Use of creatine for the preparation of a medicament or feed additive for the treatment or prevention of pathogenic diarrhoea in pigs

Abstract

This application discloses the application of creatine in the preparation of drugs or feed additives for the treatment or prevention of pathogenic diarrhea in pigs. The drugs are creatine or creatine as an active ingredient with added excipients to form oral liquids, granules, tablets, or powders. The dosage is 30–300 mg / kg of pig body weight; or it can be directly added to feed at a creatine content of 0.05%–0.5% of the total weight of the pig feed. The drugs or feed additives provided in this application are highly safe, have no toxic side effects, and can effectively prevent and treat pathogenic diarrhea caused by one or more pathogens, including porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus, and enterotoxigenic Escherichia coli. They improve the intestinal morphology and absorption function of sick pigs and reduce the morbidity and mortality of piglets.

Description

Technical Field

[0001] This application relates to the field of swine virus prevention and control technology, specifically to the application of creatine in the preparation of drugs or feed additives for the treatment or prevention of swine pathogenic diarrhea. Background Technology

[0002] Porcine diarrheal diseases mainly include porcine epidemic diarrhea (PED), transmissible gastroenteritis of swine (TGE), and rotavirus disease, all of which are caused by porcine epidemic viruses.

[0003] Porcine epidemic diarrhea (PED) is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). PED is a coronavirus that can infect pigs of all ages and breeds, but it has a high mortality rate in piglets, reaching 100% in piglets under seven days old. PEDV primarily infects and replicates in the villi cells of the small intestine, and clinical symptoms include vomiting, diarrhea, and dehydration. Transmissible gastroenteritis (TGE) is a highly contagious viral infectious disease caused by porcine transmissible gastroenteritis virus (TGEV). It is characterized by vomiting, severe diarrhea, dehydration, and high mortality in piglets under two weeks of age. TGE is particularly harmful to newly infected herds, causing 100% morbidity in pigs of all ages within a short period. The severity of the disease varies with age; younger piglets experience more severe illness and higher mortality rates, with mortality rates reaching 90%–100% in piglets under two weeks of age. Porcine rotavirus diarrhea (PoRV) is an acute intestinal infectious disease caused by porcine rotavirus (PoRV), primarily affecting piglets and causing symptoms such as vomiting, diarrhea, dehydration, and weight loss. PoRV is mainly transmitted through the fecal-oral route. The virus is present in the digestive tract of infected and carrier pigs and is excreted in their feces. Once an outbreak occurs, it can happen year after year; therefore, infected pigs and asymptomatic carriers are the main sources of infection for Porcine rotavirus diarrhea.

[0004] Furthermore, research has found that Escherichia coli can be classified into six categories based on their biological characteristics: enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterohemorrhagic Escherichia coli (EHEC), enteroadhesive Escherichia coli (EAEC), and disseminatedly adherent Escherichia coli (DAEC). Among these, ETEC is a significant pathogen causing diarrhea in piglets in the livestock industry. During infection, ETEC can enter the piglet's body through ingestion. Its fimbriae can adhere to the intestinal epithelial cells, helping to prevent ETEC from being detached by intestinal mucus and peristalsis. Subsequently, ETEC grows and multiplies on the intestinal epithelial cells, releasing large amounts of endotoxins, damaging intestinal function, leading to intestinal metabolic disorders, and disrupting water and electrolyte balance, thereby causing diarrhea, dehydration, and even death in piglets.

[0005] Swine diarrheal diseases cause massive piglet deaths, resulting in huge economic losses and severely hindering the development of the pig farming industry. Currently, the prevention and control of swine diarrheal diseases mainly relies on vaccines. However, due to the overuse of large doses of antiviral and antibacterial drugs, the mutation rate of viral (bacterial) strains far exceeds the speed of vaccine development. Furthermore, inconsistent vaccine quality, cross-infection with different pathogens, and secondary infections can also lead to unsatisfactory disease control effects after vaccination. Therefore, relying solely on vaccines is prone to immunization failure, and large-scale outbreaks of swine diarrheal diseases still frequently occur.

[0006] Therefore, developing functional nutrients with antiviral or antibacterial effects and feeding them to pigs to treat and prevent swine diarrhea will meet market demand. Summary of the Invention

[0007] In view of this, the purpose of this application is to provide the use of creatine in the preparation of drugs or feed additives for the treatment or prevention of pathogenic diarrhea in pigs. In this application, creatine and compositions including creatine have antiviral activity against various porcine viruses and bacteria that cause diarrheal diseases in pigs, such as PEDV, TGEV, PoRV, and ETEC. These drugs or feed additives can be used to prepare drugs or feed additives for the treatment or prevention of pathogenic diarrhea in pigs, which are effective in preventing and treating diarrheal diseases in pigs, and have high safety and no toxic side effects.

