CN118947816B - Application of 5-aminolevulinic acid in preparation of product for preventing and treating porcine epidemic diarrhea - Google Patents
Application of 5-aminolevulinic acid in preparation of product for preventing and treating porcine epidemic diarrhea Download PDFInfo
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- CN118947816B CN118947816B CN202411440500.7A CN202411440500A CN118947816B CN 118947816 B CN118947816 B CN 118947816B CN 202411440500 A CN202411440500 A CN 202411440500A CN 118947816 B CN118947816 B CN 118947816B
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- epidemic diarrhea
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- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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Abstract
本发明涉及5‑氨基乙酰丙酸在制备防治猪流行性腹泻的产品中的应用，对处于教槽至断奶阶段的仔猪，所述产品中5‑氨基乙酰丙酸或其药学上可接受的盐的添加量以5‑氨基乙酰丙酸计为500‑5000g/吨；对处于断奶至90日龄阶段的仔猪，所述产品中5‑氨基乙酰丙酸或其药学上可接受的盐的添加量以5‑氨基乙酰丙酸计为100‑1000g/吨饲料；对哺乳母猪，所述产品中5‑氨基乙酰丙酸或其药学上可接受的盐的添加量以5‑氨基乙酰丙酸计为100‑1000g/吨饲料。本发明首次发现5‑ALA无论是在体内还是在体外，均能够显著抑制猪流行性腹泻病毒的增殖，5‑ALA对于母猪和仔猪感染流行性腹泻都具有显著的防治效果。
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
The invention relates to the field of functional feeds, in particular to application of 5-aminolevulinic acid in preparation of a product for preventing and treating porcine epidemic diarrhea.
Background
Diarrhea is one of the most frequently occurring diseases in pig industry at home and abroad at present, and brings heavy impact to pig farming. Porcine epidemic diarrhea (Porcine EPIDEMIC DIARRHEA, PED) is a highly contagious Porcine intestinal infectious disease with diarrhea, vomiting and dehydration as main symptoms caused by Porcine epidemic diarrhea Virus (Porcine EPIDEMIC DIARRHEA Virus, PEDV), which has become a main pathogenic microorganism for diarrhea of suckling piglets in China. The virus is easy to infect pigs of various ages, has high morbidity and mortality rate for the suckling piglets, especially piglets within 7 days of age, and the mortality rate after infection is close to 100%. PEDV is transmitted primarily by faeces, by the faecal-oral route, and also by the milk of the affected lactating sow. Since 2010, a large number of PEDV strains have been outbreaked on a large scale, resulting in a significant economic loss to the pig industry.
Viral diarrhea is not specially treated with drugs, and is mainly prevented and treated by strengthening the breeding management and vaccination prevention of farms at present. However, as a single-stranded positive-strand RNA virus with a capsule, the characteristics of the genome of PEDV, replication of the virus, etc., cause the virus to be very likely to be mutated, and the vaccine prepared from classical strains is not ideal in the immunization against newly-emerging variant strains. In the existing methods, symptomatic therapy, feeding microecological preparation, selecting sensitive antibiotics and other methods for preventing bacterial secondary infection and the like are often used as auxiliary means for relieving viral diarrhea symptoms, but the curative effect is poor. Therefore, the selection of novel anti-PEDV products which are sensitive to pathogens, high in specificity, strong in inhibition effect and good in stability is an important development direction for healthy development of the pig industry.
5-Aminolevulinic acid is a natural compound produced during operation of the citric acid cycle (TCA) circuit in mitochondria of higher cells, and is a physiologically active substance which is essential for vital activities of animals and plants and is metabolically active, and which is a non-protein amino acid widely existing in living cells of living organisms such as bacteria, fungi, animals and plants. The 5-aminolevulinic acid and the derivatives thereof are widely applied to pesticides or fertilizers in the field of large agriculture, are added to animal feeds as feed additives in the field of livestock and poultry, are effective in improving animal anemia and the like, and are applied to photodynamic therapy or photodynamic therapy by using 5-ALA in the field of human clinical medicine and are also applied to using 5-ALA as a diagnostic agent of tumors. However, how to inhibit Porcine Epidemic Diarrhea Virus (PEDV) and how to prevent and treat porcine epidemic diarrhea has not been reported, the research discusses the application prospect of 5-ALA as an anti-PEDV drug through in vitro and in vivo experiments, and develops a new application of 5-ALA.
