CN115089608A - Method for establishing standard for horse excrement transplantation - Google Patents

Abstract

The invention belongs to the technical field of animal clinical treatment, and discloses a method for establishing a standard for horse excrement transplantation, which is used for selecting a donor horse; preparing excrement; preparation and transplantation of a recipient; the selection of donor horses includes clinical examination, horse master questionnaire, biological examination; the biological examination comprises a blood examination and a stool examination; the stool transplant technique of the present invention is clinically useful in horses for the treatment of a variety of diseases including diarrhea, enteritis, laminitis and irritable bowel syndrome. The fecal transplantation technique treatment of the present invention can be used in horses of different age stages. The excrement transplantation technology has excellent effect of treating diarrhea, enteritis, laminitis, irritable bowel syndrome and the like. The average treatment times of the excrement transplanting technology are 4-9 times, and the safety is high. The invention provides a standardized horse excrement transplanting technology, which has the advantages of wider application, convenient use, simplicity, quickness, low cost and good treatment effect.

Description

Method for establishing standard for horse excrement transplantation

Technical Field

The invention belongs to the technical field of animal clinical treatment, and particularly relates to a method for establishing a standard for horse excrement transplantation.

Background

At present: bacteria have a key role in the physiology of the host. Recent advances in 16S rRNA sequencing and data analysis methods have shown that every part of the body, such as the skin, mucosa, gastrointestinal tract, respiratory tract, urinary tract and reproductive tract, has certain microbiota. To date, the intestinal microbiota is the most diverse microbiota in humans and animals. The number of bacteria in the gastrointestinal tract of horses varies in view of different pH values, intestinal mobility, oxygen tension and nutrient availability. Most of the microflora colonize the intestinal microflora of horses, consisting of approximately 109 bacterial cells per gram of cecal content, and undergo microbial digestion (fermentation) to provide 60-70% of the energy to the horse population. In adult horses, most of the bacteria belong to the phylum firmicutes, Bacteroides and Proteobacteria, the largest number, and remain relatively stable in the healthy gut. There are 1015 bacterial cells throughout the gastrointestinal tract of horses, most of which are located in the colon and cecum, including at least 108 bacteria species, and 7 phyla.

The equine intestines begin to colonize at birth with microorganisms, and the composition of the intestinal microbiota affects the development of the immune system. In the early days, the microbiome of horses was similar to that of their mothers, consisting mainly of lactobacilli, suggesting that foals may obtain some of their microbiome from the birth canal. The various test methods showed that the microbial populations of foal and mare remained similar between 4 weeks and 60 days.

The intestinal flora of horses is responsible for the digestion of food and the absorption of nutrients, providing energy to the host, and is closely associated with gastrointestinal diseases such as colitis and diarrhoea. However, recent studies have shown that the gut microbiota can interact with other organs and become a major contributor to health and disease, not just the gut, because of its effects on the mental health and metabolism of the host.

The cascade between the intestinal microbiota of horses and the immune system, which is critical to overall health, fecal transplantation is aimed at restoring healthy intestinal microbiota that has been disrupted by antibiotic therapy or invasion by harmful bacteria. Stool transplantation has been used as an important option for the treatment of diarrhea and colitis, which remains the leading cause of critical illness in horses, with a mortality rate of 25.4% to 35%. Fecal transplantation helps restore the microbial balance of gut flora imbalance by transferring fecal microbial suspensions of healthy donor horses into the gut of the recipient horses to rebalance the gut flora that may be caused by specific diseases associated with gut microbiome imbalance.

In general, fecal transplantation is a safe method of treating gastrointestinal and parenteral disorders with few side effects. Despite good benefits, to date, none of the standardized methods of horse manure transplantation has been a major limitation hindering its widespread use. This is why we need a practical, safe and long-lasting guide to facilitate the clinical use of horse stool transplantation.

Through the above analysis, the problems and defects of the prior art are as follows:

(1) none of the existing methods has a standardized horse manure transplant method.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a standard establishing method for horse excrement transplantation.