[0008] To achieve, or at least partially achieve, the above objectives, this application provides the following technical solution:

[0009] Application of creatine in the preparation of drugs or feed additives for the treatment or prevention of pathogenic diarrhea in pigs.

[0010] Compared with the prior art, this application has at least the following beneficial effects:

[0011] 1. Creatine has a high safety profile and can be used as a feed additive for a long time to prevent diseases. Long-term use will not lead to the development of drug-resistant strains or other toxic side effects, which is an advantage that other anti-disease drugs do not have.

[0012] 2. Creatine effectively prevents and treats infections such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), rotavirus, and Escherichia coli by enhancing the pig's natural immune function and antiviral and antibacterial activity, thereby reducing the incidence of swine diarrhea and the mortality rate of affected pigs. Creatine has significant efficacy in treating and preventing swine viral diarrhea.

[0013] 3. In vitro experimental results showed that creatine significantly inhibited the in vitro proliferation of PEDV, TGEV, PoRV and ETEC.

[0014] 4. In vivo experimental results show that creatine enhances innate immune function, has antiviral and antibacterial activity, improves intestinal structure and absorption function in pigs, and can effectively prevent and treat infections of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, rotavirus, and Escherichia coli, thereby reducing the diarrhea rate and mortality rate of sick pigs and effectively improving the survival rate of pigs. Oral administration of creatine at 300 mg / kg body weight or addition to piglet feed can achieve a protection rate of approximately 80% against infections of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, rotavirus, and Escherichia coli. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0016] Those skilled in the art will understand that, unless otherwise stated, the terms "the," "the," and "the foregoing" used in this application may also include plural forms. It should be further understood that the word "comprising" as used in the specification of this application means the presence of the stated features, steps, or operations, but does not exclude the presence or addition of one or more other features, integers, or steps.

[0017] Those skilled in the art will understand that, where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field; and where the manufacturers of the raw materials or instruments and equipment used are not specified, they are all conventional products that can be obtained commercially.

[0018] Those skilled in the art will understand that, unless otherwise stated in this application, when numerical ranges are given in the embodiments, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application, as well as the prior art known to those skilled in the art and the descriptions in this application, can be implemented using any prior art methods, devices, and materials similar to or equivalent to those described, used, or made by means of methods, devices, and materials in the embodiments of this application.

[0019] The embodiments of this application have shown through in vivo and in vitro experiments that creatine can inhibit the proliferation of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus, and enterotoxigenic Escherichia coli, as well as inhibit their infection of cells. That is, creatine has anti-porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus, and Escherichia coli infection effects.

[0020] Based on this, embodiments of this application provide the use of creatine in the preparation of drugs or feed additives for treating or preventing pathogenic diarrhea in pigs.

[0021] In some embodiments, the swine pathogenic diarrhea is caused by infection with at least one of the pathogens PEDV, TGEV, PoRV, and ETEC.

[0022] In some embodiments, the drug or feed additive is creatine, meaning that creatine can be directly used as a drug or feed additive to treat or prevent pathogenic diarrhea in pigs.

[0023] In some embodiments, the drug or feed additive is a creatine composition comprising creatine and excipients, wherein the excipients include at least one of anhydrous ethanol, Tween-80, water for injection, lactose, soluble starch, and wheat bran.

[0024] In some embodiments, the creatine composition is a solution of creatine mixed with three solvents: anhydrous ethanol, Tween-80, and water, wherein the concentration of creatine is 50–100 mg / mL.

[0025] In some embodiments, the creatine composition is a solid mixture of creatine, lactose, and soluble starch, wherein the mass fraction of creatine in the solid mixture is 45% to 55%.

[0026] In some embodiments, the creatine composition is a powdered mixture of creatine and soluble starch or bran, wherein the mass fraction of creatine in the powdered mixture is 45% to 55%.

[0027] In some embodiments, when the drug or feed additive is a creatine composition, its formulation type includes at least one of oral liquid, granules, tablets, and powders, and the formulation can be prepared using conventional pharmaceutical formulation methods.

[0028] In some embodiments, the method of using the drug or feed additive includes at least one of the following methods:

[0029] (1) Add the creatine composition to the feed at a dosage of 30-300 mg / kg of pig body weight;

[0030] (2) Creatine is administered alone as a drug at a dose of 30 to 300 mg / kg of pig body weight; or creatine is added directly to feed as a feed additive at a dose of 0.05% to 0.5% of the total feed weight.

[0031] (3) When used for daily prevention of swine pathogenic diarrhea, it can be added to the feed at a dose of 30 mg / kg pig body weight or 0.05% of total feed weight and used throughout the feeding cycle.

