Application of creatine in preparation of medicine or feed additive for treating or preventing swine pathogenic diarrhea

Abstract

The invention discloses an application of creatine in preparation of a medicine or a feed additive for treating or preventing pig pathogenic diarrhea, the medicine is creatine or is prepared into oral liquid, granules, tablets and powder by taking the creatine as an active ingredient and adding auxiliary materials, and the use dosage is that the content of the creatine is 30-300mg / kg of pig body weight; or the feed additive is directly added into the feed, and the adding amount is that the content of creatine accounts for 0.05-0.5% of the total weight of the pig feed. The medicine or the feed additive provided by the invention is high in safety and free from toxic and side effects, can effectively prevent and treat pathogenic diarrhea caused by infection of one or more pathogens of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus and enterotoxigenic escherichia coli, improves the intestinal morphological structure and the absorption function of diseased pigs, and improves the immunity of the pigs. The morbidity and mortality of piglets are reduced.

Description

Technical Field

[0001] The present application relates to the technical field of pig virus prevention and treatment, and specifically to the use of creatine in the preparation of drugs or feed additives for treating or preventing pig pathogenic diarrhea. Background Art

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

[0003] PED is a highly contagious enteric disease. Porcine epidemic diarrhea virus (PEDV) is the main cause of PED. It is a coronavirus. Pigs of different ages and breeds may be infected with PEDV and become ill, but it has a high mortality rate for piglets. The mortality rate of piglets under seven days old can reach 100%. PEDV mainly infects and replicates in the villus enterocytes of the small intestine. The clinical symptoms are vomiting, diarrhea and dehydration in pigs. TGE is a highly contagious viral infectious disease caused by porcine transmissible gastroenteritis virus (TGEV). The disease is characterized by vomiting, severe diarrhea, dehydration and high mortality in piglets within two weeks of age. TGE causes significant harm to pigs infected for the first time. It can cause 100% of pigs of all ages to become ill in a short period of time, and the severity of the disease varies with age. The younger the age, the more severe the disease and the higher the mortality rate. The mortality rate of piglets within two weeks of age can reach 90% to 100%. Porcine rotavirus diarrhea is an acute intestinal infectious disease caused by porcine rotavirus (PoRV), which mainly affects piglets, causing symptoms such as vomiting, diarrhea, dehydration and weight loss. PoRV is mainly transmitted through the fecal-oral route. The virus exists in the digestive tract of sick and infected pigs and is excreted from the body with feces. Once an outbreak occurs, it may occur year after year. Therefore, sick pigs and latently infected pigs are the main sources of infection for porcine rotavirus diarrhea.

[0004] In addition, studies have found that E. coli can be divided into six categories according to their biological characteristics: enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), enteroadhesive E. coli (EAEC) and diffusely adherent E. coli (DAEC). Among them, ETEC is an important pathogen that causes diarrhea in piglets in the breeding industry. During the infection of piglets, ETEC can enter the piglet's body through ingestion, and its pili can adhere to the pig's intestinal epithelial cells, which helps ETEC avoid falling off due to the flushing of intestinal mucus and intestinal peristalsis; then ETEC grows and multiplies on the intestinal epithelial cells, and releases a large amount of endotoxins, which damages intestinal function, causes intestinal metabolic disorders, and breaks the water and electrolyte balance, thus causing diarrhea, dehydration, and even death in piglets.

[0005] Swine diarrheal diseases have caused the death of a large number of piglets, resulting in huge economic losses and seriously hindering the development of the pig farming industry. At present, the prevention and control measures for swine diarrheal diseases mainly rely on vaccines, but due to the abuse of large doses of antiviral and antibacterial drugs, the mutation rate of virus (bacteria) strains far exceeds the speed of vaccine development. In addition, the uneven quality of vaccines, cross-infection of different pathogens, secondary infection, etc., can also lead to unsatisfactory prevention and control effects of diseases after vaccination. Therefore, relying solely on vaccines can easily lead to immune failure, and large-scale swine diarrheal diseases still often break out.

[0006] Therefore, developing functional nutrients with antiviral or antibacterial effects and feeding them to pigs to treat and prevent porcine diarrheal diseases will meet market needs. Summary of the invention

[0007] In view of this, the purpose of the present application is to provide the use of creatine in the preparation of a drug or feed additive for treating or preventing pathogenic diarrhea in pigs. In this application, creatine and a composition comprising creatine have resistance to a variety of pig viruses and bacteria that cause diarrheal diseases in pigs, such as PEDV, TGEV, PoRV and ETEC, and can be used to prepare a drug or feed additive for treating or preventing pathogenic diarrhea in pigs. The drug or feed additive has a good preventive and therapeutic effect on diarrheal diseases in pigs, and is highly safe and has no toxic side effects.