The invention is realized by a standard establishing method for horse excrement transplantation, which comprises the following steps:

(1): selection of donor horses;

(2): preparing excrement;

(3): preparation of the recipient and transplantation.

Further, the selection of donor horses includes clinical examination, horse master questionnaire, biological examination.

Further, the clinical examination is to examine whether the donor horse has a pathogen, has a bad microorganism, and will adversely react with the gastrointestinal tract of the recipient.

Further, the horse owner questionnaire includes asking the horse owner the following questions:

(1) whether or not there is an infectious disease from exposure to coronavirus, syphilis, malaria, trypanosomiasis, tuberculosis, brucellosis, Equine Infectious Anemia (EIA), equine herpes virus (EHV-1, EHV 4), equine adenovirus, rotavirus, equine influenza, equine rabies, herpes zoster, tetanus, west nile virus;

(2) whether the age is less than 12 months or greater than 10 years;

(3) whether a history of systemic infectious disease exposure;

(4) whether or not Botomaku pestilence exists;

(5) whether there is a history of overseas travel within 30 days;

(6) whether to use an unknown drug or to use an experimental study drug;

(7) male horses with or without dangerous behavior, including but not limited to, sexual contact with mares with EIA, viral hepatitis, syphilis;

(8) whether or not a tissue transplant has been received, including but not limited to skin, bone marrow;

(9) whether a fecal microbiota transplant has been received without proper techniques, i.e., fecal material of unknown origin, has transplanted non-pre-screened feces;

(10) horses with a history of bites, including but not limited to infection and/or use of antibiotic drugs;

(11) whether horses have a history of laminitis;

(12) whether horses had participated in a clinical study history of the gastrointestinal tract;

(13) whether or not a horse has received a blood transplant;

(14) whether horses have acupuncture treatment history within 30 days;

(15) whether abnormal behaviors appear within 30 days or not is judged, and the physical and mental stress is indicated;

(16) whether treatment is performed without proper aseptic conditions within 30 days, including but not limited to, reuse of the injection needle;

(17) whether there is a parasitic disease or viral and/or bacterial infection within 30 days, and gastrointestinal tract involvement, such as diarrhea, colic and colitis;

(18) whether there was a history of live attenuated vaccination within 30 days, if there is a risk of transmission;

(19) whether a patient has a hospital history within 30 days or not and the risk of spreading multidrug-resistant organisms;

(20) whether or not there is metabolic syndrome in horses, including but not limited to metabolic disorders;

(21) whether or not there is a history of irritable bowel syndrome, inflammatory bowel disease, chronic constipation;

(22) whether or not there is salmonella infection;

(23) whether there are overweight and obese horses;

(24) whether horses have been exposed to antibiotics and chemotherapy within 30 days;

(25) whether horses with fever and lymphadenectasis exist;

(26) whether or not there is an irregular history of vaccination in horses;

(27) horses with irregular history of repelling insects.

Further, the biological examination includes a blood examination and a stool examination.

Further, the blood examination comprises a general examination and a specific examination, wherein the general examination comprises EHV-1, EHV-4, equine adenovirus, equine influenza, equine rabies, tetanus, equine influenza, West Nile virus, and the specific examination comprises EHV-1 and EHV-4 antibodies;

the stool examination comprises general examination and specific examination, wherein the general examination comprises clostridium difficile, detection pathogens such as salmonella, drug-resistant escherichia coli and other multi-drug-property bacteria, norwalk virus, stool occult blood test, protozoa and spirochetes, and the specific examination is rotavirus;

when the excrement examination is carried out, excrement culture is recommended to be carried out on a selective flat plate, and a standard antibiotic susceptibility test is carried out, a disc diffusion test is the first choice, and a cultured excrement sample is prepared for microbial culture and antibiotic susceptibility test;

in the case of stool examination, stool culture on selective plates is recommended, as follows:

culture medium	Pathogenic bacteria	Growth characteristics
			Campylobacter blood agar plate	Campylobacter genus	Red wine
Trypsin Soy Agar (TSA) plate	Salmonella	Green
			TSA (T A-site of origin) plate with 5% sheep blood	Streptococcus equi	Erythema
TSA and Macconkey II agar plates	Escherichia coli (drug resistance)	Pink to red
			TSA (T A-site of origin) plate with 5% sheep blood	Pseudomonas aeruginosa	White colour (Bai)
TSA and McKai II agarFat plate	Klebsiella pneumoniae	Pink (pink)