[0032] (4) When used for the clinical treatment of pathogenic diarrhea in pigs, the oral solution is administered orally at a dose of 300 mg / kg of pig body weight of creatine; or the powder, granules or tablets are fed directly at a dose of 300 mg / kg of pig body weight of creatine; or the powder, granules or tablets are mixed with the diet at a dose of 300 mg / kg of pig body weight of creatine and added to the feed; or creatine is mixed directly with the diet at a dose of 0.5% of the total feed weight, added three times a day in the morning, noon and evening, for a continuous week.

[0033] The technical solution of this application and the technical effects achieved will be described in detail below through more specific embodiments.

[0034] Example 1

[0035] This embodiment provides a method for preparing creatine oral solution, including the following steps:

[0036] (1) Weigh out 10g of creatine, 20mL of anhydrous ethanol and 30mL of Tween-80 and set aside;

[0037] (2) Dissolve creatine in anhydrous ethanol, then add Tween-80, and finally add water for injection to make up to 100 mL. Mix well to obtain a creatine solution with a concentration of 100 mg / mL, namely the creatine oral solution J1.

[0038] Example 2

[0039] This embodiment provides a method for preparing creatine oral solution, including the following steps:

[0040] (1) Weigh out 5g of creatine, 20mL of anhydrous ethanol and 30mL of Tween-80 and set aside;

[0041] (2) Dissolve creatine in anhydrous ethanol, then add Tween-80, and finally add water for injection to make up to 100 mL. Mix well to obtain a creatine solution with a concentration of 50 mg / mL, namely the creatine oral solution J2.

[0042] Example 3

[0043] This embodiment provides a method for preparing creatine granules, including the following steps:

[0044] (1) Weigh out 50g creatine, 15g lactose and 35g soluble starch and set aside;

[0045] (2) Mix creatine, lactose and soluble starch and granulate, dry and granulate to obtain granules with a creatine mass fraction of 50%, namely the creatine granules J3.

[0046] Example 4

[0047] This embodiment provides a method for preparing creatine powder, including the following steps:

[0048] (1) Weigh out 50g of creatine and 50g of soluble starch and set aside;

[0049] (2) Mix creatine and soluble starch evenly, pass through a 60-mesh sieve to obtain a powder with a creatine mass fraction of 50%, namely the creatine powder J4.

[0050] Example 5

[0051] This embodiment provides a method for preparing creatine tablets, including the following steps:

[0052] (1) Weigh out 50g creatine, 15g lactose and 35g soluble starch and set aside;

[0053] (2) Mix creatine, lactose and soluble starch evenly, compress into tablets using a tablet press, and dry to obtain tablets with a creatine mass fraction of 50%, namely the creatine tablet J5.

[0054] The formulations and parameters of the various creatine preparations prepared in the above embodiments are shown in Table 1 below:

[0055] Table 1

[0056] Example number Composition Number Components Creatine concentration Formulation type Example 1 J1 Creatine, anhydrous ethanol, Tween-80 100mg / mL oral liquid Example 2 J2 Creatine, anhydrous ethanol, Tween-80 50mg / mL oral liquid Example 3 J3 Creatine, lactose, soluble starch 50% (mass fraction) Granules Example 4 J4 Creatine, soluble starch 50% (mass fraction) powder Example 5 J5 Creatine, lactose, soluble starch 50% (mass fraction) tablet

[0057] Inhibitory effect of creatine on the in vitro proliferation of PEDV, TGEV, PoRV, and ETEC

[0058] This application's embodiments validated the inhibitory effect of creatine on the in vitro proliferation of four porcine diarrhea pathogens: PEDV, TGEV, PoRV, and ETEC. The validation method is as follows:

[0059] 1. Materials and Methods

[0060] 1.1 Viruses: Porcine epidemic diarrhea virus (PEDV) YN strain, porcine transmissible gastroenteritis virus (TGEV) WH1 strain, and porcine rotavirus (PoRV) TM-a strain were isolated, preserved, and donated by the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University; Escherichia coli (ETEC) K88 strain was donated by the Guangdong Academy of Agricultural Sciences.

[0061] 1.2 Cells: African green monkey kidney cells (Vero cells) were used for the proliferation of PEDV YN strain; porcine kidney cells (PK-15) were used for the proliferation of TGEV WH1 strain; rhesus monkey embryonic kidney passaged cell line (MA-104 cells) was used for the proliferation of PoRV TM-a strain. All cells were kindly provided by Huazhong Agricultural University; LB liquid and semi-solid media were used for the culture of ETEC K88.

[0062] 1.3 Test drug: Creatine (Maclean's, 98% purity).

[0063] 1.4 Reagents: DMEM medium, EDTA, trypsin, fetal bovine serum and penicillin-streptomycin were purchased from GIBCO; tryptone, yeast extract, sodium chloride and agar were purchased from SIGMA.