[0008] In order to achieve or at least partially achieve the above objectives, the present application provides the following technical solutions:

[0009] Application of creatine in preparing medicine or feed additive for treating or preventing pathogenic diarrhea in pigs.

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

[0011] 1. Creatine is highly safe and can be used as a feed additive for a long time to prevent diseases. Long-term use will not lead to the production of drug-resistant strains and will not have other toxic side effects. This is an advantage that other anti-disease drugs do not have.

[0012] 2. Creatine can effectively prevent and treat porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus, and Escherichia coli infection by enhancing the pig's natural immune function and antiviral and antibacterial activity, thereby reducing the incidence of porcine diarrhea and the mortality rate of infected pigs. Creatine has significant efficacy in treating and preventing porcine viral diarrhea.

[0013] 3. The results of in vitro experiments show that creatine has a significant inhibitory effect on the in vitro proliferation of PEDV, TGEV, PoRV and ETEC.

[0014] 4. In vivo experimental results show that creatine can enhance natural immune function, antiviral and antibacterial activity, improve pig intestinal structure and absorption function, and effectively prevent and treat porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, rotavirus and Escherichia coli infection, thereby reducing the diarrhea rate and mortality rate of diseased pigs and effectively improving the survival rate of pigs. Oral administration of creatine at 300 mg / kg pig body weight or adding it to piglet feed can achieve a protection rate of about 80% against porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus and Escherichia coli infection. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described here are only used to explain the present application and are not used to limit the present application.

[0016] Those skilled in the art will appreciate that, unless otherwise stated, the words "said", "the", and "the aforementioned" used in the text of the present application may also include plural forms. It should be further understood that the wording "including" used in the specification of the present application means the presence of the features, steps, and operations, but does not exclude the presence or addition of one or more other features, integers, and steps.

[0017] Those skilled in the art will appreciate that, for any specific experimental steps or conditions not specified in the examples, the experiments may be carried out according to the conventional experimental steps or conditions described in the literature in the art; any raw materials or instruments used without specifying the manufacturer are conventional products that can be purchased commercially.

[0018] Those skilled in the art will understand that, unless otherwise specified in the present application, when a numerical range is given in an embodiment, 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 are consistent with the prior art mastery of those skilled in the art and the records of this application, and any methods, equipment and materials of the prior art similar or equivalent to the methods, equipment and materials described in the embodiments of this application may also be used to implement this application.

[0019] The examples of the present application found 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, and 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, the embodiments of the present application provide the use of creatine in the preparation of a drug or feed additive for treating or preventing pathogenic diarrhea in pigs.

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

[0022] In certain embodiments, the drug or feed additive is creatine, that is, creatine can be directly used as a drug or feed additive for treating or preventing pathogenic diarrhea in pigs.

[0023] In certain 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 bran.

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

[0025] In certain 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 certain embodiments, the creatine composition is a powdery mixture of creatine and soluble starch or bran, wherein the mass fraction of creatine in the powdery mixture is 45% to 55%.

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

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

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

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

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

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

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

[0034] Example 1

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

[0036] (1) Weigh 10 g creatine, 20 mL anhydrous ethanol, and 30 mL 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, and obtain a creatine solution with a concentration of 100 mg / mL, i.e., the creatine oral solution J1.

[0038] Example 2

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

[0040] (1) Weigh 5 g creatine, 20 mL anhydrous ethanol, and 30 mL 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, and obtain a creatine solution with a concentration of 50 mg / mL, i.e., the creatine oral solution J2.

[0042] Example 3

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

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

[0045] (2) creatine, lactose and soluble starch are mixed and granulated, and then dried and granulated to obtain granules with a mass fraction of creatine of 50%, i.e., the creatine granules J3.

[0046] Example 4

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

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

[0049] (2) The creatine and soluble starch are mixed uniformly, and passed through a 60-mesh sieve to obtain a powder with a mass fraction of creatine of 50%, i.e., the creatine powder J4.

[0050] Example 5

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

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

[0053] (2) The creatine, lactose and soluble starch are mixed evenly, compressed into tablets using a tablet press, and dried to obtain tablets with a mass fraction of creatine of 50%, namely the creatine tablets J5.