Disclosure of Invention
In order to solve the problems, the invention provides application of 5-aminolevulinic acid in preparing a product for preventing and treating porcine epidemic diarrhea.
The invention provides an application of 5-aminolevulinic acid or pharmaceutically acceptable salt thereof in preparing a product for preventing and treating porcine epidemic diarrhea, wherein the addition amount of the 5-aminolevulinic acid or pharmaceutically acceptable salt thereof in the product is 10-1500 g/ton calculated by 5-aminolevulinic acid for piglets in a period from weaning to weaning, the addition amount of the 5-aminolevulinic acid or pharmaceutically acceptable salt thereof in the product is 10-1500 g/ton feed calculated by 5-aminolevulinic acid for piglets in a period from weaning to 90 days, and the addition amount of the 5-aminolevulinic acid or pharmaceutically acceptable salt thereof in the product is 10-1000 g/ton feed calculated by 5-aminolevulinic acid for lactating sows.
Further, for piglets in the creep to weaning stage, the amount of 5-aminolevulinic acid or a pharmaceutically acceptable salt thereof added in the product is 1000 g/ton calculated as 5-aminolevulinic acid.
Further, for piglets at 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 as 5-aminolevulinic acid.
Further, the amount of 5-aminolevulinic acid or a pharmaceutically acceptable salt thereof added to the lactating sow is 500 g/ton as 5-aminolevulinic acid.
Further, the product also comprises one or more of lactoferrin, organic iron, IGF-1 (insulin-like growth factor-1) and antibacterial peptide.
Further, the organic iron is ferrous glycinate or ferrous fumarate.
The beneficial effects of the invention are as follows:
1. The invention discovers for the first time that 5-ALA can obviously inhibit the proliferation of porcine epidemic diarrhea virus both in vivo and in vitro. The 5-aminolevulinic acid has remarkable control effect on the epidemic diarrhea of sow and piglet infection.
2. Animal test researches show that when the porcine epidemic diarrhea virus infection is prevented, the 5-aminolevulinic acid has better effect when being cooperated with other beneficial components including lactoferrin, organic iron, IGF-1 and antibacterial peptide, wherein the organic iron has better effect when adopting iron glycinate or ferrous fumarate.
3. According to the technical concept of the invention, 5-ALA and one or more of other beneficial components including lactoferrin, organic iron, IGF-1 and antibacterial peptide can be combined together to form a product for preventing and treating porcine epidemic diarrhea. The product can be a feed additive, premix, complete feed or functional water agent, and can be added by mixing as a feed additive or can be fed by drinking water.
Drawings
FIG. 1 is a screen for safe concentrations of 5-aminolevulinic acid of example 1;
FIG. 2 shows the results of a first test of qRT-PCR for detecting the replication level of PEDV virus according to example 2;
FIG. 3 shows the results of a second test of qRT-PCR for detecting the replication level of PEDV virus according to example 2;
FIG. 4 is the results of the IFA test of example 2;
FIG. 5 is the results of two assays of the virus TCID50 of example 2;
FIG. 6 is a graph showing the qRT-PCR assay of example 3 for the comparison of the level of PEDV replication in piglet manure;
FIG. 7 is a graph comparing the qRT-PCR detection of the replication level of PEDV in the jejunum of piglets of example 3.
Detailed Description
The invention is further illustrated by the following examples.
Example 1, safety evaluation of 5-aminolevulinic acid-acting cells:
1.1, materials and reagents:
CCK-8 kit and reverse transcription reagent are purchased from Vazyme company, and Probe qPCR Mix is purchased from TaKaRa company;
serum (Fetal Bovine Serum, FBS) required for cell culture was purchased from LONSERA;
Pancreatin (ET) for digestion and detoxification was purchased from GIBCO;
DMEM is available from CORNING corporation;
24 well cell culture plates were purchased from NEST company;
LLC-PK1 (porcine kidney proximal tubular epithelial cells) and Vero cells were stored in the laboratory;
PEDV MS strain (GenBank accession NO: MT 683617) was supplied by the present laboratory preservation.
