medicine compoundfermented preparationgroupTraditional Chinese2001%99%80.689.2812402medicine compoundgroup

After 7 days of feeding, the daily weight gain of the traditional Chinese medicine compound fermented preparation group is higher than that of the traditional Chinese medicine compound group and the blank group, and the mortality rate of sows is reduced, the survival rate and the daily weight gain of sows are significantly improved.

Embodiment 4 Actual Treatment Effect of the Tetra-Strain Fermentation Product of the Traditional Chinese Medicine Compound on the ASFV

A traditional Chinese medicine compound fermented preparation for treating ASFV is provided. The traditional Chinese medicine compound fermented preparation is made from the following raw materials in parts by weight: 20 g ofCodonopsis pilosula,20 g ofAtractylodes macrocephala,20 g ofWolfiporia cocos,9 g ofAuckiandialappa LappaDecne., 15 g ofTerminalia chebula,9 g ofAconitum carmichaeli,15 g ofMyristica fragrans,15 g ofCynanchum otophyllum,40 g ofDioscorea polystachyaand 5 g ofZiziphus jujuba. The raw materials are mixed and crushed to obtain mixed powder. The mixed powder is placed into a primary fermenter, and theBacillus subtilisand theSaccharomyces cerevisiaeare inoculated in the primary fermenter to obtain a mixture; the mixture is stirred continuously at 38° C. for 48 h. During stirring, air is introduced into the primary fermenter containing the mixture for aerobic fermentation to obtain an intermediate product. Half of the intermediate product is placed into a secondary fermenter, anaerobic fermentation microorganismsLactobacillus acidophilusandBifidobacteriumare added into the secondary fermenter with half of the intermediate product, and the secondary fermenter is placed into a fermentation bag with one-way exhaust valve for anaerobic fermentation at 28° C. for 5 d to obtain a fermented mixture. Contents of theBacillus subtilis, theSaccharomyces cerevisiae, theBifidobacteriumand theLactobacillus acidophilusare greater than or equal to 1.0×107CFU/L. The fermented mixture is directly aseptically filled in a fluid dosage form, and low-temperature spray drying treatment is performed on the fermented mixture to obtain the traditional Chinese medicine preparation fermented with probiotics.

During the peak period of the ASFV from April to June 2021, the traditional Chinese medicine compound fermented preparation was used in a pig farm in Zhumadian, Henan Province, for pigs with symptoms. The traditional Chinese medicine compound fermented preparation was mixed with pig feed to feed the pig based on the weight of freeze-dried traditional Chinese medicine compound fermented preparation once a day. The feeding ratio of the frozen-dried traditional Chinese medicine compound and the pig weights is 0.2 g: 1 kg. During the ASFV, it was fed continuously for 7 days, and after symptom control, it was consolidated for 5 days. In the initial stage of this study, 300 live pigs were selected for the experiment and divided into three groups. The first group was a blank group, the second group was a traditional Chinese medicine compound fermented preparation control group, and the third was a traditional Chinese medicine compound group. Observation and recording were conducted at any time.

TABLE 2Treatment effect of traditional Chinese medicine compoundand fermentation preparation on sows in pig farmNumberDeathof testingSurvivaltollGroupanimalsMortalityrate(head)Blank group100982%98Traditional Chinese1004852%48medicine compoundfermentedpreparation groupTraditional Chinese1006238%62medicine compoundgroup

After 12 days of treatment, the traditional Chinese medicine compound fermented reparation group significantly reduced the mortality rate of live pigs and improved their survival rate.

It should be noted that the above-mentioned implementation schemes should be understood as explanatory and not limiting a scope of protection of the disclosure. The scope of protection of the disclosure shall be subject to claims. For those skilled in the art, some non-essential improvements and adjustments made to the disclosure, without departing from its essence and scope of the disclosure, still fall within the scope of protection of the disclosure.