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

[0055] Table 1

[0056] Example No. Composition No. Components Creatine concentration Formulation type Example 1 J1 Creatine, anhydrous ethanol, Tween-80 100mg / mL Oral solution Example 2 J2 Creatine, anhydrous ethanol, Tween-80 50mg / mL Oral solution 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 proliferation of PEDV, TGEV, PoRV and ETEC in vitro

[0058] The present application example verifies the inhibitory effect of creatine on the in vitro proliferation of four swine diarrhea pathogens, PEDV, TGEV, PoRV, and ETEC, and the verification 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 National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University; Escherichia coli (ETEC) K88 strain was donated by 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 cell line (MA-104 cells) were used for the proliferation of PoRV TM-a strain. All cells were donated by Huazhong Agricultural University; LB liquid and semi-solid medium were used for the culture of ETEC K88.

[0062] 1.3 Experimental drug: Creatine (McLean, purity 98%).

[0063] 1.4 Reagents: DMEM culture medium, EDTA, trypsin, fetal bovine serum and double antibody (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 test: PEDV YN strain virus liquid was diluted 10 times in DMEM maintenance medium, and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions were inoculated into 96-well plates with cultured Vero cell monolayers, 100 μL / well. DMEM maintenance solution containing different concentrations of creatine was added to each group, and the cells were cultured in a 37°C incubator with 5% CO2 for 36 hours to observe the cell lesions. The number of lesion-positive wells in each dilution was counted, and the TCID of each creatine group was calculated using the Reed-Muench method. 50 , compare different groups of TCID 50 The inhibitory effect of creatine on the proliferation of PEDV in vitro was determined by the value of , and three replicates were performed in each group.

[0066] (2) TGEV in vitro proliferation inhibition test: The TGEV WH1 strain virus liquid was diluted 10 times in DMEM maintenance medium, and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions were inoculated into 96-well plates with cultured PK15 cell monolayers, 100 μL / well. DMEM maintenance solution containing different concentrations of creatine was added to each group, and the cells were cultured in a 37°C incubator with 5% CO2 for 36 hours to observe the cell lesions. The number of lesion-positive wells in each dilution was counted, and the TCID of each creatine group was calculated using the Reed Muench method. 50 , compare different groups of TCID 50 The inhibitory effect of creatine on the proliferation of TGEV in vitro was judged by the value of , and three replicates were performed in each group.

[0067] (3) PoRV in vitro proliferation inhibition test: The PoRV TM-a strain virus solution was diluted 10-fold in DMEM maintenance medium, and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions were inoculated into the cultured MA-104 cell monolayer in a 96-well plate, 100 μL / well. DMEM maintenance solution containing different concentrations of creatine was added to each group, and the cells were cultured in a 37°C incubator with 5% CO2 for 36 hours to observe the cell lesions. The number of lesion-positive wells in each dilution was counted, and the TCID of each creatine group was calculated using the Reed-Muench method. 50 , compare different groups of TCID 50 The inhibitory effect of creatine on the proliferation of PoRV in vitro was determined by the value of , and three replicates were performed in each group.

[0068] (4) ETEC in vitro proliferation inhibition test: Add the ETEC bacterial suspension to the plate count agar medium that has been sterilized by high pressure and cooled to 45°C, take 15 mL and pour it into a sterilized glass plate, and place the Oxford cup in the plate; after it solidifies, use a pipette to draw 100 μL of liquid containing different concentrations of creatine and place it in the Oxford cup; place the plate in a 37°C incubator for 24 hours, and then measure the diameter of the inhibition zone. Use LB liquid medium as the blank control, and repeat three times for each creatine concentration.

[0069] 1.6 Data analysis: The experimental data were analyzed using one-way ANOVA and Duncan's multiple comparison method in SPSS26.0 statistical software, with P < 0.05 as the standard for significant difference and P < 0.01 as the standard for extremely significant difference. The results were expressed as mean ± standard deviation.

[0070] 2. Test results:

[0071] Table 2 shows the TCID of different doses of creatine against different viruses 50 Influence results, where a, b, c, and d are marks of significant difference.

[0072] Table 2

[0073]

[0074]

[0075] As shown in Table 2, with the increase of creatine concentration, the TCID 50 The results showed that creatine has a certain inhibitory effect on the proliferation of PEDV in Vero cells cultured in vitro. With the increase of creatine addition, the TCID 50 The TCID of PoRV on MA-104 cells decreased significantly with the increase of creatine concentration. 50 It showed that creatine had a significant inhibitory effect on the proliferation of PoRV in MA-104 cells cultured in vitro.

