Application of 5-aminolevulinic acid in preparing products for preventing and treating porcine epidemic diarrhea

Abstract

The present invention relates to the use of 5-aminolevulinic acid in the preparation of a product for preventing and treating porcine epidemic diarrhea. For piglets in the creep to weaning stage, the addition amount of 5-aminolevulinic acid or a pharmaceutically acceptable salt thereof in the product is 500-5000 g / ton calculated as 5-aminolevulinic acid; for piglets in the weaning to 90-day-old stage, the addition amount of 5-aminolevulinic acid or a pharmaceutically acceptable salt thereof in the product is 100-1000 g / ton of feed calculated as 5-aminolevulinic acid; for lactating sows, the addition amount of 5-aminolevulinic acid or a pharmaceutically acceptable salt thereof in the product is 100-1000 g / ton of feed calculated as 5-aminolevulinic acid. The present invention firstly finds that 5-ALA can significantly inhibit the proliferation of porcine epidemic diarrhea virus both in vivo and in vitro, and 5-ALA has a significant preventive and therapeutic effect on sows and piglets infected with epidemic diarrhea.

Description

Technical Field

[0001] The invention relates to the field of functional feed, and in particular to application of 5-aminolevulinic acid in preparing products for preventing and treating porcine epidemic diarrhea. Background Art

[0002] Porcine diarrhea is one of the most common diseases in the pig industry at home and abroad, and it has dealt a heavy blow to the pig farming industry. Porcine Epidemic Diarrhea (PED) is a highly contagious enteric disease of pigs caused by Porcine Epidemic Diarrhea Virus (PEDV), with diarrhea, vomiting and dehydration as the main symptoms. PEDV has become the main pathogenic microorganism of diarrhea in suckling piglets in my country. The virus easily infects pigs of all ages, and has a high morbidity and high mortality rate for suckling piglets, especially piglets under 7 days old, with a mortality rate of nearly 100% after infection. PEDV is mainly transmitted through feces, infected through the fecal-oral route, and can also be transmitted through the milk of sick lactating sows. Since 2010, a large-scale outbreak of multiple PEDV strains has caused huge economic losses to the pig industry.

[0003] There is no specific drug for viral diarrhea. Currently, it is mainly prevented and treated by strengthening feeding management on farms and vaccination. However, as an enveloped single-stranded positive-strand RNA virus, the characteristics of PEDV's genome and viral replication make the virus very easy to mutate, resulting in vaccines prepared with classic strains to prevent newly emerging mutant strains, and the immune effect is not ideal. Among the existing methods, symptomatic therapy, feeding microecological preparations, and the selection of sensitive antibiotics to prevent secondary bacterial infections are often used as auxiliary means to relieve the symptoms of viral diarrhea, but the cure effect is very poor. Therefore, the selection of new anti-PEDV products that are sensitive to pathogens, have high specificity, strong inhibitory effects, and good stability is an important research and development direction for the healthy development of the pig industry.

[0004] 5-aminolevulinic acid is a natural compound produced during the operation of the citric acid cycle (TCA) circuit in the mitochondria of higher cells. It is widely present in the living cells of bacteria, fungi, animals and plants. It is a non-protein amino acid and is a metabolically active physiologically active substance that is essential for the life activities of animals and plants. 5-aminolevulinic acid and its derivatives have been widely used in pesticides or fertilizers in the field of large-scale agriculture. In the field of livestock and poultry, they are also added to animal feed as feed additives, which is effective in improving animal anemia. In the field of human clinical medicine, 5-ALA is used for photodynamic therapy or photodynamic therapy, and 5-ALA is also used as a diagnostic agent for tumors. However, its inhibition of porcine epidemic diarrhea virus (PEDV) and its application effect in the prevention and treatment of porcine epidemic diarrhea have not been reported. This study explored the application prospects of 5-ALA as an anti-PEDV drug through in vitro and in vivo experiments, and developed a new use of 5-ALA. Summary of the invention

[0005] In order to solve the above problems, the present invention proposes the use of 5-aminolevulinic acid in the preparation of products for preventing and treating porcine epidemic diarrhea.