[0064] 1.5 In vitro proliferation inhibition:

[0065] (1) PEDV in vitro proliferation inhibition assay: PEDV YN strain virus solution was serially diluted 10-fold with DMEM maintenance medium, and 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 Six dilutions were seeded into 96-well plates containing pre-cultured Vero cell monolayers at 100 μL / well. DMEM maintenance medium containing different concentrations of creatine was added to each group, and the plates were incubated at 37°C with 5% CO2 for 36 h. Cytopathic effects were observed. The number of wells with positive cytopathic effects for each dilution was counted, and the TCID for each creatine group was calculated using the Reed-Muench method. 50 Comparing TCIDs of different groups 50 The value was used to determine the inhibitory effect of creatine on PEDV proliferation in vitro, and three replicates were performed for each group.

[0066] (2) In vitro proliferation inhibition assay of TGEV: The TGEV WH1 strain virus solution was serially diluted 10-fold with DMEM maintenance medium, and 10... -2 10 -3 10 -4 10 -5 10 -6 10 -7 Six dilutions were seeded into 96-well plates containing pre-cultured PK15 cell monolayers at 100 μL / well. DMEM maintenance medium containing different concentrations of creatine was added to each group, and the plates were incubated at 37°C with 5% CO2 for 36 h. Cytopathic effects were observed. The number of wells with positive cytopathic effects for each dilution was counted, and the TCID for each creatine group was calculated using the Reed-Muench method. 50 Comparing TCIDs of different groups 50 The value was used to determine the inhibitory effect of creatine on the in vitro proliferation of TGEVs, and three replicates were performed for each group.

[0067] (3) PoRV in vitro proliferation inhibition assay: The PoRV TM-a strain virus solution was serially diluted 10-fold with DMEM maintenance medium, and 10 liters were taken. -2 10 -3 10 -4 10 -5 10 -6 10 -7 Six dilutions were seeded into 96-well plates containing pre-cultured MA-104 cell monolayers at 100 μL / well. DMEM maintenance medium containing different concentrations of creatine was added to each group, and the plates were incubated at 37°C with 5% CO2 for 36 h. Cytopathic effects were observed. The number of wells with positive cytopathic effects for each dilution was counted, and the TCID for each creatine group was calculated using the Reed-Muench method. 50 Comparing TCIDs of different groups 50 The value was used to determine the inhibitory effect of creatine on the in vitro proliferation of PoRV. Three replicates were performed for each group.

[0068] (4) ETEC in vitro proliferation inhibition test: ETEC bacterial suspension was added to plate counting agar medium that had been autoclaved and cooled to 45°C. 15 mL of the agar was poured into a sterilized glass petri dish, and an Oxford cup was placed in the dish. After solidification, 100 μL of liquid containing different concentrations of creatine was pipetted into the Oxford cup. The petri dish was placed in a 37°C incubator for 24 h, and the diameter of the inhibition zone was measured. LB liquid medium was used as a blank control, and each creatine concentration was repeated in triplicate.

[0069] 1.6 Data Analysis: Experimental data were analyzed using one-way ANOVA and Duncan's method for multiple comparisons in SPSS 26.0 statistical software. P < 0.05 was considered significant, and P < 0.01 was considered highly significant. Results are expressed as mean ± standard deviation.

[0070] 2. Experimental Results:

[0071] Table 2 shows the TCID of different doses of creatine against different viruses. 50 The results are affected by the differences, where a, b, c, and d are indicators of significant differences.

[0072] Table 2

[0073]

[0074]

[0075] Table 2 shows that with increasing creatine concentration, the TCID of PEDV on Vero cells... 50 The significant decrease indicates that creatine has a certain inhibitory effect on the proliferation of PEDV in cultured Vero cells. With increasing creatine dosage, the TCID of TGEV on PK15 cells... 50 The significant decrease indicates that creatine has a significant inhibitory effect on the proliferation of TGEV in PK15 cells cultured in vitro. With increasing creatine concentration, the TCID of PoRV on MA-104 cells also decreased. 50 The significant decrease indicates that creatine has a significant inhibitory effect on the proliferation of PoRV in MA-104 cells cultured in vitro.

[0076] Table 3 shows the results of creatine inhibition of ETEC at different doses, where a, b, and c are indicators of significant differences.

[0077] Table 3

[0078] Group Diameter of the inhibition zone (cm) Blank group <![CDATA[0.00±0.00 c ]]> 10 μg / mL group <![CDATA[0.80±0.24 b ]]> 50 μg / mL group <![CDATA[1.23±0.37 bc ]]> 100 μg / mL group <![CDATA[2.24±0.19 a ]]>

[0079] As shown in Table 3, the diameter of the inhibition zone increases with the increase of creatine concentration, indicating that creatine has an inhibitory effect on the proliferation of ETEC.