1.2, Test method:
Dissolving 5-aminolevulinic acid in DMEM according to the specification to prepare mother solution with concentration of 10 mM, fully dissolving, and sub-packaging at-80 ℃ for standby. LLC-PK1 cells were plated uniformly in 96-well plates and incubated in a cell incubator (37 ℃ C., 5% CO 2) to a density of about 90% to begin the assay. After washing 3 times with PBS solution, 6 test groups were added with the same volume (200. Mu.L) of 5-aminolevulinic acid at different concentrations, the concentrations of 5-aminolevulinic acid added were 0.125. Mu.M, 0.2. Mu.M, 1. Mu.M, 5. Mu.M, 25. Mu.M and 125. Mu.M, the control group was a maintenance solution (pure DMEM containing 6. Mu.g/mlET), 6 replicates were set for each concentration, 10. Mu.L of CCK-8 solution was added per well after 24h treatment at 37℃and the cell viability was calculated by reading with an enzyme-labeled instrument after 2h action at 37℃and then the difference between the test group and the control group was calculated with GRAPHPAD PRISM 6. The test was performed in two separate replicates.
1.3, Test results:
Referring to FIG. 1, when the maintenance solution is 6. Mu.g/mlET, the maximum drug safety concentration of 5-aminolevulinic acid on LLC-PK1 cells is 25. Mu.M, namely, the 5-aminolevulinic acid is nontoxic to cells at the concentration of 25. Mu.M, and the toxic effect on cells is more remarkable when the concentration reaches 125. Mu.M, and the results of two independent repeated experiments are shown as the same in FIG. 1A and FIG. 1B, so that the maximum drug safety concentration of 5-aminolevulinic acid selected in the experiment is 25. Mu.M.
Example 2, inhibition effect of 5-aminolevulinic acid on PEDV:
According to the safety concentration of the 5-aminolevulinic acid selected, the concentration of the 5-aminolevulinic acid is set to be 1 mu M, 5 mu M and 25 mu M,3 test groups are added with the same volume (200 mu L) of 5-aminolevulinic acid with different concentrations to pretreat LLC-PK1 cells for 2 h, and then the PEDV MS strain (MOI=1) is inoculated, and meanwhile, 6 mu g/mlET of a maintenance solution control group is set. Supernatants were harvested after 24h incubation at 37 ℃ for TCID 50, and cell samples were harvested for qRT-PCR and indirect immunofluorescence assay (IFA) to detect changes in the replication level of PEDV at different dosing concentrations.
2.1, Fluorescence quantitative Real-time PCR method to detect the effect of 5-aminolevulinic acid on PEDV replication:
2.1.1, sample preparation and handling:
Sample preparation by spreading LLC-PK1 cells in 24-well cell culture plates, adding 5-aminolevulinic acid with a safe concentration selected for pretreatment, pretreating for 2 hours at 37 ℃ and inoculating PEDV MS strain (MOI=1, maintaining solution is 6 mug/mlET), culturing for 24 hours at 37 ℃ and collecting supernatant to measure TCID 50, and collecting cell samples to extract total RNA.
2.1.2, Extracting total RNA:
According to the steps on the Omega RNA extraction kit instruction, the total RNA of the cell sample is extracted, and the specific steps are as follows:
(1) Discarding the original liquid in the 24-well plate, adding about 500 mu LPBS to wash the cells in each well, and washing for three times;
(2) Adding 350 mu L TRK LYSIS Buffer into each hole, standing for 2-3min, scraping off a cell sample, and transferring into an EP tube;
(3) Adding 70% absolute ethyl alcohol with equal volume into an EP pipe, and standing for 2-3min after vortex mixing;
(4) Transferring the mixed solution into HiBind RNA Mini Columns columns, standing for 1min, and centrifuging for 1min at room temperature 10000 rcf;
(5) After the centrifugal liquid is discarded, 500 mu L of Wash Buffer I is added into the column, and the column is centrifuged for 30s at room temperature 10000 rcf;
(6) After the centrifugal liquid is removed, 500 mu L of Wash Buffer II is added into the column, and the column is centrifuged for 1min at room temperature 10000 rcf;
(7) Repeating step (6);
(8) After discarding the centrifugate, the mixture is centrifugated for 4min at room temperature of 14000 rcf;
(9) Transferring HiBind RNA Mini Columns to a new EP pipe, uncovering and air drying for 5min;
(10) Adding 30 mu L of preheated DEPC water into the column, standing for 5min, and centrifuging at room temperature of 14000rcf for 2min;
(11) The collected EP tube was added again to HiBind RNA Mini Columns and centrifuged at 14000rcf for 2min at room temperature.