[0006] The present invention provides an application of 5-aminolevulinic acid or a pharmaceutically acceptable salt thereof in the preparation of a product for preventing and treating porcine epidemic diarrhea. For piglets in the creep stage to weaning stage, the amount of 5-aminolevulinic acid or a pharmaceutically acceptable salt thereof added to the product is 10-1500 g / ton of feed calculated as 5-aminolevulinic acid; for piglets in the weaning to 90-day-old stage, the amount of 5-aminolevulinic acid or a pharmaceutically acceptable salt thereof added to the product is 10-1500 g / ton of feed calculated as 5-aminolevulinic acid; for lactating sows, the amount of 5-aminolevulinic acid or a pharmaceutically acceptable salt thereof added to the product is 10-1000 g / ton of feed calculated as 5-aminolevulinic acid.

[0007] Furthermore, for piglets in the creep to weaning stage, the amount of 5-aminolevulinic acid or a pharmaceutically acceptable salt thereof added to the product is 1000 g / ton, calculated as 5-aminolevulinic acid.

[0008] Furthermore, for piglets from weaning to 90 days of age, the amount of 5-aminolevulinic acid or a pharmaceutically acceptable salt thereof added to the product is 500 g / ton, calculated as 5-aminolevulinic acid.

[0009] Furthermore, for lactating sows, the added amount of 5-aminolevulinic acid or a pharmaceutically acceptable salt thereof in the product is 500 g / ton in terms of 5-aminolevulinic acid.

[0010] Furthermore, the product also includes one or more of lactoferrin, organic iron, IGF-1 (insulin-like growth factor-1), and antimicrobial peptides.

[0011] Furthermore, the organic iron is glycine iron or ferrous fumarate.

[0012] The beneficial effects of the present invention are as follows:

[0013] 1. The present invention first discovered that 5-ALA can significantly inhibit the proliferation of porcine epidemic diarrhea virus both in vivo and in vitro. 5-aminolevulinic acid has a significant preventive and therapeutic effect on porcine epidemic diarrhea in sows and piglets.

[0014] 2. Animal experiments have shown that 5-aminolevulinic acid is more effective when it works synergistically with other beneficial components including lactoferrin, organic iron, IGF-1, and antimicrobial peptides in the prevention and treatment of porcine epidemic diarrhea virus infection. Among them, the effect of organic iron using glycine iron or ferrous fumarate is better.

[0015] 3. According to the technical concept of the present invention, 5-ALA can be combined with one or more of other beneficial components including lactoferrin, organic iron, IGF-1, and antimicrobial peptides to form a product for preventing and treating porcine epidemic diarrhea. The product can be a feed additive, a premix, a complete feed, or a functional aqueous solution. As a feed additive, it can be added to the feed or fed through drinking water. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Screening of safe concentration of 5-aminolevulinic acid in Example 1;

[0017] Figure 2 This is the first test result of qRT-PCR detection of PEDV virus replication level in Example 2;

[0018] Figure 3 This is the second test result of qRT-PCR detection of PEDV virus replication level in Example 2;

[0019] Figure 4 It is the IFA test result of Example 2;

[0020] Figure 5 The results of two determination tests of the virus TCID50 of Example 2;

[0021] Figure 6 This is a comparison chart of the PEDV replication level in piglet feces detected by qRT-PCR in Example 3;

[0022] Figure 7 This is a comparison chart of the PEDV replication level in the jejunum of piglets detected by qRT-PCR in Example 3. DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to the embodiments.

[0024] Example 1, Safety evaluation of 5-aminolevulinic acid acting on cells:

[0025] 1.1 Materials and reagents:

[0026] CCK-8 kit and reverse transcription reagent were purchased from Vazyme, and Probe qPCR Mix was purchased from TaKaRa;

[0027] The serum (Fetal Bovine Serum, FBS) required for cell culture was purchased from LONSERA;

[0028] Trypsin-EDTA (ET) used for digestion and inoculation was purchased from GIBCO;

[0029] DMEM was purchased from CORNING;

[0030] The 24-well cell culture plates were purchased from NEST;

[0031] LLC-PK1 (porcine kidney proximal tubule epithelial cells) and Vero cells were maintained in our laboratory;

[0032] PEDV MS strain (GenBank accession NO: MT683617) was provided by our laboratory.

