Yak diarrhea comprehensive treatment system and method based on Tibetan medicine compound

Abstract

The invention relates to the technical field of animal disease prevention and treatment, in particular to a comprehensive treatment system and method for yak diarrhea based on a Tibetan medicine compound.The method comprises the steps that a yak excrement sample is collected, and the diarrhea pathogen type is judged through multiple PCR detection; calling a corresponding compound proportion in a Tibetan medicine compound database according to a judgment result, and blending a powdery compound composition consisting of herpetospermum pedunculosum, rhododendron parvifolium, pterocephalus hookeri and herba aconiti; dynamically extracting with a low-temperature buffer solution, mixing with lactobacillus plantarum freeze-dried powder, and preparing a sustained-release microcapsule through a sodium alginate-chitosan two-phase embedding technology; the yaks with diarrhea are drenched with the micro-capsules firstly, and then the remaining micro-capsules are mixed with fermented highland barley feed in proportion to be continuously fed. According to the invention, the integrated treatment process of pathogen recognition, Tibetan medicine matching, bacterial medicine synergy and continuous administration is realized, the curative effect, compliance and Tibetan medicine application stability are effectively improved, and the Tibetan medicine is suitable for scientific prevention and treatment of diarrhea of yaks in plateau pasturing areas.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal disease prevention and treatment, and in particular to a yak diarrhea comprehensive treatment system and method based on a Tibetan medicine compound. BACKGROUND

[0002] With the continuous development of animal husbandry in the Qinghai-Tibet Plateau, yak, as an important economic and ecological species in alpine regions, its health status directly affects the sustainable development of animal husbandry. In recent years, due to climate anomalies, changes in feed and transmission of epidemic sources, the incidence of yak diarrhea has shown an increasing trend, seriously affecting its growth performance and economic benefits. Due to the complexity of yak diarrhea, it can be caused by bacterial, viral, parasitic or mixed infection of multiple pathogens, and traditional treatment methods rely on antibiotics, antidiarrheal drugs and electrolyte replacement, etc., which have certain limitations. At the same time, the potential of Tibetan medicine resources in the prevention and treatment of yak diseases has gradually attracted attention, especially in the aspects of intestinal regulation and anti-inflammatory and antibacterial, which shows significant advantages.

[0003] However, the existing yak diarrhea treatment methods still have many technical bottlenecks in practical application. First, most treatment methods cannot achieve precise identification of the type of pathogen, resulting in lack of targeting of Tibetan medicine use and unstable drug efficacy; second, the taste problem of Tibetan medicine components, low bioavailability and loss of probiotic activity in the gastrointestinal environment all restrict the exertion of its synergistic treatment potential; in addition, current methods rely on artificial feeding at regular intervals, lack of scientific and standardized continuous intervention mechanism, especially in the plateau grazing scene, the drug compliance is poor and the treatment process is controllable. SUMMARY

[0004] The present application provides a yak diarrhea comprehensive treatment system and method based on a Tibetan medicine compound, which provides a new comprehensive treatment scheme integrating pathogen identification, compound customization, sustained release delivery and continuous feeding, to improve the intervention efficiency and promotion feasibility of yak diarrhea.

[0005] A yak diarrhea comprehensive treatment method based on a Tibetan medicine compound, comprising the following steps: S1: Collecting yak fecal samples, determining the type of diarrhea pathogens by multiplex PCR detection, the type of pathogens including bacterial type, viral type, parasitic type and mixed infection type; S2: According to the determined type of pathogen, retrieve the corresponding Tibetan medicine compound composition from the pre-established Tibetan medicine compound database, the Tibetan medicine compound composition is composed of Bo Longgu Zi, Small Leaf Azalea, Wings First Grass and Bangga in a specific ratio; S3: The obtained Tibetan medicine compound composition is extracted by low-temperature dynamic extraction to obtain an active extraction liquid, mixed with Lactobacillus plantarum CGMCC 1.555 freeze-dried powder, and prepared into slow-release microcapsules with a diameter of 1-2 mm by using a sodium alginate-chitosan two-phase embedding technology; S4: The slow-release microcapsules prepared are first administered to the diarrhea yak at a dose of 20 g per head, and after 8 hours, the remaining microcapsules are mixed into fermented highland barley feed at a ratio of 1:10 for continuous feeding for 3 days.

[0006] Optionally, the S1 comprises: S11: Collecting yak fecal samples, and simultaneously extracting pathogen DNA and RNA in the yak fecal samples by using the CTAB method; S12: Based on the extracted pathogen DNA and RNA, performing multiplex PCR detection to generate a multiplex PCR detection result; S13: Analyzing the multiplex PCR detection result, and determining the type of diarrhea pathogens according to the analysis result.

[0007] Optionally, the performing multiplex PCR detection comprises: Using bacterial universal primers to detect bacterial pathogens; Using rotavirus gene primers to detect viral pathogens; Using cryptosporidium gene primers to detect parasitic pathogens.

[0008] Optionally, the determining the type of diarrhea pathogens according to the analysis result comprises: If only the bacterial universal primers amplify a 1500 bp band, it is determined that the type of diarrhea pathogens is bacterial; If only the rotavirus primers amplify a 650 bp band, it is determined that the type of diarrhea pathogens is viral; If only the cryptosporidium primers amplify a 550 bp band, it is determined that the type of diarrhea pathogens is parasitic; If ≥2 target bands are amplified at the same time, it is determined that the type of diarrhea pathogens is mixed infection.