The total RNA extracted was reverse transcribed with HISCIPT REVERSE TRANSCRIPATASE, by mixing 8. Mu.L of the RNA sample extracted with 2. Mu.L of 5X qRT SuperMixII, and reverse transcribing the cDNA obtained by the procedure of 15min at 50℃and 5s at 85℃and preserving at-20 ℃.
2.1.3 Fluorescent quantitative Real-time PCR:
the laboratory has established a method for quantitative detection of PEDV by single fluorescence, can carry out quantitative RT-PCR amplification of PEDV by single fluorescence, can calculate copy number through Ct value, thus reflecting the content of PEDV virus in a sample to be detected, and reflects the inhibition effect of 5-aminolevulinic acid on the two viruses through the virus content of PEDV in the sample to be detected. The reaction system is shown in Table 1. The test was performed in two separate replicates.
The PCR cycle system comprises 95 ℃ pre-denaturation for 30s, 95 ℃ denaturation for 10s and 60 ℃ denaturation for 34s, 40 cycles total, and the annealing temperature is 60 ℃.
2.2, Indirect immunofluorescence assay (IFA) method to detect the effect of 5-aminolevulinic acid on replication of PEDV:
(1) LLC-PK1 cells were plated in 24-well plates, cultured for 24h, added with 5-aminolevulinic acid at a safe concentration that had been screened, pretreated for 2h at 37 ℃, inoculated with PEDV MS (MOI=1), and added with 6 μg/ml ET maintenance solution, cultured for 24h at 37 ℃;
(2) Discarding culture solution in the original hole, adding PBS, washing for three times, 5min times each time, repeating for three times;
(3) Fixing, namely adding 250 mu L of 4% paraformaldehyde solution into each hole, fixing at room temperature for 15 min, and washing with PBS for three times, each time for 5min;
(4) Permeation by adding 0.1% Triton solution into the well, allowing permeation at room temperature for 13min, and washing with PBS three times for 5min each time;
(5) Incubation for primary antibody, namely adding 200 mu L of PEDV N protein monoclonal antibody diluted by BSA (diluted 1:200) into each well, incubating for 12h at 4 ℃ overnight, and washing with PBS for three times, wherein each time is 5min;
(6) The secondary antibody is incubated, 200 mu L of goat anti-mouse-Alexa Flou diluted by BSA (1:200 dilution) is added to each hole under the dark condition, the mixture is incubated for 1h at 37 ℃ under dark condition, and PBS is used for washing three times, each time is 5: 5min;
(7) Cell nucleus staining, namely adding a proper amount of DAPI dye, incubating for 5-10min at room temperature in a dark place, and washing for 3 times by PBS (phosphate buffer solution) for 5min each time;
(8) The samples were placed under an inverted fluorescence microscope (Axiovert 200, zeiss, germany) and the samples were observed for the amount of fluorescence and photographed, wherein green fluorescence represented PEDV N protein, blue fluorescence represented nuclei, and the difference in the amount of green fluorescence was compared between the control and test groups.
2.3, TCID 50 determination:
Vero cells were spread evenly in 96 well cell culture plates, after cell culture to confluence of monolayer, the culture broth was aspirated, and the collected supernatant samples were serially 10-fold gradient diluted with maintenance fluid from 10 -1-10-6, respectively. 100 μl of each gradient of PEDV MS strain virus dilution was added to a 96-well cell culture plate, and 8 replicate wells were placed per gradient of virus dilution. Cytopathy is observed after incubation at 37℃for 24 h and the disease is recorded. TCID 50 was calculated according to the Reed-Muench method. The test was performed in two separate replicates.
2.4, Test results:
2.4.1, qRT-PCR method to detect the effect of 5-aminolevulinic acid on PEDV replication:
As can be seen from fig. 2 and 3, as the concentration of 5-aminolevulinic acid administered increases, the CT value increases (fig. 2A, 3A) and the corresponding virus copy number of PEDV MS strain decreases (fig. 2B, 3B) compared with the control group, indicating that 5-aminolevulinic acid has a significant inhibitory effect on replication of PEDV in LLC-PK1 cells, and that the inhibitory effect is dose-dependent with the concentration of 5-aminolevulinic acid administered. The test was repeated independently for two times, and the results of both tests were consistent.
2.4.2 Detection of the Effect of 5-aminolevulinic acid on PEDV replication by the IFA method
The IFA assay results are shown in fig. 4, where green fluorescence represents PEDV N protein and blue fluorescence represents nucleus. The result also shows that 5-aminolevulinic acid has a significant inhibitory effect on replication of PEDV on LLC-PK1 cells and is dose dependent, as the green fluorescence amount of PEDV N protein is significantly reduced with increasing 5-aminolevulinic acid dosing concentration compared to the control group.