[0033] 1.2 Test methods:

[0034] 5-aminolevulinic acid was dissolved in DMEM according to the instructions to prepare a mother solution with a concentration of 10 mM. After fully dissolved, it was aliquoted and stored at -80°C for later use. LLC-PK1 cells were evenly plated in a 96-well plate and cultured in a cell culture incubator (37°C, 5% CO2) until the density was about 90%. The experiment was started when the cells were washed three times with PBS solution. The same volume (200 μL) of 5-aminolevulinic acid with different concentrations was added to the six experimental groups. The concentrations of 5-aminolevulinic acid added were 0.125 μM, 0.2 μM, 1 μM, 5 μM, 25 μM and 125 μM. The control group was maintained with maintenance solution (pure DMEM containing 6 μg / ml ET). Six replicates were set for each concentration. After being treated at 37°C for 24 hours, 10 μL of CCK-8 solution was added to each well. After being treated at 37°C for 2 hours, the cells were read with an ELISA reader to calculate the cell survival rate. The difference between the experimental group and the control group was then calculated using GraphPad Prism 6. This experiment was repeated twice independently.

[0035] 1.3 Test results:

[0036] See Figure 1 When the maintenance medium was 6 μg / ml ET, the maximum safe drug concentration of 5-aminolevulinic acid on LLC-PK1 cells was 25 μM, that is, it was non-toxic to cells at a concentration of 25 μM, and its toxic effect on cells was more significant when its concentration reached 125 μM. Figure 1 A and Figure 1 B is the result of two independent repeated experiments, and the results were consistent. Therefore, the maximum safe concentration of 5-aminolevulinic acid screened out in this experiment is 25 μM.

[0037] Example 2, the inhibitory effect of 5-aminolevulinic acid on PEDV:

[0038] According to the safe concentration of 5-aminolevulinic acid screened out, the concentration of 5-aminolevulinic acid was set to 1μM, 5μM, and 25μM. The three experimental groups added 5-aminolevulinic acid of the same volume (200μL) with different concentrations to pretreat LLC-PK1 cells for 2h, and then inoculated with PEDV MS strain (MOI=1). At the same time, a 6μg / ml ET maintenance solution control group was set. After culturing at 37℃ for 24h, the supernatant was collected to determine the TCID 50 Cell samples were collected for qRT-PCR and indirect immunofluorescence assay (IFA) to detect changes in the replication level of PEDV under different administration concentrations.

[0039] 2.1. Fluorescence quantitative real-time PCR method to detect the effect of 5-aminolevulinic acid on PEDV replication:

[0040] 2.1.1. Sample preparation and processing:

[0041] Sample preparation: LLC-PK1 cells were plated in 24-well cell culture plates, and the selected safe concentration of 5-aminolevulinic acid was added for pretreatment. After pretreatment at 37°C for 2 hours, the PEDV MS strain (MOI = 1, maintenance medium 6 μg / ml ET) was inoculated, and the supernatant was collected after culturing at 37°C for 24 hours to determine the TCID 50 , and collected cell samples to extract total RNA.

[0042] 2.1.2 Extraction of total RNA:

[0043] According to the steps in the Omega RNA extraction kit manual, extract total RNA from cell samples. The specific steps are as follows:

[0044] (1) Discard the original liquid in the 24-well plate and add about 500 μL PBS to each well to wash the cells for a total of three times;

[0045] (2) Add 350 μL TRK Lysis Buffer to each well and let stand for 2-3 minutes. Scrape the cell sample and transfer it to an EP tube.

[0046] (3) Add an equal volume of 70% anhydrous ethanol to the EP tube, vortex to mix, and let stand for 2-3 minutes;

[0047] (4) Transfer the above mixture to HiBind RNA Mini Columns, let it stand for 1 min, and centrifuge at 10000 rcf for 1 min at room temperature;

[0048] (5) After discarding the centrifuged liquid, add 500 μL Wash Buffer I to the column and centrifuge at 10,000 rcf for 30 s at room temperature;

[0049] (6) After discarding the centrifuged liquid, add 500 μL Wash Buffer II to the column and centrifuge at 10,000 rcf for 1 min at room temperature;

[0050] (7) Repeat step (6);

[0051] (8) After discarding the centrifuged liquid, centrifuge at room temperature at 14000 rcf for 4 minutes;

[0052] (9) Transfer the HiBind RNA Mini Columns to a new EP tube and air-dry for 5 minutes with the lid open.