[0076] Table 3 shows the results of different doses of creatine inhibiting ETEC, where a, b, and c are marks of significant difference.

[0077] Table 3

[0078] Group Diameter of 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, with the increase of creatine concentration, the diameter of the inhibition zone increased, indicating that creatine has an inhibitory effect on the proliferation of ETEC.

[0080] Protective effect of creatine preparation on piglets infected with PEDV

[0081] The present application example verifies the protective effect of creatine preparation on piglets infected with PEDV, and the verification method is as follows:

[0082] 1. Materials and methods:

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

[0084] 1.2 Experimental drug: Creatine preparation J1.

[0085] 1.3 Experimental animals: 7-day-old healthy piglets of the same breed and origin, similar weight and without maternal antibodies were selected for the challenge test. The experimental piglets were divided into three experimental groups (low-, medium- and high-dose groups of creatine preparation) and a blank group. The creatine preparation was dissolved in milk and the volume was made up to 3 mL for oral administration, including the low-dose group of creatine preparation (added at a creatine content of 30 mg / kg pig body weight), the medium-dose group of creatine preparation (added at a creatine content of 100 mg / kg pig body weight), the high-dose group of creatine preparation (added at a creatine content of 300 mg / kg pig body weight) and the blank group (gavagely fed with the same dose of milk without creatine preparation), with 10 piglets in each group.

[0086] 1.4 Challenge test: After feeding for one week, the piglets in each group were fed with 10 7 TCID 50 / mL PEDV (NY strain) was used for infection. Each head was orally infused with 5mL of virus solution. The control group was infused with the same dose of cell culture medium (DMEM). Then, the mice were isolated and raised and observed for 7 consecutive days until the end of the experiment.

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

[0088] 1.6 Data analysis: The experimental data were analyzed using one-way ANOVA and Duncan's multiple comparison method in SPSS26.0 statistical software, with P < 0.05 as the standard for significant difference and P < 0.01 as the standard for extremely significant difference. The results are expressed as mean ± standard deviation.

[0089] 2. Test results:

[0090] After different doses of creatine preparations were added to milk and fed to piglets infected with PEDV, the morbidity and mortality rates of piglets in each group are shown in Table 4, where a, b, c, and d are 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 protect PEDV-infected piglets. Compared with the blank group, the diarrhea rate of PEDV-infected piglets decreased to 25%-75%, and the mortality rate of diseased pigs decreased to 20%-65%.

[0094] The expression levels of PEDV structural gene N or M in the intestinal mucosa of the jejunum and ileum tissues of piglets were detected respectively. The test results are shown in Table 5 below, where a, b, c, and d are significant difference marks.

[0095] Table 5

[0096]

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

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

[0099] Table 6

[0100]

[0101]

[0102] As shown in Table 6, creatine preparations can significantly increase the height and surface area of ​​villi in the jejunum and ileum, and reduce the height of villi / 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 results of the effects of creatine preparations on the absorption function of PEDV-infected piglets.

[0104] Table 7

[0105] Test 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 the plasma D-xylose content, indicating that the addition of creatine preparations can improve the small intestinal absorption function of PEDV-infected piglets.

[0107] Protective effect of creatine preparation on piglets infected with TGEV

[0108] The present application example verifies the protective effect of creatine preparation on TGEV-infected piglets, and the verification method is as follows:

[0109] 1. Materials and Methods

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

[0111] 1.2 Experimental drug: Creatine preparation J3;

[0112] 1.3 Experimental animals: 7-day-old healthy piglets of the same breed and origin, similar weight and without maternal antibodies were selected for the challenge test. The experimental piglets were divided into three experimental groups (low-, medium- and high-dose groups of creatine preparation) and a blank group. The creatine preparation was dissolved in milk and the volume was made up to 3 mL for oral administration, including the low-dose group of creatine preparation (added at a creatine content of 30 mg / kg pig body weight), the medium-dose group of creatine preparation (added at a creatine content of 100 mg / kg pig body weight), the high-dose group of creatine preparation (added at a creatine content of 300 mg / kg pig body weight) and the blank group (gavagely fed with the same dose of milk without creatine preparation), with 10 piglets in each group.

[0113] 1.4 Challenge test: After feeding for one week, the piglets in each group were fed with 10 6 The mice were challenged with TGEV WH1 with CID50 / mL, and 5 mL was orally infused to each head. The control group was infused with the same dose of cell culture medium (DMEM), and then isolated and raised, and observed for 7 consecutive days until the end of the experiment.