2.4.3 Determination of virus TCID 50:
The results of TCID 50 for viral titres (fig. 5) showed that 5-aminolevulinic acid had a significant inhibitory effect on PEDV replication compared to the control group and exhibited dose dependency. The test was repeated independently twice (fig. 5A and 5B), and the results of both tests were identical.
Example 3, effectiveness study of 5-aminolevulinic acid in controlling PEDV virus infection in piglets:
In order to study the effect of 5-aminolevulinic acid on preventing and treating piglet PEDV infection, the effect of 5-ALA on piglet infection virus is further verified by monitoring the piglet state and death number in the test process of different treatments and the influence of 5-ALA on anti-PEDV virus load in piglet bodies.
The method comprises the steps of selecting 15 piglets with 14-day-old creep feed into three groups (5 piglets/group), namely a 1-healthy control group, a 2-challenge group and a 3-group 5-ALA group, feeding the piglets in three independent rooms respectively, confirming that the piglets are negative to PEDV, transmissible gastroenteritis virus and porcine rotavirus through a colloidal gold rapid detection kit (BioNote), feeding 3-group 5-ALA group piglets with creep feed containing 1000 g/ton of 5-ALA dose 3 days before challenge, simultaneously taking the same amount of DMEM by the 1-group piglets and the 2-group piglets, taking 3mL DMEM containing PEDV MS strains orally by the 2-group piglets and the 3-group piglets after the test starts, collecting feces of the piglets every 6 hours after the test starts, monitoring the toxin expelling condition of the piglets in each group through a PCR method, and recording the number of the dead piglets in each group. All piglets were sacrificed 48h after infection, the effect of 5-ALA on the infection of piglets was counted, all piglets were dissected and small intestine tissues of piglets were collected, and viral loads in the faeces, jejunum and ileum of each group of piglets were detected by real-time fluorescent quantitative PCR (detection method was the same as in example 2).
As shown in Table 2, severe watery diarrhea (5/5) occurred in all piglets in the 5 piglets of the challenge group, 1 piglet died at 24hpi, two piglets at 30hpi died, all piglets at 5 had died at 48hpi, physiological and mental state changes were not obvious in the 5 piglets 24hpi pre-oral administration of 5-ALA, diarrhea occurred in 1 piglet at 30hpi and died, diarrhea occurred in 2 piglets up to 36hpi and died, 3 piglets survived at 48hpi, the surviving piglets had good status, no abnormal physiological or pathological changes occurred in the 5 piglets of the healthy control group, and thus test results revealed that the protection rate of 5-ALA to piglets infected with PEDV was 60%.
From the results shown in fig. 6 and 7, it is evident that 5-ALA can significantly inhibit the virus content in the feces of the sick pigs compared with the PEDV-infected control group, and effectively inhibit the replication of the virus in the small intestine tissues (jejunum and ileum), which indicates that 5-ALA can inhibit the replication of PEDV in the bodies of piglets, and thus effectively prevent and treat the PEDV infection of piglets.
Example 4, application effect study of 5-aminolevulinic acid in controlling PEDV virus infection in piglets:
By adding 5-aminolevulinic acid with different contents into piglet (suckling piglet) and weaned piglet feeds in the creep period, the application effect of the 5-aminolevulinic acid in preventing and treating the PEDV virus infection of the piglets in different periods and the optimal addition amount are examined.
The test has 90 test piglets, including the piglets in the creep stage (suckling piglets) and the weaned piglets in the two stages of susceptibility to PEDV.