The cultivation of the fecal sample specifically comprises the following steps:

s1: taking 10 grams of feces from a potential donor;

s2: stool was diluted with 10 ml Phosphate Buffered Saline (PBS) sterilized pH =7 or NaCl;

s3: centrifuging the cocktail at 4 deg.C for 6000 Xg for 10 min;

s4: an upper liquid fraction is obtained.

Further, the preparation of the stool comprises stool collection and stool storage;

the feces collection is directly obtained from rectum under anaerobic condition or performed by using fresh feces and sterilized plastic gloves; the method comprises the following specific steps:

(a) collecting newly excreted feces at appropriate time points, preferably in the morning, noon and sunset, using a sterile glove;

(b) sealing the container and placing the container in a clean sealed bag;

(c) delivering the bag together with a refrigerator and an ice bag within 1 hour after defecation, wherein the fecal material can be stored for 8 hours at 4 ℃;

the stool storage includes fresh storage and frozen storage, depending on when the stool transplant procedure is performed;

the fresh storage, for instant fecal transplantation, can prepare feces with several 50 ml centrifuge tubes after the feces discharge, for screening and storage respectively to preserve the viability of the microbiota, the centrifuge tubes prepared with feces are placed in a cooling box and before an ice bag in a larger storage bag and sealed, so that the centrifuge tubes are preferably sent to a laboratory for preparation within 1 hour after the feces discharge, horse feces can be stored for 6 hours at room temperature, no obvious influence is caused on the survival of bacteria, but bacterial colonies at room temperature can change after more than 6 hours.

The cryopreservation, performed in a refrigerator at-80 ℃ to liquid nitrogen at-196 ℃, should be prepared in a single "dose", not allowing to refreeze once thawed, preparing a "dose" of stool transplant for horses using 200 g of fresh stool.

Fresh feces should be homogenized with 0.9% normal saline and filtered through a 1.0 mm stainless steel sieve or a four-fold sterile gauze to remove undigested food and smaller materials. The mixture was then centrifuged at 6000 Xg for 10 minutes at 4 ℃. The fecal material obtained was mixed with sterile pharmaceutical grade glycerol (Sigma, St Louis, MO) to a final concentration of 10%, or using a maltodextrin-trehalose mix formulation at a ratio of 3:1 as a cryopreservative, and after proper mixing, the fecal mixture was individually filled into sterile containers with easily identifiable markers and immediately frozen at-80 ℃, leaving at least 2 fecal samples in 50 ml centrifuge tubes for future analysis.

Further, the preparation of the feces also comprises the establishment of a feces library.

Further, the preparation of the recipient includes antibiotic treatment and laxative treatment;

the antibiotic treatment is carried out, wherein 5 g of ampicillin, 5 g of neomycin sulfate, 5 g of metronidazole and 2.5 g of vancomycin are injected into a nasogastric tube 24 hours before fecal transplantation, and 5 liters of antibiotic mixed liquid diluted by sterile normal saline is used;

in the laxative treatment, a 10% PEG mixed solution is given to a nasogastric tube once every 0.5 hour for 4 times, and excrement is transplanted after 2 hours while free water is provided;

the PEG solution can be prepared by the following steps:

(1) 100 g of PEG is weighed;

(2) dissolving in water bath at 95 deg.c for 10 min with 1L NaCl;

(3) the PEG solution was allowed to cool to about 37 ℃ prior to administration, should be used fresh, and the above steps could be repeated 4 times.