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

[0115] 1.6 Data analysis: The experimental data were analyzed using one-way ANOVA and Duncan's multiple comparison method in SPSS26.0 statistical software, with P < 0.05 as the standard for significant difference and P < 0.01 as the standard for extremely significant difference. The results are expressed as mean ± standard deviation.

[0116] 2. Test results:

[0117] After different doses of creatine preparations were added to milk and fed to piglets infected with TGEV, the morbidity and mortality rates of piglets in each group are shown in Table 8, where a, b, c, and d are 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] As shown in Table 8, different doses of creatine preparations can significantly reduce the diarrhea rate and mortality of TGEV-infected piglets, indicating that creatine preparations can protect TGEV-infected piglets. Compared with the blank group, the diarrhea rate of TGEV-infected piglets decreased to 10% to 70%, and the mortality rate of diseased pigs decreased to 0% to 50%.

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

[0122] Table 9

[0123]

[0124] As shown in Table 9, creatine preparations can significantly increase the villus height and villus surface area of ​​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 results of the effects of creatine preparations on the absorption function of TGEV-infected piglets.

[0126] Table 10

[0127] Test 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] As shown in Table 10, creatine preparations can significantly increase the plasma D-xylose content, indicating that the addition of creatine preparations can improve the small intestinal absorption function of TGEV-infected piglets.

[0129] Protective effect of creatine preparation on piglets infected with PoRV

[0130] The present application example verifies the protective effect of creatine preparation on piglets infected with PoRV, and the verification method is as follows:

[0131] 1. Materials and Methods

[0132] 1.1 Experimental 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: 7-day-old healthy piglets of the same breed and origin, similar weight and without maternal antibodies were selected for the challenge test. The experimental piglets were divided into three experimental groups (low-, medium- and high-dose groups of creatine preparation) and a blank group. The creatine preparation was dissolved in milk and the volume was made up to 3 mL for oral administration, including the low-dose group of creatine preparation (added at a creatine content of 30 mg / kg pig body weight), the medium-dose group of creatine preparation (added at a creatine content of 100 mg / kg pig body weight), the high-dose group of creatine preparation (added at a creatine content of 300 mg / kg pig body weight) and the blank group (gavagely fed with the same dose of milk without creatine preparation), with 10 piglets in each group.

[0135] 1.4 Challenge test: After feeding for one week, the piglets in each group were fed with 10 7 TCID 50 / mL PoRV TM-a was used for infection. Each head was orally infused with 5mL of virus solution. The control group was infused with the same dose of cell culture medium (DMEM). Then, the mice were isolated and raised and observed for 7 consecutive days until the end of the experiment.

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

[0137] 1.6 Data analysis: The experimental data were analyzed using one-way ANOVA and Duncan's multiple comparison method in SPSS26.0 statistical software, with P < 0.05 as the standard for significant difference and P < 0.01 as the standard for extremely significant difference. The results are expressed as mean ± standard deviation.

[0138] 2. Test results:

[0139] After different doses of creatine preparations were added to milk and fed to piglets infected with PoRV, the morbidity and mortality rates of piglets in each group are shown in Table 11, where a, b, c, and d are 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] As shown in Table 11, different doses of creatine preparations can significantly reduce the diarrhea rate and mortality of PoRV-infected piglets, indicating that creatine preparations can protect PoRV-infected piglets. Compared with the blank group, the diarrhea rate of PoRV-infected piglets decreased to 15% to 75%, and the mortality of diseased pigs decreased to 0% to 60%.

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

[0144] Table 12

[0145]

[0146] As shown in Table 12, creatine preparations can significantly increase the height and surface area of ​​the villi 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 results of the effects of creatine preparations on the absorption function of PoRV-infected piglets.

[0148] Table 13

[0149] Test 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] As shown in Table 13, creatine preparations can significantly increase the plasma D-xylose content, indicating that the addition of creatine preparations can improve the small intestinal absorption function of PoRV-infected piglets.

[0151] Protective effect of creatine preparation on piglets infected with Escherichia coli

[0152] The present application example verifies the protective effect of creatine preparation on piglets infected with E. coli, and the verification method is as follows:

[0153] 1. Materials and Methods

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

[0155] 1.2 Experimental drug: Creatine preparation J5.