The 45 healthy creep stage piglets (14 days old) are randomly divided into 9 groups (5 piglets per group), wherein the 1 st group is a healthy control group, the 2 nd group is a virus attack group, and the 3 rd to 9 th groups are 5-ALA groups (specific groups are shown in Table 3) added with different dosages and are respectively fed into 6 independent rooms;
The average weight of 45 healthy weaned pigs is (7.30+/-0.15) kg, and the weight difference is not obvious through statistical analysis. Randomly dividing into 9 groups (5 heads in each group), wherein the 1 st group is a healthy control group, the 2 nd group is a virus attack group, and the 3 rd to 9 th groups are 5-ALA groups added with different dosages (specific groups are shown in Table 4) respectively and are fed into 6 independent rooms;
Before the beginning of the experiment, all piglets in the 2 stages are confirmed to be negative to PEDV, transmissible gastroenteritis virus and porcine rotavirus by a colloidal gold rapid detection kit (BioNote), 5-ALA groups are added 3 days before the challenge, feeds containing different doses of 5-ALA are fed (the doses are shown in table 3), and a control group and a challenge group are simultaneously fed with feeds containing the same dose of DMEM. The challenge and 5-ALA groups were fed 5mLDMEM containing 10 7 TCID50/mL of PEDV MS strain after the start of the test. And (5) observing diarrhea conditions of the test piglets every day, simultaneously counting the number of dead pigs, and calculating diarrhea rate and death rate. And (5) isolated feeding, and continuously observing for 5 days until the test is finished. The effect of 5-ALA on each group of piglets infected with virus is counted.
From Table 3, it is clear that 5-aminolevulinic acid (5-ALA) has remarkable control effect on piglets in the creep stage of PEDV infection. The daily gain, diarrhea rate and death rate of piglets infected by PEDV can be obviously reduced by adding different doses of 5-ALA into piglet feed in the creep period, and the effect is different with different addition amounts. In the creep feed stage, when the addition amount of 10-1500g/T feed in the feed is about 1000g/T feed, the diarrhea rate and the death rate of piglets infected with PEDV are reduced, when the addition amount of 40% and 20% are achieved, the comprehensive state of the piglets is optimal, daily gain is highest, and when the addition amount is increased to 1500g/T feed, the diarrhea rate and the death rate of the piglets are not reduced, but the daily gain of the piglets is slightly worse than that of the piglets with 1000g/T feed. Therefore, the therapeutic effect on PEDV-infected creep piglets is optimal when the feed is added in an amount of 1000 g/T.
From Table 4, it is clear that 5-aminolevulinic acid (5-ALA) also has a remarkable control effect on weaned pigs infected with PEDV. The daily gain, diarrhea rate and death rate of the piglets infected by PEDV can be obviously reduced by adding different doses of 5-ALA into the feed for the weaned piglets, and the effect is different along with the different addition amounts. In the weaned piglet stage, when the feed additive amount is 10-1500 g/T, the feed additive has obvious effects on reducing daily gain, diarrhea rate and death rate of piglets infected with PEDV, when the feed additive amount is 500 g/T, the diarrhea rate is 40%, the death rate is 20%, the comprehensive state of the piglets is optimal, the daily gain is highest, when the feed additive amount is increased to 1000-1500 g/T, the diarrhea rate and the death rate of the piglets are not reduced, but the daily gain of the piglets is slightly worse than that of the feed additive amount of 500 g/T. Therefore, the treatment effect on weaned pigs infected with PEDV is optimal when the feed is added in an amount of 500 g/T.
Example 5, application effect study of 5-aminolevulinic acid in controlling PEDV virus infection in sow:
In 2023, in month 4, 45 sows with epidemic diarrhea are selected from a certain sow farm in Guangdong, and randomly divided into 9 groups (5 sows in each group), the 1 st diseased sow is a control group, and is not fed with 5-ALA, the 2 nd to 9 test subjects are also diseased sows, the 2 nd to 9 th test subjects are respectively fed with 5-ALA feeds with different contents (specific dosages are shown in Table 5), and the 9 treatment groups are respectively fed in 9 independent rooms. The test period was 7 days. The method comprises the steps of observing feeding condition of sows during each feeding, observing mental state and diarrhea condition of the sows after treatment of the sows with diseases, and evaluating curative effect evaluation indexes adopted by evaluation, wherein 1) the curative effect is that clinical symptoms completely disappear after medical treatment, spirit and diet are recovered to be normal, the curative rate is calculated by counting the curative rate, 2) the curative effect is that the clinical symptoms are obviously lightened after medical treatment, the spirit and diet recovery are improved, the curative effect is calculated by counting the curative rate, and 3) the curative effect is the sum of the curative rate and the curative rate.