[0053] (10) Add 30 μL of preheated DEPC water to the column, let it stand for 5 min, and then centrifuge at room temperature at 14,000 rcf for 2 min;

[0054] (11) Collect the centrifuged liquid in the EP tube and add it back into the HiBind RNA Mini Columns. Centrifuge at 14000 rcf for 2 min at room temperature.

[0055] The extracted total RNA was reverse transcribed using HiScipt Reverse Transcripatase, and the steps were as follows: 8 μL of the extracted RNA sample was mixed with 2 μL of 5×qRT SuperMixII, and reverse transcription was performed according to the program of 50°C for 15 min and 85°C for 5 s. The obtained cDNA was stored at -20°C for future use.

[0056] 2.1.3. Fluorescence quantitative real-time PCR:

[0057] This laboratory has established a single fluorescence quantitative detection method for PEDV, which can perform single fluorescence quantitative RT-PCR amplification on PEDV. The copy number can be calculated by the Ct value, thereby reflecting the content of PEDV virus in the sample to be tested. The virus content of PEDV in the sample to be tested reflects the inhibitory effect of 5-aminolevulinic acid on the above two viruses. The reaction system is shown in Table 1. This experiment was repeated twice independently.

[0058]

[0059] The PCR cycle system was: 95°C pre-denaturation for 30 s, 95°C denaturation for 10 s, 60°C for 34 s, for a total of 40 cycles, and the annealing temperature was 60°C.

[0060] 2.2. Indirect immunofluorescence assay (IFA) to detect the effect of 5-aminolevulinic acid on PEDV replication:

[0061] (1) LLC-PK1 cells were plated in 24-well plates and cultured for 24 h. 5-aminolevulinic acid was added at a safe concentration that had been screened, and pretreated at 37°C for 2 h. PEDV MS (MOI = 1) was inoculated, and 6 μg / ml ET maintenance solution was added, and cultured at 37°C for 24 h.

[0062] (2) Discard the culture medium in the original well and add PBS to wash three times, 5 min each time, and repeat three times;

[0063] (3) Fixation: Add 250 μL of 4% paraformaldehyde solution to each well and fix at room temperature for 15 min. Wash three times with PBS, 5 min each time.

[0064] (4) Permeabilization: Add 0.1% Triton solution to the wells, permeabilize for 13 min at room temperature, and wash three times with PBS, 5 min each time;

[0065] (5) Primary antibody incubation: Add 200 μL of PEDV N protein monoclonal antibody diluted with BSA (1:200) to each well, incubate overnight at 4°C for 12 h, and wash three times with PBS, each time for 5 min;

[0066] (6) Secondary antibody incubation: Add 200 μL of goat anti-mouse-Alexa Fluor diluted in BSA (1:200) to each well in the dark, incubate at 37°C in the dark for 1 h, and wash three times with PBS, 5 min each time;

[0067] (7) Nuclear staining: Add an appropriate amount of DAPI dye and incubate at room temperature in the dark for 5-10 min. Wash with PBS three times, 5 min each time.

[0068] (8) The samples were placed under an inverted fluorescence microscope (Axiovert 200, Zeiss, Germany) to observe the fluorescence of the samples and take pictures. The green fluorescence represents the PEDV N protein and the blue fluorescence represents the cell nucleus. The difference in the amount of green fluorescence between the control group and the experimental group was compared.

[0069] 2.3 TCID 50 Determination:

[0070] Vero cells were evenly plated in a 96-well cell culture plate. After the cells were cultured to a single layer, the culture medium was aspirated and the collected supernatant samples were washed with maintenance medium from 10 -1 -10 -6 Make a 10-fold gradient dilution. Take 100 μL of each gradient of PEDV MS strain virus dilution and add it to a 96-well cell culture plate. Set up 8 replicate wells for each gradient virus dilution. After culturing at 37°C for 24 h, observe the cell lesions and record the pathological changes. Calculate the TCID according to the Reed-Muench method 50 This experiment was repeated twice independently.