Further, the transplantation includes fresh stool transplantation and frozen stool transplantation;

the fresh fecal transplantation comprises the following steps:

(1) nasogastric tube route, injecting prepared fecal mixture wine into the stomach of the recipient using nasogastric tube;

(2) capsules and colonoscopes deliver stool;

(3) performing fecal perfusion by using a colonoscope;

in the frozen fecal transplantation, frozen fecal material should be thawed overnight at 4 ℃ or in a water bath at 37 ℃ for 5 minutes, and the frozen 'fecal pellets' can be directly delivered to the intestinal tract using rectal techniques if colonoscopy cannot be performed;

the subject is medically tracked and monitored after transplantation.

By combining all the technical schemes, the invention has the advantages and positive effects that: the fecal transplantation method of the present invention helps restore the microbial balance of gut flora imbalance by transferring fecal microbial suspensions of healthy donor horses into the gut of the donor horses to rebalance the gut flora that may be caused by specific diseases associated with gut microbiome imbalance; the invention provides a standardized horse excrement transplanting technology, so that the horse excrement transplanting technology is more widely applied and is not hindered; the stool transplant technique of the present invention can be used for treating various diseases including diarrhea, enteritis, laminitis and irritable bowel syndrome in horse clinics; the fecal transplantation technology of the invention can be used in horses of different age stages; the excrement transplantation technology has excellent effect of treating diarrhea, enteritis, laminitis, irritable bowel syndrome and the like; the average treatment times of the excrement transplanting technology are 4-9 times, and the safety is high.

Drawings

FIG. 1 is a schematic illustration of a method of delivery of a fresh or frozen fecal microbiota transplant as provided by an embodiment of the present invention;

in the figure: A. conveying through a nasogastric tube; B. colonoscope delivery; C. rectal methods;

FIG. 2 is a schematic view of a horse dung grading system provided by an embodiment of the present invention;

FIG. 3 is a schematic diagram of the visual observation result of horse dung of case 1 provided by the embodiment of the invention;

FIG. 4 is a schematic diagram showing the visual observation of the stool structure 3 days after the stool transplant treatment of case 1 according to the example of the present invention;

FIG. 5 is a schematic illustration of a visual inspection of horse stool from case 2 provided by an embodiment of the present invention;

FIG. 6 is a schematic illustration of the visual observation of stool structure 3 days after stool transplant treatment for case 2 provided by an example of the present invention;

FIG. 7 is a schematic illustration of a distribution of stool transplant cases provided by an embodiment of the present invention;

FIG. 8 is a schematic illustration of the age distribution of a stool transplant case provided by an embodiment of the present invention;

FIG. 9 is a schematic illustration of the sex distribution of a stool transplant case provided by an embodiment of the present invention;

FIG. 10 is a schematic illustration of the cure rate of a fecal transplantation therapy provided by an embodiment of the present invention;

FIG. 11 is a graph showing the average number of fecal transplants under various disease conditions provided by an embodiment of the present invention;

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In view of the problems of the prior art, the present invention provides a method for establishing a standard for horse feces transplantation, and the present invention is described in detail below with reference to the accompanying drawings.

Main scheme and effect description section:

FIG. 1 shows a method of transport for fresh or frozen fecal microbiota transplantation;

fresh stool transplantation, (1) nasogastric tube route, as shown in fig. 1A, prepared stool mix alcohol is injected into the stomach of the recipient using nasogastric tube; (2) capsule and colonoscope fig. 1B delivers stool; (3) colonoscopy performed stool perfusion fig. 1C.

Frozen fecal transplants, frozen fecal material should be thawed overnight at 4 ℃ or in a water bath at 37 ℃ for 5 minutes, and using the rectal technique, FIG. 1C, the frozen "fecal pellets" can be delivered directly into the intestinal tract if colonoscopy is not available.