[0080] The protective effect of creatine preparations against PEDV infection in piglets

[0081] This application's embodiments validated the protective effect of creatine preparations against PEDV-infected piglets, and the validation method is as follows:

[0082] 1. Materials and Methods:

[0083] 1.1 Test virus: PEDV YN strain, donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0084] 1.2 Test drug: Creatine preparation J1.

[0085] 1.3 Experimental Animals: Healthy 7-day-old piglets of the same breed and origin, with similar weight and no maternal antibodies, were selected for the challenge experiment. The piglets were divided into three experimental groups (low, medium, and high dose groups of creatine preparation) and one blank group. Creatine preparation was dissolved in milk and diluted to 3 mL before oral administration. The low-dose group (adding creatine at a dose of 30 mg / kg pig body weight), the medium-dose group (adding creatine at a dose of 100 mg / kg pig body weight), the high-dose group (adding creatine at a dose of 300 mg / kg pig body weight), and the blank group (feeding the same dose of milk without creatine preparation) consisted of 10 piglets in each group.

[0086] 1.4 Challenge Test: After feeding for one week, piglets in each group were challenged with 10... 7 TCID 50 Each animal was challenged with PEDV (NY strain) at a dose of / mL and orally administered 5mL of virus solution. The control group was orally administered the same dose of cell culture medium (DMEM). The animals were then isolated and observed for 7 consecutive days until the end of the experiment.

[0087] 1.5 Observation indicators: The diarrhea status of experimental piglets was observed daily. At the end of the experimental period, the piglets were slaughtered to observe intestinal lesions. At the same time, the number of dead pigs was counted, and the diarrhea rate, mortality rate, and protection rate were calculated. The inhibitory effect of creatine preparation on PEDV virus was determined by real-time quantitative PCR detection of the expression level of PEDV structural gene N or M in the intestinal mucosa of piglets. The detection of changes in intestinal morphology and structure was determined by hematoxylin-eosin staining. Intestinal absorption function was determined by detecting plasma D-xylose content.

[0088] 1.6 Data Analysis: Experimental data were analyzed using one-way ANOVA and Duncan's method in SPSS 26.0 statistical software. P < 0.05 was considered significant, and P < 0.01 was considered highly significant. Results are expressed as mean ± standard deviation.

[0089] 2. Experimental Results:

[0090] Table 4 shows the morbidity and mortality rates of piglets infected with PEDV after adding different doses of creatine to milk and feeding it to them. A, B, C, and D are indicators of significant differences.

[0091] Table 4

[0092] Group Diarrhea rate (%) mortality rate(%) Protection rate (%) Blank group <![CDATA[100 a ]]> <![CDATA[100 a ]]> <![CDATA[0 c ]]> 30mg / kg group <![CDATA[75 b ]]> <![CDATA[65 b ]]> <![CDATA[35 b <!-- 6 -->]]> 100mg / kg group <![CDATA[55 c ]]> <![CDATA[30 c ]]> <![CDATA[70 a ]]> 300mg / kg group <![CDATA[25 d ]]> <![CDATA[20 c ]]> <![CDATA[80 a ]]>

[0093] As shown in Table 4, different doses of creatine preparations can significantly reduce the diarrhea rate and mortality rate of PEDV-infected piglets, indicating that creatine preparations can play a protective role against PEDV-infected piglets. Compared with the control group, the diarrhea rate of PEDV-infected piglets decreased to 25%–75%, while the mortality rate of the affected piglets decreased to 20%–65%.

[0094] The expression levels of PEDV structural genes N and M in the intestinal mucosa of the jejunum and ileum of piglets were detected. The results are shown in Table 5 below, where a, b, c, and d are statistically significant markers.

[0095] Table 5

[0096]

[0097] As shown in Table 5, creatine preparations can significantly reduce the expression levels of the N and M genes of PEDV, indicating that creatine preparations can inhibit PEDV replication in vivo. Among them, a, b, c, and d are the markers of significant difference.

[0098] Table 6 shows the results of changes in intestinal morphology and structure in piglets of each group, where a, b, c, and d are indicators of significant differences.

[0099] Table 6

[0100]

[0101]

[0102] As shown in Table 6, creatine preparations can significantly increase the villus height and villus surface area in the jejunum and ileum, and reduce the villus height / crypt depth, indicating that creatine preparations can improve the intestinal morphology and structure of PEDV-infected piglets and promote intestinal health.

[0103] Table 7 shows the effects of creatine preparations on the absorption function of PEDV-infected piglets.

[0104] Table 7

[0105] Testing items control group Blank group 30mg / kg group 100mg / kg group 300mg / kg group D-xylose content (mmol / L) <![CDATA[0.67±0.12 a ]]> <![CDATA[0.15±0.08 c ]]> <![CDATA[0.21±0.11 bc ]]> <![CDATA[0.39±0.12 b ]]> <![CDATA[0.57±0.18 a ]]>

[0106] As shown in Table 7, creatine preparations can significantly increase plasma D-xylose content, indicating that adding creatine preparations can improve the small intestinal absorption function of PEDV-infected piglets.