[0071] 2.4 Test results:

[0072] 2.4.1. qRT-PCR detection of the effect of 5-aminolevulinic acid on PEDV replication:

[0073] Depend on Figure 2 and Figure 3 It can be seen that compared with the control group, as the concentration of 5-aminolevulinic acid increased, the CT value increased accordingly ( Figure 2 A. Figure 3 A), and the corresponding PEDV MS strain virus copy number decreased accordingly ( Figure 2 B. Figure 3 B) shows that 5-aminolevulinic acid has a significant inhibitory effect on the replication of PEDV in LLC-PK1 cells, and the inhibitory effect is dose-dependent with the administration concentration of 5-aminolevulinic acid. This experiment was repeated twice independently, and the results of the two experiments were consistent.

[0074] 2.4.2. IFA method to detect the effect of 5-aminolevulinic acid on PEDV replication

[0075] The results of the IFA test are shown in Figure 4, where green fluorescence represents PEDV N protein and blue fluorescence represents cell nuclei. Compared with the control group, the amount of green fluorescence of PEDV N protein decreased significantly with the increase of 5-aminolevulinic acid administration concentration. This result also shows that 5-aminolevulinic acid has a significant inhibitory effect on the replication of PEDV in LLC-PK1 cells and is dose-dependent.

[0076] 2.4.3 Virus TCID 50 Determination of:

[0077] TCID 50 The results of virus titer detection ( Figure 5 ) It can be seen that compared with the control group, 5-aminolevulinic acid has a significant inhibitory effect on the replication of PEDV and is dose-dependent. This experiment was repeated twice independently ( Figure 5 A. Figure 5 B), and the results of the two tests were consistent.

[0078] Example 3, Study on the effectiveness of 5-aminolevulinic acid in preventing and treating PEDV virus infection in piglets:

[0079] In order to study the effect of 5-aminolevulinic acid in preventing and treating PEDV virus infection in piglets, the status and number of deaths of piglets under different treatments and the effect of 5-ALA on the anti-PEDV virus load in piglets were monitored during the experiment, and the effect of 5-ALA on piglets infected with the virus was further verified.

[0080] Fifteen 14-day-old creep feed piglets were selected and randomly divided into three groups (5 piglets / group), namely, Group 1 healthy control group, Group 2 challenge group and Group 3 5-ALA group, and they were kept in three separate rooms respectively; the piglets were confirmed to be negative for PEDV, transmissible gastroenteritis virus and porcine rotavirus by colloidal gold rapid test kit (BioNote); the piglets in Group 3 5-ALA group were fed with creep feed containing 1000g / ton of 5-ALA starting from 3 days before challenge; the piglets in Group 1 and Group 2 took the same amount of DMEM orally at the same time; after the start of the experiment, the piglets in Group 2 and Group 3 took 3mL DMEM containing PEDV MS strain orally; after the start of the experiment, the piglet feces were collected every 6 hours, and the detoxification of the piglets in each group was monitored by PCR method; and the number of piglets that died in each group was recorded at the same time. All piglets were killed 48 hours after infection, and the effect of 5-ALA on the virus infection of piglets was counted. All piglets were autopsied and the small intestinal tissues were collected. The viral load in the feces, jejunum and ileum of each group of piglets was detected by real-time fluorescence quantitative PCR (the detection method was the same as that in Example 2).

[0081]

[0082] As shown in Table 2, all 5 piglets in the challenge group developed severe watery diarrhea (5 / 5) within 24 hpi (infection lasted 24 hours), 1 piglet died at 24 hpi, 2 piglets died at 30 hpi, and all 5 piglets died at 48 hpi; the 5 piglets that had been orally administered 5-ALA had no obvious changes in their physiological and mental states within 24 hpi, 1 piglet developed diarrhea and died at 30 hpi, and 2 piglets developed diarrhea and died by 36 hpi; 3 piglets survived at 48 hpi, and the surviving piglets were in good condition; the 5 piglets in the healthy control group had no abnormal physiological and pathological changes; from the test results, it was known that the protection rate of 5-ALA against piglets infected with PEDV was 60%.