The invention is further illustrated in detail below with reference to specific examples:

case 1:

colitis

An 11 year old Mongolian horse was treated for anorexia and weight loss over several weeks. The horses had good appetite for the grain, but did not eat hay. Periodic anthelmintic regimens (4-6 times per year), most recently 2 weeks prior to admission. Fecal flotation was negative for parasites. The physical condition of the other 12 horses in the farm was well reported by the owner and the referring veterinarian. In clinical examinations, body temperature, pulse and respiratory rate were all normal (37.4 ℃, 48/min and 20/min, respectively). The horse looked badly (body score 2.5/9), 250 kg body weight, painless punctate edema under the mandible and in the abdomen. Except for the thickening of the small intestinal wall, ultrasound examination of the thorax and abdomen was normal. The only abnormal results of Complete Blood Count (CBC) and serum chemistry examinations were mild neutrophilia (8.3 × 103 cells/microliter), low plasma protein (36 g/l, albumin 17 g/l), hypocalcemia (2.6 mmol/l), hypomagnesemia (0.42 mmol/l), and low iron binding capacity (TTBC =34.0 μmol/l). The plasma osmolality is very low at 10.4 mm Hg (normally 18-22 mm Hg). The renal function examination is normal (BUN 5.6 mu mol/l, creatinine 140.4 mu mol/l), and the urine analysis is also normal. Colitis was primarily diagnosed based on clinical symptoms, laboratory results and ultrasound examination results.

The horse did not eat overnight and a progressive treatment regimen of dexamethasone (20 mg per day) was initiated. During the treatment period, the horses had returned to normal appetite, however after one week they began losing most of their body weight. Thus, the horse is referral to my hospital.

We first evaluated the feces according to the horse feces grading system (fig. 2). Observations indicated that the feces of this horse were graded as 2 (fig. 3).

The treatment procedure is as follows: we have performed a fecal transplantation treatment according to the method described in the present invention. After 3 consecutive days of treatment, the horse feces structure was significantly improved as shown in fig. 4, and the fecal grading system score was changed to 5.

Case 2:

diarrhea (diarrhea)

A2-year-old blood-mixing horse is treated due to poor appetite and diarrhea. The horse is called by a master, the horse is always healthy, and the parasite expelling and the vaccine are all in place. Local veterinarians have undergone antibiotic and infusion treatments, but have not improved. After transfer to my home, the owner refuses further laboratory examinations. In clinical examinations, the body temperature, pulse and respiratory rate results were 38.4 ℃, 62/min and 31/min, respectively. The horse looks bad (body score 3.5/9) and weighs 300 kg. According to clinical symptoms, a primary diagnosis is diarrhea.

We first evaluated the feces according to the horse feces grading system (fig. 2). Observations indicated that the feces of this horse were graded as 2 (fig. 5).

Procedure we performed a fecal transplantation treatment according to the method described in the present invention. After 7 consecutive days of treatment, the horse feces structure was significantly improved as shown in fig. 6, and the fecal grading system score was changed to 5.

In combination with different disorders, we performed 264 additional clinical trials, the summary of which is shown in FIGS. 7-11.

In summary, the comparison of the data in FIGS. 7-11 shows that the stool transplant technique of the present invention can be used to treat a variety of diseases including diarrhea, enteritis, laminitis, and irritable bowel syndrome in equine clinics. The fecal transplantation technique treatment of the present invention can be used in horses of different age stages. The excrement transplantation technology has excellent effect of treating diarrhea, enteritis, laminitis, irritable bowel syndrome and the like. The average treatment times of the excrement transplanting technology are 4-9, and the safety is high. The invention provides a standardized horse excrement transplanting technology, which has the advantages of wider application, low cost and good treatment effect.

In the description of the present invention, "a plurality" means two or more unless otherwise specified; the terms "upper", "lower", "left", "right", "inner", "outer", "front", "rear", "head", "tail", and the like, indicate orientations or positional relationships that are based on the orientations or positional relationships shown in the drawings, are merely for convenience in describing the present invention and to simplify the description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," "third," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

It should be noted that the embodiments of the present invention can be realized by hardware, software, or a combination of software and hardware. The hardware portion may be implemented using dedicated logic; the software portions may be stored in a memory and executed by a suitable instruction execution system, such as a microprocessor or specially designed hardware. Those skilled in the art will appreciate that the apparatus and methods described above may be implemented using computer executable instructions and/or embodied in processor control code, such code being provided on a carrier medium such as a disk, CD-or DVD-ROM, programmable memory such as read only memory (firmware), or a data carrier such as an optical or electronic signal carrier, for example. The apparatus of the present invention and its modules may be implemented by hardware circuits such as very large scale integrated circuits or gate arrays, semiconductors such as logic chips, transistors, or programmable hardware devices such as field programmable gate arrays, programmable logic devices, or software executed by various types of processors, or a combination of hardware circuits and software, e.g., firmware.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made by those skilled in the art within the technical scope of the present invention disclosed in the present invention should be covered within the scope of the present invention.