As can be seen from Table 5, 5-aminolevulinic acid (5-ALA) also has significant control effects on lactating sows infected with PEDV. The effective rate of treating the infectious PEDV of the sow can be remarkably increased by adding different doses of 5-ALA into the feed for the sick lactating sow, and the effect is different according to the different addition amounts. When the feed is added into the feed with the addition amount of 10-1000 g/T, the curative effect, including cure rate and obvious efficiency, of the infected PEDV sow is improved, when the feed with the addition amount of 500 g/T, the effective rate reaches 80%, the cure rate reaches 40%, the antifeeding phenomenon of the sow is obviously reduced, and when the feed with the addition amount of 750-1000 g/T is improved, the curative effect of the infected sow is not improved, and the cure rate is slightly poor. Therefore, the therapeutic effect on the PEDV infected sows is optimal when the feed is added in an amount of 500 g/T.
Example 6 application effect study of different beneficial components in synergy with 5-aminolevulinic acid in the control of porcine PEDV virus infection:
By adding different beneficial components and 5-aminolevulinic acid into weaned pig feed for cooperative application, the cooperative application effect of the different beneficial components and the 5-aminolevulinic acid in preventing and treating porcine PEDV virus infection is examined.
100 Weaned pigs are selected in the test, the average weight is 7.25+/-0.15 kg, and the weight difference is not obvious through statistical analysis. Randomly dividing into 10 groups (10 heads in each group), wherein the 1 st group is a healthy control group, the 2 nd group is a virus attack group, and the 3 rd to 10 th groups are test groups, wherein different beneficial components are cooperatively added on the basis of adding 5-ALA (the specific grouping is shown in Table 6), and the 10 independent rooms are respectively fed;
Before the start of the piglet test, the rapid detection kit of colloidal gold confirms that the piglets are negative to PEDV, transmissible gastroenteritis virus and porcine rotavirus, 5-ALA group is added 3 days before the virus attack, the feed containing 5-ALA and different beneficial components is fed, wherein the 5-ALA dose is 1000 g/T feed, the synergistic beneficial components and the dose are shown in Table 6, and the control group and the virus attack group are simultaneously fed with feed containing the same dose of DMEM. The challenge and test groups were fed 5mLDMEM containing 10 7TCID50/mL PEDV MS strain after the start of the test. And (5) observing diarrhea conditions of the test piglets every day, simultaneously counting the number of dead pigs, and calculating diarrhea rate and death rate. And (5) isolated feeding, and continuously observing for 5 days until the test is finished. The effect of 5-ALA on each group of piglets infected with virus is counted.
As can be seen from Table 6, the synergistic effect of the different beneficial components was different from that of 5-aminolevulinic acid in porcine PEDV virus infection.
Compared with a control group added with 5-aminolevulinic acid alone, after lactoferrin is added in a feed in a synergistic way, the diarrhea rate and the death rate of the piglets infected with PEDV are reduced, the diarrhea rate is 30%, the death rate is 20%, and the synergistic effect is good;
Compared with a control group in which 5-aminolevulinic acid is added singly, the diarrhea rate and the death rate of the piglets infected with PEDV are reduced by cooperatively adding organic iron into the feed, the diarrhea rate is 30-40%, the death rate is 20%, and the synergistic effect is good, wherein when the 5-aminolevulinic acid is used together with iron glycinate or ferrous fumarate, the synergistic effect is optimal, and the lysine is secondary;
Compared with a control group added with 5-aminolevulinic acid alone, the synergistic addition of IGF-1 (insulin-like growth factor-1) reduces the diarrhea rate and death rate of piglets infected with PEDV by 30%, and has good synergistic effect, wherein the death rate is 20%;
Compared with a control group added with 5-aminolevulinic acid alone, the synergistic addition of the antibacterial peptide reduces the diarrhea rate and the death rate of piglets infected with PEDV by 30%, and has 20% of diarrhea rate and good synergistic effect;
The experiment shows that the 5-aminolevulinic acid has remarkable control effect on piglets infected by PEDV, has synergistic effect with organic iron, particularly has optimal use effect of lactoferrin, iron glycine or ferrous fumarate together with the organic iron, and can obtain good synergistic effect of IGF-1, antibacterial peptide and 5-aminolevulinic acid. The above beneficial components can be used singly or together to cooperate with 5-aminolevulinic acid to exert the effect of preventing and treating PEDV infection
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (6)
- The application of 5-aminolevulinic acid in preparing a product for preventing and treating porcine epidemic diarrhea is characterized in that the addition amount of 5-aminolevulinic acid in the product is 10-1500 g/ton for piglets in the period from creep to weaning, the addition amount of 5-aminolevulinic acid in the product is 10-1500 g/ton of feed for piglets in the period from weaning to 90 days, and the addition amount of 5-aminolevulinic acid in the product is 10-1000 g/ton of feed for lactating sows.