[0083] Depend on Figure 6 and Figure 7 The results showed that compared with the PEDV-infected control group, 5-ALA could significantly inhibit the viral content in the feces of sick pigs; and effectively inhibit the replication of the virus in the small intestinal tissue (jejunum and ileum), indicating that 5-ALA could inhibit the replication of PEDV in piglets, thereby effectively preventing and treating PEDV infection in piglets.

[0084] Example 4, Study on the application effect of 5-aminolevulinic acid in preventing and treating PEDV virus infection in piglets:

[0085] By adding different amounts of 5-aminolevulinic acid to the feed of piglets (suckling piglets) and weaned piglets in the creep stage, the application effect and optimal addition amount of 5-aminolevulinic acid in preventing and treating PEDV virus infection in piglets at different stages were investigated.

[0086] A total of 90 piglets were tested in the experiment, including piglets in the above-mentioned creep stage (suckling piglets) and weaned piglets, which are two stages susceptible to PEDV.

[0087] 45 healthy piglets (14 days old) in the crutch stage were randomly divided into 9 groups (5 piglets in each group), with Group 1 as the healthy control group, Group 2 as the challenge group, and Groups 3-9 as the groups supplemented with different doses of 5-ALA (see Table 3 for specific groups), and they were housed in 6 separate rooms;

[0088] 45 healthy weaned piglets, with an average weight of (7.30±0.15) kg, had no significant difference in weight after statistical analysis. They were randomly divided into 9 groups (5 pigs in each group), with Group 1 being the healthy control group, Group 2 being the challenge group, and Groups 3-9 being the groups with different doses of 5-ALA added (see Table 4 for specific groups), and were kept in 6 separate rooms;

[0089] Before the start of the experiment, all piglets in the above two stages were confirmed to be negative for PEDV, transmissible gastroenteritis virus and porcine rotavirus by colloidal gold rapid test kit (BioNote); the groups supplemented with 5-ALA were fed feed containing different doses of 5-ALA (doses are shown in Table 3) starting from 3 days before the challenge, and the control group and the challenge group were fed feed containing the same dose of DMEM at the same time. After the start of the experiment, the challenge group and the 5-ALA group were fed feed containing 10 7 5 mL DMEM of PEDV MS strain with TCID50 / mL. Observe the diarrhea of ​​the experimental piglets every day, count the number of dead pigs, and calculate the diarrhea rate and mortality rate. Isolate and raise them, and observe them for 5 consecutive days until the end of the experiment. Statistically analyze the effect of 5-ALA on the virus-infected piglets in each group.

[0090]

[0091] As shown in Table 3, 5-aminolevulinic acid (5-ALA) has a significant preventive and therapeutic effect on PEDV-infected piglets in the creep stage. Adding different doses of 5-ALA to piglet feed in the creep stage can significantly reduce the daily weight gain, diarrhea rate and mortality of PEDV-infected piglets, and the effect varies with the amount of addition. In the creep stage, when the amount of 10-1500g / T of feed is added to the feed, it has a significant effect on reducing the diarrhea rate and mortality of PEDV-infected piglets. When the amount of addition is 1000g / T of feed, the diarrhea rate is 40%, the mortality rate is 20%, and the overall condition of this group of piglets is the best, and the daily weight gain is the highest; when the amount of addition is increased to 1500g / T of feed, the diarrhea rate and mortality of piglets are not reduced, but the daily weight gain of piglets is slightly worse than that of 1000g / T of feed. Therefore, the treatment effect of 1000g / T of feed on PEDV-infected creep piglets is the best.