[0156] 1.3 Experimental animals: 7-day-old healthy piglets of the same breed and origin, similar weight, and no exposure to antibiotics were selected for the challenge test. The experimental piglets were divided into three experimental groups (low-, medium-, and high-dose groups of creatine preparations) and a blank group. Creatine preparations were added to the feed of the experimental groups, including the low-dose group of creatine preparations (added at a creatine content of 30 mg / kg pig body weight), the medium-dose group of creatine preparations (added at a creatine content of 100 mg / kg pig body weight), the high-dose group of creatine preparations (added at a creatine content of 300 mg / kg pig body weight) and the blank group (no creatine preparations added to the feed), with 10 piglets in each group.

[0157] 1.4 Challenge test: After feeding for one week, the piglets in each group were fed with 10 9CFU of K88 were used for infection, and 4 mL of bacterial solution was orally infused into each head. The control group was infused with the same dose of PBS, and then they were isolated and raised, and observed for 7 consecutive days until the end of the experiment.

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

[0159] 1.6 Data analysis: The experimental data were analyzed using one-way ANOVA and Duncan's multiple comparison method in SPSS26.0 statistical software, with P < 0.05 as the standard for significant difference and P < 0.01 as the standard for extremely significant difference. The results are expressed as mean ± standard deviation.

[0160] 2. Test results:

[0161] After adding different doses of creatine preparations to the feed, the morbidity and mortality of piglets in each group are shown in Table 14, where a, b, c, and d are 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 protect ETEC infected piglets. Compared with the blank group, the diarrhea rate of ETEC infected piglets decreased to 10% to 75%.

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

[0166] Table 15

[0167]

[0168]

[0169] As shown in Table 15, creatine preparations can significantly increase the height and surface area of ​​villi in the jejunum and ileum, and reduce the height of villi / 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 results of the effects of creatine preparations on the absorption function of PEDV-infected piglets.

[0171] Table 16

[0172] Test 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 the plasma D-xylose content, indicating that the addition of 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 treating or preventing pathogenic diarrhea in pigs. The prepared drugs or feed additives have the ability to resist various pig viruses and bacteria that cause diarrheal diseases in pigs, such as PEDV, TGEV, PoRV and ETEC. The dosage forms of the drugs and feed additives include oral liquid, granules, powder, tablets and other dosage forms.

[0175] The present application is described in detail above. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the present application and its core ideas. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. Application of creatine in the preparation of medicines or feed additives for treating or preventing pathogenic diarrhea in pigs.

2. The use according to claim 1, characterized in that: The swine pathogenic diarrhea is caused by infection with at least one of PEDV, TGEV, PoRV and ETEC pathogens.

3. The use according to claim 1, characterized in that: The medicine or feed additive is creatine.

4. The use according to claim 1, characterized in that: The medicine or feed additive is a composition comprising creatine and auxiliary materials, wherein the auxiliary materials comprise at least one of anhydrous ethanol, Tween-80, water for injection, lactose, soluble starch and bran.

5. The use according to claim 4, characterized in that: The creatine composition is a solution formed by mixing creatine with three solvents, namely, anhydrous ethanol, Tween-80 and water, wherein the concentration of creatine is 50-100 mg / mL.

6. The use according to claim 4, 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%.

7. The use according to claim 4, characterized in that: The creatine composition is a powdery mixture of creatine and soluble starch or bran, wherein the mass fraction of creatine in the powdery mixture is 45% to 55%.

8. The use according to claim 4, characterized in that: When the medicine or feed additive is a creatine composition, its preparation type includes at least one of oral liquid, granules, tablets and powders.

9. The use according to claim 1, characterized in that: The method of using the drug or feed additive includes at least one of the following (1) to (4): (1) adding the creatine composition to feed at a dosage of 30 to 300 mg / kg of pig body weight; (2) Creatine is administered as a single drug at a dose of 30 to 300 mg / kg of pig body weight; or creatine is directly added to the feed as a feed additive at a dose of 0.05% to 0.5% of the total weight of the feed; (3) When used for daily prevention of pathogenic diarrhea in pigs, it can be added to feed at a dosage of 30 mg / kg of pig body weight or 0.05% of the total weight of feed and used throughout the entire feeding cycle; (4) When used for the clinical treatment of pathogenic diarrhea in pigs, the oral solution is orally administered at a dosage of 300 mg / kg of pig body weight; or the powder, granules or tablets are directly fed at a dosage of 300 mg / kg of pig body weight; or the powder, granules or tablets are mixed with the daily diet at a dosage of 300 mg / kg of pig body weight and added to the feed; or creatine is directly mixed with the daily diet at a dosage of 0.5% of the total weight of the feed and added three times a day in the morning, noon and evening for one week.

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