[0107] The protective effect of creatine preparations against TGEV infection in piglets

[0108] This application's embodiments verified the protective effect of creatine preparations against TGEV-infected piglets, and the verification method is as follows:

[0109] 1. Materials and Methods

[0110] 1.1 Test virus: TGEV WH1 strain, donated by the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University;

[0111] 1.2 Test drug: Creatine preparation J3;

[0112] 1.3 Experimental Animals: Healthy 7-day-old piglets of the same breed and origin, with similar weight and no maternal antibodies, were selected for the challenge experiment. The piglets were divided into three experimental groups (low, medium, and high dose groups of creatine preparation) and one blank group. Creatine preparation was dissolved in milk and diluted to 3 mL before oral administration. The low-dose group (adding creatine at a dose of 30 mg / kg pig body weight), the medium-dose group (adding creatine at a dose of 100 mg / kg pig body weight), the high-dose group (adding creatine at a dose of 300 mg / kg pig body weight), and the blank group (feeding the same dose of milk without creatine preparation) consisted of 10 piglets in each group.

[0113] 1.4 Challenge Test: After feeding for one week, piglets in each group were challenged with 10... 6 The animals were challenged with TGEV WH1 with a CID50 / mL, and each animal was orally administered 5mL of the culture medium (DMEM) while the control group was orally administered the same dose. They were then isolated and observed for 7 days until the end of the experiment.

[0114] 1.5 Observation indicators: The diarrhea of ​​experimental piglets was observed daily. At the end of the experimental period, the experimental piglets were slaughtered to observe intestinal lesions. At the same time, the number of dead pigs was counted, and the diarrhea rate, mortality rate and protection rate were calculated. The detection of changes in intestinal morphology and structure was determined by hematoxylin-eosin staining. Intestinal absorption function was determined by detecting plasma D-xylose content.

[0115] 1.6 Data Analysis: Experimental data were analyzed using one-way ANOVA and Duncan's method for multiple comparisons in SPSS 26.0 statistical software. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered highly significant. Results are expressed as mean ± standard deviation.

[0116] 2. Experimental Results:

[0117] Table 8 shows the morbidity and mortality rates of piglets infected with TGEV after adding different doses of creatine to milk and feeding them to piglets. A, B, C, and D are indicators of significant differences.

[0118] Table 8

[0119] Group Diarrhea rate (%) mortality rate(%) Protection rate (%) Blank group <![CDATA[100 a ]]> <![CDATA[100 a ]]> <![CDATA[0 d ]]> 30mg / kg group <![CDATA[70 b ]]> <![CDATA[50 b ]]> <![CDATA[50 c ]]> 100mg / kg group <![CDATA[30 c ]]> <![CDATA[20 c ]]> <![CDATA[80 b ]]> 300mg / kg group <![CDATA[10 d ]]> <![CDATA[0 d ]]> <![CDATA[100 a ]]>

[0120] Table 8 shows that different doses of creatine preparations significantly reduced the diarrhea rate and mortality rate of TGEV-infected piglets, indicating that creatine preparations can protect piglets infected with TGEV. Compared with the control group, the diarrhea rate of TGEV-infected piglets decreased to 10%–70%, while the mortality rate of affected piglets decreased to 0%–50%.

[0121] Table 9 shows the results of changes in intestinal morphology and structure in piglets of each group, where a, b, c, and d are indicators of significant differences.

[0122] Table 9

[0123]

[0124] As shown in Table 9, creatine preparations can significantly increase the villus height and villus surface area in the jejunum and ileum, and reduce the villus height / crypt depth, indicating that creatine preparations can improve the intestinal morphology and structure of TGEV-infected piglets and promote intestinal health.

[0125] Table 10 shows the effects of creatine preparations on the absorption function of TGEV-infected piglets.

[0126] Table 10

[0127] Testing items control group Blank group 30mg / kg group 100mg / kg group 300mg / kg group D-xylose content (mmol / L) <![CDATA[0.87±0.23 a ]]> <![CDATA[0.20±0.15 c ]]> <![CDATA[0.31±0.09 c ]]> <![CDATA[0.49±0.18 b ]]> <![CDATA[0.79±0.22 a ]]>

[0128] Table 10 shows that creatine preparations can significantly increase plasma D-xylose content, indicating that adding creatine preparations can improve the small intestinal absorption function of TGEV-infected piglets.

[0129] The protective effect of creatine preparations against PoRV infection in piglets

[0130] This application's embodiments validated the protective effect of creatine preparations against PoRV-infected piglets, and the validation method is as follows:

[0131] 1. Materials and Methods

[0132] 1.1 Test virus: PoRV TM-a strain, donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0133] 1.2 Creatine: Creatine preparation J4.