[0092]

[0093] As shown in Table 4, 5-aminolevulinic acid (5-ALA) also has a significant preventive and therapeutic effect on weaned piglets infected with PEDV. Adding different doses of 5-ALA to the feed of weaned piglets can significantly reduce the daily weight gain, diarrhea rate and mortality of PEDV-infected piglets, and the effect varies with the amount of addition. In the weaned piglet stage, when the amount of 10-1500 g / T of feed is added to the feed, it has a significant effect on reducing the daily weight gain, diarrhea rate and mortality of PEDV-infected piglets. When the amount of addition is 500 g / T of feed, the diarrhea rate is 40%, the mortality rate is 20%, and the comprehensive state of this group of piglets is the best, and the daily weight gain is the highest; when the amount of addition is increased to 1000-1500 g / T of feed, the diarrhea rate and mortality of piglets are not reduced, but the daily weight gain of piglets is slightly worse than that of 500 g / T of feed. Therefore, the therapeutic effect of 500 g / T of feed on weaned piglets infected with PEDV is the best.

[0094] Example 5, Study on the application effect of 5-aminolevulinic acid in preventing and treating PEDV virus infection in sows:

[0095] In April 2023, 45 lactating sows with epidemic diarrhea from the same batch were selected from a sow farm in Guangdong and randomly divided into 9 groups (5 in each group). The sick sows in Group 1 were the control group and were not fed 5-ALA; the test subjects in Groups 2-9 were also sick sows, and Groups 2-9 were fed with feeds with different 5-ALA contents (see Table 5 for specific doses); the 9 treatment groups were kept in 9 separate rooms. The test period was 7 days. The feeding situation of the sows was observed at each feeding; after the sick sows were treated, their mental state and diarrhea were observed; the efficacy evaluation indicators used for evaluation were as follows: 1) Cure: After drug treatment, the clinical symptoms completely disappeared, and the spirit and diet returned to normal, which was considered cured, and the cure rate was calculated by counting the number of cures; 2) Significant effect: After drug treatment, the clinical symptoms were significantly alleviated, and the spirit and diet recovered and improved, which was considered as a marked effect of treatment, and the marked effect number was calculated by counting the number of marked effects; 3) Effective: It is the sum of the cure rate and the marked effect rate.

[0096]

[0097] As shown in Table 5, 5-aminolevulinic acid (5-ALA) also has a significant preventive and therapeutic effect on lactating sows infected with PEDV. Adding different doses of 5-ALA to the feed of sick lactating sows can significantly increase the effective rate of treating sows infected with PEDV, and the effect varies with the amount of addition. When the amount of 10-1000 g / T of feed is added to the feed, the efficacy of sows infected with PEDV, including the cure rate and the marked efficacy, increases. When the amount of addition is 500 g / T of feed, the effective rate reaches 80%, the cure rate reaches 40%, and the feeding refusal phenomenon of sows in this group is significantly reduced; when the amount of addition is increased to 750-1000 g / T of feed, the effective rate of treatment of sick sows is not improved, and the cure rate is slightly worse. Therefore, the treatment effect on sows infected with PEDV is optimal when the amount of addition is 500 g / T of feed.

[0098] Example 6: Study on the effect of different beneficial components and 5-aminolevulinic acid in the prevention and treatment of PEDV virus infection in pigs:

[0099] By adding different beneficial components to the feed of weaned piglets and using them in synergistic combination with 5-aminolevulinic acid, the synergistic effect of different beneficial components and 5-aminolevulinic acid in preventing and treating porcine PEDV virus infection was investigated.

[0100] The experiment selected 100 weaned piglets with an average weight of 7.25±0.15kg. The statistical analysis showed no significant difference in weight. They were randomly divided into 10 groups (10 pigs in each group), with the first group being the healthy control group, the second group being the toxicity-challenging group, and the third to tenth groups being the experimental groups, which were supplemented with different beneficial components based on the addition of 5-ALA (see Table 6 for specific groups), and were raised in 10 separate rooms;

[0101] Before the piglet experiment began, the colloidal gold rapid test kit confirmed that the piglets were negative for PEDV, transmissible gastroenteritis virus, and porcine rotavirus; the groups supplemented with 5-ALA were fed feed containing 5-ALA mixed with different beneficial components (the dose of 5-ALA was 1000 g / T feed, and the beneficial components and doses synergized with it are shown in Table 6) from 3 days before the challenge, and the control group and the challenge group were fed feed containing the same dose of DMEM. After the start of the experiment, the challenge group and the experimental group were fed with 10 7 TCID 50 / mL of PEDV MS strain 5mL DMEM. Observe the diarrhea of ​​the experimental piglets every day, count the number of dead pigs, and calculate the diarrhea rate and mortality rate. Isolate and raise them, and observe them for 5 consecutive days until the end of the experiment. Statistic the effect of 5-ALA on the virus-infected piglets in each group.