Claims (10)

1. A method for establishing a standard for horse stool transplantation, comprising:

(1) selection of donor horses;

(2) preparing excrement;

(3) preparation of the recipient and transplantation.

2. The method of creating criteria for horse stool transplant according to claim 1 wherein said selection of donor horses includes clinical examination, horse master questionnaire, biological examination.

3. A method of establishing criteria for use in equine fecal transplantation as set forth in claim 2 wherein said clinical examination is of a donor horse for the presence of pathogens, the presence of necrotic microorganisms and the adverse effects of the gastrointestinal tract of the recipient.

4. A method of establishing criteria for horse stool transplant as set forth in claim 2 wherein said horse owner questionnaire comprises asking the horse owner the following questions:

(1) whether or not there is an infectious disease from exposure to coronavirus, syphilis, malaria, trypanosomiasis, tuberculosis, brucellosis, Equine Infectious Anemia (EIA), equine herpes virus (EHV-1, EHV 4), equine adenovirus, rotavirus, equine influenza, equine rabies, herpes zoster, tetanus, west nile virus;

(2) whether the age is less than 12 months or greater than 10 years;

(3) whether a systemic infectious disease exposure history;

(4) whether or not a bordeaux is present;

(5) whether there is a history of overseas travel within 30 days;

(6) whether to use an unknown drug or to use an experimental study drug;

(7) male horses with or without dangerous behavior, including but not limited to, sexual contact with mares with EIA, viral hepatitis, syphilis;

(8) whether or not a tissue transplant has been received, including but not limited to skin, bone marrow;

(9) whether fecal microbiota transplantation has been previously accepted without proper techniques, i.e., fecal material of unknown origin, transplantation of non-pre-screened fecal material;

(10) horses with or without a history of bites, including but not limited to infection and/or use of antibiotic drugs;

(11) whether horses have a history of laminitis;

(12) whether horses had participated in a history of clinical studies of the gastrointestinal tract;

(13) whether or not a horse has received a blood transplant;

(14) whether horses with acupuncture treatment history exist within 30 days;

(15) whether abnormal behaviors appear within 30 days or not is judged, and the physical and mental stress is indicated;

(16) whether treatment was performed without proper sterility for 30 days, including but not limited to, reuse of the injection needle;

(17) whether there is a parasitic disease or viral and/or bacterial infection within 30 days, and gastrointestinal tract involvement, such as diarrhea, colic and colitis;

(18) whether there was a history of live attenuated vaccination within 30 days, if there is a risk of transmission;

(19) whether a patient has a hospital history within 30 days or not and the patient is at risk of transmitting multidrug-resistant organisms;

(20) whether or not there is metabolic syndrome in horses, including but not limited to metabolic disorders;

(21) whether or not there is a history of irritable bowel syndrome, inflammatory bowel disease, chronic constipation;

(22) whether there is salmonella infection;

(23) whether there are overweight and obese horses;

(24) whether horses have been exposed to antibiotics and chemotherapy within 30 days;

(25) whether horses with fever and lymphadenectasis exist;

(26) whether or not there is an irregular history of vaccination in horses;

(27) whether or not there is a horse with an irregular history of repelling insects.