- 2. Use of 5-aminolevulinic acid according to claim 1 for the preparation of a product for controlling porcine epidemic diarrhea, wherein the amount of 5-aminolevulinic acid added to the product is 1000 g/ton for piglets in the creep-to-weaning stage.
- 3. Use of 5-aminolevulinic acid according to claim 1 for the preparation of a product for controlling porcine epidemic diarrhea, wherein the amount of 5-aminolevulinic acid added to the product is 500 g/ton for piglets at weaning to 90 days of age.
- 4. Use of 5-aminolevulinic acid according to claim 1 for the preparation of a product for controlling porcine epidemic diarrhea, wherein the amount of 5-aminolevulinic acid added to the product for lactating sows is 500 g/ton.
- 5. The use of 5-aminolevulinic acid according to claim 1 for the preparation of a product for the prevention and treatment of porcine epidemic diarrhea, wherein the product further comprises one or more of lactoferrin, organic iron, IGF-1, and antimicrobial peptides.
- 6. The use of 5-aminolevulinic acid according to claim 5 for the preparation of a product for the prevention and treatment of porcine epidemic diarrhea, wherein the organic iron is iron glycinate or ferrous fumarate.
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| CN113170840A (en) * | 2021-05-20 | 2021-07-27 | 河南邑鸿善成生物技术有限公司 | Feed additive containing 5-aminolevulinic acid and ferrous glycinate as well as preparation method and application thereof |
| CN113475634A (en) * | 2021-06-28 | 2021-10-08 | 河南邑鸿善成生物技术有限公司 | Pig feed containing 5-aminolevulinic acid, preparation method and application of pig feed to piglets |
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| JP2000014329A (en) * | 1998-07-02 | 2000-01-18 | Natl Fedelation Of Agricult Coop Assoc | Agent for preventing and treating swine viral diarrhea |
| KR100459918B1 (en) * | 2002-06-19 | 2004-12-03 | (주)엔바이로젠 | A livestock feed additive comprising δ-aminolevulinic acid for enhancing viability of livestock |
| KR101314806B1 (en) * | 2011-06-10 | 2013-10-08 | 제너럴바이오(주) | Animal feed containing powder and extracts of 5-Aminolevulinic acid, Sanguisorba officinalis L. and Rhus japonica L. |
| JP6395855B2 (en) * | 2014-04-03 | 2018-09-26 | ベーリンガー インゲルハイム フェトメディカ インコーポレイテッド | Porcine epidemic diarrhea virus vaccine |
| GB201905940D0 (en) * | 2019-04-29 | 2019-06-12 | Intract Pharma Ltd | 5-aminolevulinic acid for the local treatment of inflammatory bowel disease |
| JP7497790B2 (en) * | 2019-12-27 | 2024-06-11 | 国立大学法人北海道大学 | Treatment and/or prevention agent for swine cholera |
| US11883376B2 (en) * | 2020-04-22 | 2024-01-30 | Nadimpally Satyavarahala Raju | Viral infection treatment with 5-aminolevulinic acid |
| WO2022014654A1 (en) * | 2020-07-15 | 2022-01-20 | ネオファーマジャパン株式会社 | Therapeutic and/or prophylactic agent for african swine fever (asf) |
| CN114773457B (en) * | 2022-03-10 | 2023-06-23 | 上海交通大学 | Recombinant adenovirus expressing pig-derived anti-porcine epidemic diarrhea virus N protein single-chain antibody and its construction and use |
| CN116549429B (en) * | 2023-07-06 | 2023-09-15 | 北京挑战生物技术有限公司 | Application of 5-aminolevulinic acid in preparation of products for preventing and treating porcine reproductive and respiratory syndrome |
| CN117243293B (en) * | 2023-11-13 | 2024-01-30 | 北京挑战生物技术有限公司 | Application of 5-aminolevulinic acid in preparation of product for preventing and treating constipation of sow |
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| CN113170840A (en) * | 2021-05-20 | 2021-07-27 | 河南邑鸿善成生物技术有限公司 | Feed additive containing 5-aminolevulinic acid and ferrous glycinate as well as preparation method and application thereof |
| CN113475634A (en) * | 2021-06-28 | 2021-10-08 | 河南邑鸿善成生物技术有限公司 | Pig feed containing 5-aminolevulinic acid, preparation method and application of pig feed to piglets |
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