[0134] 1.3 Experimental Animals: Healthy 7-day-old piglets of the same breed and origin, with similar weight and no maternal antibodies, were selected for the challenge experiment. The piglets were divided into three experimental groups (low, medium, and high dose groups of creatine preparation) and one blank group. Creatine preparation was dissolved in milk and diluted to 3 mL before oral administration. The low-dose group (adding creatine at a dose of 30 mg / kg pig body weight), the medium-dose group (adding creatine at a dose of 100 mg / kg pig body weight), the high-dose group (adding creatine at a dose of 300 mg / kg pig body weight), and the blank group (feeding the same dose of milk without creatine preparation) consisted of 10 piglets in each group.

[0135] 1.4 Challenge Test: After feeding for one week, piglets in each group were challenged with 10... 7 TCID 50 Animals were challenged with PoRV™-a at a dose of 5 mL / mL, and each animal was orally administered 5 mL of the virus solution. The control group was orally administered the same dose of cell culture medium (DMEM). The animals were then isolated and observed for 7 consecutive days until the end of the experiment.

[0136] 1.5 Observation indicators: The diarrhea of ​​experimental piglets was observed daily. At the end of the experimental period, the experimental piglets were slaughtered to observe intestinal lesions. At the same time, the number of dead pigs was counted, and the diarrhea rate, mortality rate and protection rate were calculated. The detection of changes in intestinal morphology and structure was determined by hematoxylin-eosin staining. Intestinal absorption function was determined by detecting plasma D-xylose content.

[0137] 1.6 Data Analysis: Experimental data were analyzed using one-way ANOVA and Duncan's method for multiple comparisons in SPSS 26.0 statistical software. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered highly significant. Results are expressed as mean ± standard deviation.

[0138] 2. Experimental Results:

[0139] Table 11 shows the morbidity and mortality rates of piglets infected with PoRV after adding different doses of creatine to milk and feeding it to them. A, B, C, and D are indicators of significant differences.

[0140] Table 11

[0141] Group Diarrhea rate (%) mortality rate(%) Protection rate (%) Blank group <![CDATA[100 a ]]> <![CDATA[100 a ]]> <![CDATA[0 d ]]> 30mg / kg group <![CDATA[75 b ]]> <![CDATA[60 b ]]> <![CDATA[40 c ]]> 100mg / kg group <![CDATA[35 c ]]> <![CDATA[30 c ]]> <![CDATA[70 b ]]> 300mg / kg group <![CDATA[15 d ]]> <![CDATA[0 d ]]> <![CDATA[100 a ]]>

[0142] Table 11 shows that different doses of creatine preparations significantly reduced the diarrhea rate and mortality rate of PoRV-infected piglets, indicating that creatine preparations can protect against PoRV-infected piglets. Compared with the control group, the diarrhea rate of PoRV-infected piglets decreased to 15%–75%, while the mortality rate of affected piglets decreased to 0%–60%.

[0143] Table 12 shows the results of changes in intestinal morphology and structure in piglets of each group, where a, b, c, and d are indicators of significant differences.

[0144] Table 12

[0145]

[0146] As shown in Table 12, creatine preparations can significantly increase the villus height and villus surface area in the jejunum and ileum of piglets, and reduce the villus height / crypt depth, indicating that creatine preparations can improve the intestinal morphology and structure of PoRV-infected piglets and promote intestinal health.

[0147] Table 13 shows the effects of creatine preparations on the absorption function of PoRV-infected piglets.

[0148] Table 13

[0149] Testing items control group Blank group 30mg / kg group 100mg / kg group 300mg / kg group D-xylose content (mmol / L) <![CDATA[0.82±0.14 a ]]> <![CDATA[0.25±0.23 c ]]> <![CDATA[0.33±0.12 c ]]> <![CDATA[0.52±0.26 b ]]> <![CDATA[0.78±0.26 a ]]>

[0150] Table 13 shows that creatine preparations can significantly increase plasma D-xylose content, indicating that adding creatine preparations can improve the small intestinal absorption function of PoRV-infected piglets.

[0151] The protective effect of creatine preparations against Escherichia coli infection in piglets

[0152] This application's embodiments verified the protective effect of creatine preparations against Escherichia coli-infected piglets, and the verification method is as follows:

[0153] 1. Materials and Methods

[0154] 1.1 Test strain: K88 strain, donated by Guangdong Academy of Agricultural Sciences.

[0155] 1.2 Test drug: Creatine preparation J5.