[0102]

[0103] As shown in Table 6, the synergistic effects of different beneficial components and 5-aminolevulinic acid in porcine PEDV virus infection are different.

[0104] Compared with the control group that only added 5-aminolevulinic acid, the diarrhea rate and mortality rate of piglets infected with PEDV were reduced by adding lactoferrin to the feed, with the diarrhea rate being 30% and the mortality rate being 20%, indicating a good synergistic effect.

[0105] Compared with the control group that added 5-aminolevulinic acid alone, the diarrhea rate and mortality rate of piglets infected with PEDV were reduced by adding organic iron to the feed, with the diarrhea rate being 30%-40% and the mortality rate being 20%, and the synergistic effect was good. Among them, when 5-aminolevulinic acid was used together with glycine iron or ferrous fumarate, the synergistic effect was the best, followed by lysine iron; the synergistic effect of inorganic iron (ferrous sulfate) and 5-aminolevulinic acid was not as good as that of organic iron;

[0106] Compared with the control group that only added 5-aminolevulinic acid, the synergistic addition of IGF-1 (insulin-like growth factor-1) reduced the diarrhea rate and mortality rate of piglets infected with PEDV, with a diarrhea rate of 30% and a mortality rate of 20%, showing a good synergistic effect.

[0107] Compared with the control group with 5-aminolevulinic acid alone, the synergistic addition of antimicrobial peptides reduced the diarrhea rate and mortality of piglets infected with PEDV, with a diarrhea rate of 30% and a mortality rate of 20%, indicating a good synergistic effect.

[0108] Through this experiment, we know that 5-aminolevulinic acid has a significant preventive and therapeutic effect on piglets infected with PEDV, and has a synergistic effect with organic iron, especially lactoferrin, glycine iron or ferrous fumarate, which has the best effect when used together with it; at the same time, it can be found that IGF-1, antimicrobial peptides and 5-aminolevulinic acid also have a good synergistic effect. The above beneficial components can be used alone or together to synergize with 5-aminolevulinic acid to exert the preventive and therapeutic effect on PEDV infection.

[0109] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0110] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

The use of 1.5-aminolevulinic acid in the preparation of a product for preventing and treating porcine epidemic diarrhea is characterized in that: For piglets in the creep to weaning stage, the amount of 5-aminolevulinic acid added to the product is 10-1500 g / ton of feed; for piglets in the weaning to 90-day-old stage, the amount of 5-aminolevulinic acid added to the product is 10-1500 g / ton of feed; for lactating sows, the amount of 5-aminolevulinic acid added to the product is 10-1000 g / ton of feed.

2. The use of 5-aminolevulinic acid according to claim 1 in the preparation of a product for preventing and treating porcine epidemic diarrhea, characterized in that: For piglets in the creep to weaning stage, the added amount of 5-aminolevulinic acid in the product is 1000 g / ton.

3. The use of 5-aminolevulinic acid according to claim 1 in the preparation of a product for preventing and treating porcine epidemic diarrhea, characterized in that: For piglets from weaning to 90 days of age, the added amount of 5-aminolevulinic acid in the product is 500 g / ton.

4. The use of 5-aminolevulinic acid according to claim 1 in preparing a product for preventing and treating porcine epidemic diarrhea, characterized in that: For lactating sows, the added amount of 5-aminolevulinic acid in the product is 500g / ton.

5. The use of 5-aminolevulinic acid according to claim 1 in preparing a product for preventing and treating porcine epidemic diarrhea, characterized in that: The product also includes one or more of lactoferrin, organic iron, IGF-1, and antimicrobial peptides.

6. The use of 5-aminolevulinic acid in the preparation of a product for preventing and treating porcine epidemic diarrhea according to claim 5, characterized in that: The organic iron is glycine iron or ferrous fumarate.

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