5. The method of claim 2 wherein said biological tests comprise blood tests and stool tests.

6. The method of claim 5, wherein the blood test comprises a general test and a specific test, the general test comprises EHV-1, EHV-4, equine adenovirus, equine influenza, equine rabies, tetanus, equine influenza, west nile virus, and the specific test comprises EHV-1 and EHV-4 antibodies;

the fecal test comprises general and specific tests, the general test comprises clostridium difficile, pathogens such as salmonella, drug-resistant escherichia coli and other multi-drug-property bacteria, norwalk virus, fecal occult blood test, protozoa and spirochete, and the specific test is rotavirus;

when the excrement examination is carried out, excrement culture is recommended to be carried out on a selective flat plate, and a standard antibiotic susceptibility test is carried out, a disc diffusion test is the first choice, and a cultured excrement sample is prepared for microbial culture and antibiotic susceptibility test;

the cultivation of the fecal sample specifically comprises the following steps:

s1: taking 10 grams of feces from a potential donor;

s2: diluting the feces with 10 ml of Phosphate Buffered Saline (PBS) sterilized at pH =7 or NaCl;

s3: centrifuging the cocktail at 4 deg.C at 6000 Xg for 10 min;

s4: an upper liquid fraction is obtained.

7. A method of establishing standards for equine fecal transplantation as set forth in claim 1 wherein said fecal preparation includes fecal collection and fecal storage;

the feces collection is directly obtained from rectum under anaerobic condition or performed by using fresh feces and sterilized plastic gloves; the method comprises the following specific steps:

(a) collecting newly excreted feces at appropriate time points, preferably in the morning, noon and sunset, using a sterile glove;

(b) sealing the container and placing the container in a clean sealed bag;

(c) delivering the bag together with a refrigerator and an ice bag within 1 hour after defecation, wherein the fecal material can be stored for 8 hours at 4 ℃;

the stool storage includes fresh storage and frozen storage, depending on when the stool transplant procedure is performed;

the fresh storage, for the instant fecal transplantation, can prepare the fecal with several 50 ml centrifuge tubes after the fecal is discharged, respectively used for screening and storing to keep the vitality of microbial flora, the centrifuge tubes prepared with the fecal are placed in a cooling box and an ice bag in a larger storage bag and sealed, so that the centrifuge tubes are preferably sent to a laboratory for preparation within 1 hour after the fecal is discharged, horse feces can be stored for 6 hours at room temperature, the survival of bacteria is not obviously affected, but bacterial colonies at room temperature can change after more than 6 hours;

the cryopreservation, performed in a refrigerator at-80 ℃ to liquid nitrogen at-196 ℃, should be prepared in a single "dose", not allowing to refreeze once thawed, preparing a "dose" of stool transplant for horses using 200 g of fresh stool.

8. A method of establishing standards for horse stool transplantation as set forth in claim 1, wherein said stool preparation further comprises establishment of a stool pool.

9. A method of establishing standards for equine fecal transplantation as set forth in claim 1 wherein said preparation of the recipient includes antibiotic treatment and laxative treatment;

the antibiotic treatment is carried out by injecting 5 g of ampicillin, 5 g of neomycin sulfate, 5 g of metronidazole and 2.5 g of vancomycin into a nasogastric tube 24 hours before the excrement transplantation, and diluting the mixed solution with 5 liters of sterile normal saline to obtain an antibiotic mixed solution;

in the laxative treatment, a 10% PEG mixed solution is given to a nasogastric tube once every 0.5 hour for 4 times, and excrement is transplanted after 2 hours while free water is provided;

the PEG solution can be prepared by the following steps:

(1) weighing 100 g of PEG;

(2) dissolving with 1L NaCl in water bath at 95 deg.C for 10 min;

(3) the PEG solution was allowed to cool to about 37 ℃ prior to administration, and should be used fresh, and the above steps were repeated 4 times.

10. A method of establishing standards for equine fecal transplantation as set forth in claim 1 wherein said transplantation includes fresh fecal transplantation and frozen fecal transplantation;

the fresh fecal transplantation comprises the following steps:

(1) nasogastric tube route, injecting prepared fecal mixture wine into the stomach of the recipient using nasogastric tube;

(2) capsules and colonoscopes deliver stool;

(3) performing fecal perfusion by using a colonoscope;

in the frozen fecal transplantation, frozen fecal material should be thawed overnight at 4 ℃ or in a water bath at 37 ℃ for 5 minutes, and the frozen 'fecal pellets' can be directly delivered to the intestinal tract using rectal techniques if colonoscopy cannot be performed;

the subject is medically tracked and monitored after transplantation.

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