[0156] 1.3 Experimental Animals: Healthy 7-day-old piglets of the same breed and origin, with similar weights and no prior exposure to antibiotics were selected for the challenge experiment. The piglets were divided into three experimental groups (low, medium, and high dose groups of creatine preparation) and a control group. Creatine preparation was added to the feed of the experimental groups. The low-dose group (creatine preparation was added at a dose of 30 mg / kg pig body weight), the medium-dose group (creatine preparation was added at a dose of 100 mg / kg pig body weight), the high-dose group (creatine preparation was added at a dose of 300 mg / kg pig body weight), and the control group (no creatine preparation was added to the feed) had 10 piglets in each group.

[0157] 1.4 Challenge Test: After feeding for one week, piglets in each group were challenged with 10... 9K88 animals were challenged with CFU, and each animal was orally administered 4 mL of bacterial solution. The control group was administered the same dose of PBS. The animals were then isolated and observed for 7 days until the end of the experiment.

[0158] 1.5 Observation indicators: The diarrhea of ​​experimental piglets was observed daily. At the end of the experimental period, the experimental piglets were slaughtered to observe intestinal lesions and calculate the diarrhea rate. Changes in intestinal morphology and structure were determined by hematoxylin-eosin staining. Intestinal absorption function was determined by detecting plasma D-xylose content.

[0159] 1.6 Data Analysis: Experimental data were analyzed using one-way ANOVA and Duncan's method for multiple comparisons in SPSS 26.0 statistical software. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered highly significant. Results are expressed as mean ± standard deviation.

[0160] 2. Experimental Results:

[0161] Table 14 shows the morbidity and mortality rates of piglets in each group after adding different doses of creatine preparations to the feed, where a, b, c, and d are indicators of significant differences.

[0162] Table 14

[0163] Group Diarrhea rate (%) Blank group <![CDATA[100 a ]]> 30mg / kg group <![CDATA[75 b ]]> 100mg / kg group <![CDATA[45 c ]]> 300mg / kg group <![CDATA[10 d ]]>

[0164] As shown in Table 14, different doses of creatine preparations can significantly reduce the diarrhea rate of ETEC-infected piglets, indicating that creatine preparations can play a protective role against ETEC-infected piglets. Compared with the control group, the diarrhea rate of ETEC-infected piglets decreased to 10%–75%.

[0165] Table 15 shows the results of changes in intestinal morphology and structure in piglets of each group, where a, b, c, and d are indicators of significant differences.

[0166] Table 15

[0167]

[0168]

[0169] As shown in Table 15, creatine preparations can significantly increase the villus height and villus surface area in the jejunum and ileum, and reduce the villus height / crypt depth, indicating that creatine can improve the intestinal morphology and structure of ETEC-infected piglets and promote intestinal health.

[0170] Table 16 shows the effects of creatine preparations on the absorption function of PEDV-infected piglets.

[0171] Table 16

[0172] Testing items control group Blank group 30mg / kg group 100mg / kg group 300mg / kg group D-xylose content (mmol / L) <![CDATA[0.76±0.21 a ]]> <![CDATA[0.29±0.13 c ]]> <![CDATA[0.32±0.26 c ]]> <![CDATA[0.56±0.19 b ]]> <![CDATA[0.74±0.14 a ]]>

[0173] As shown in Table 16, creatine preparations can significantly increase plasma D-xylose content, indicating that adding creatine preparations can improve the small intestinal absorption function of ETEC-infected piglets.

[0174] In summary, creatine can be used to prepare drugs or feed additives for the treatment or prevention of pathogenic diarrhea in pigs. The prepared drugs or feed additives possess resistance to various porcine viruses and bacteria that cause diarrheal diseases, including PEDV, TGEV, PoRV, and ETEC. The dosage forms of these drugs and feed additives include oral liquids, granules, powders, and tablets.

[0175] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. The use of creatine in the preparation of drugs or feed additives for the treatment or prevention of swine pathogenic diarrhea, wherein the swine pathogenic diarrhea is caused by infection with at least one of porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), and enterotoxigenic Escherichia coli (ETEC).

2. The application according to claim 1, characterized in that, The drug or feed additive is a composition including creatine and excipients, wherein the excipients include at least one of anhydrous ethanol, Tween-80, water for injection, lactose, soluble starch, and wheat bran.

3. The application according to claim 2, characterized in that, The creatine composition is a solution of creatine mixed with three solvents: anhydrous ethanol, Tween-80, and water, wherein the concentration of creatine is 50~100 mg / mL.

4. The application according to claim 2, characterized in that, The creatine composition is a solid mixture of creatine, lactose, and soluble starch, wherein the mass fraction of creatine in the solid mixture is 45% to 55%.

5. The application according to claim 2, characterized in that, The creatine composition is a powdered mixture of creatine and soluble starch or wheat bran, wherein the mass fraction of creatine in the powdered mixture is 45% to 55%.

6. The application according to claim 2, characterized in that, The pharmaceutical preparation type includes at least one of oral liquid, granules, tablets, and powder.

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