[0009] Optionally, the S2 comprises: S21: According to the determined type of diarrhea pathogens (bacterial / viral / parasitic / mixed infection), matching a preset compound calling rule in a pre-established Tibetan medicine compound database; S22: Extracting corresponding quality ratios of Trichosanthes kirilowii Maxim, Ledum palustre, Petrosavia petrosa and Ban ga from a medicinal material storage of the Tibetan medicine compound database according to the matched compound calling rule; S23: Mixing and crushing the extracted Trichosanthes kirilowii Maxim, Ledum palustre, Petrosavia petrosa and Ban ga through an 80-mesh sieve to generate a powder-like Tibetan medicine compound composition.

[0010] Optionally, the compound calling rule is specifically: If the diarrhea pathogen type is bacterial, calling rule B is used: Bolenguazi: Xiaoye Dujuan: Yishoucao: Bangga = 5:3:1:1; If the diarrhea pathogen type is viral, calling rule V is used: Bolenguazi: Xiaoye Dujuan: Yishoucao: Bangga = 3:4:2:1; If the diarrhea pathogen type is parasitic, calling rule P is used: Bolenguazi: Xiaoye Dujuan: Yishoucao: Bangga = 4:2:1:2; If the diarrhea pathogen type is mixed infection, calling rule M is used: Bolenguazi: Xiaoye Dujuan: Yishoucao: Bangga = 4:3:2:1.

[0011] Optionally, the S3 comprises: S31: Place the powdered Tibetan medicine compound composition prepared in S2 into a dynamic extraction tank, add 10 times the mass of pH 5.5 citric acid-disodium hydrogen phosphate buffer, and stir at 200 rpm at 40°C for 2 hours. Filter through a 0.22 μm filter membrane to obtain an active extract; S32: Mix the active extract with Lactobacillus plantarum (CGMCC 1.555) freeze-dried powder at a mass ratio of 2:1, and uniformly oscillate at 25°C in a sterile environment for 30 minutes to form a uniform mixed slurry; S33: Treat the mixed slurry using a sodium alginate-chitosan two-phase embedding technique to generate sustained-release microcapsules.

[0012] Optionally, the treatment of the mixed slurry using a sodium alginate-chitosan two-phase embedding technique is specifically: Add the mixed slurry to a 3% (w / v) sodium alginate solution, and form droplets through an electrostatic atomizer at a flow rate of 0.5 mL / min; Collect the droplets in a 2% (w / v) CaCl2 solution for solidification for 10 minutes to form sodium alginate gel cores; Transfer the gel cores to a 0.8% (w / v) chitosan (molecular weight 80 kDa) acetic acid solution for coating for 15 minutes, wash with deionized water, and then freeze-dry to obtain sustained-release microcapsules with a diameter of 1-2 mm.

[0013] Optionally, the S4 comprises: S41: Administer the prepared sustained-release microcapsules to the diarrhea yak at a dose of 20 g per head by directly pouring into the mouth using a oral gavage; S42: After 8 hours of pouring, evenly mix the remaining sustained-release microcapsules into fermented barley feed at a mass ratio of 1:10 to form a mixed feed containing microcapsules; The remaining slow-release microcapsules = total amount of prepared slow-release microcapsules - 20 g / head * number of yak heads to be treated; S43: Place the microcapsule-containing mixed feed in the manger for the diarrhea yak to freely eat, and continuously feed for 3 days.

[0014] A yak diarrhea comprehensive treatment system based on Tibetan medicine compound is used to implement the above-mentioned yak diarrhea comprehensive treatment method based on Tibetan medicine compound, and comprises the following modules: A sample collection and detection module is used to collect yak fecal samples, extract pathogen DNA and RNA, and detect and identify bacterial, viral, parasitic and mixed infection pathogen types by multiplex PCR; A compound matching and combination module is used to call preset compound calling rules in the Tibetan medicine compound database according to the detection results, extract Bolenguazi, Xiaoye Dujuan, Yishoucao and Bangga, and mix them in proportion to generate a powder-shaped Tibetan medicine compound composition; An active extraction and microcapsule preparation module is used to extract Tibetan medicine active ingredients with a buffer and mix them with Lactobacillus plantarum freeze-dried powder, and prepare slow-release microcapsules with a diameter of 1-2 mm by using a sodium alginate-chitosan double-phase embedding technology; A gavage and feeding module is used to first gavage the slow-release microcapsules into the yak at a dose of 20 g / head, then mix the remaining slow-release microcapsules into fermented highland barley feed at a mass ratio of 1:10 for continuous feeding for 3 days, so as to realize continuous treatment intervention.

[0015] The beneficial effects of the present application are as follows: In the present application, by performing multiplex PCR detection on yak diarrhea fecal samples, bacterial, viral, parasitic and mixed infection pathogens are identified, a compound calling rule database is set, corresponding Tibetan medicine compound proportions are matched according to different pathogen profiles, one-to-one matching of 'pathogen-compound' is realized, the problems of 'unsuitable medication' or 'broad-spectrum ineffectiveness' in traditional empirical medication are avoided, and the accuracy and targeting of clinical treatment are improved. Experiments have proved that this method has excellent efficacy differentiation for different types of diarrhea, especially the symptom relief time for mixed infection diarrhea is significantly shortened.

[0016] In the present application, by setting a low-temperature dynamic extraction + probiotic synergistic co-loading + double-layer embedding controlled release strategy, the active Tibetan medicine extract and Lactobacillus plantarum CGMCC 1.555 freeze-dried powder are compounded, a double-phase slow-release microcapsule is formed by using a sodium alginate gel core and a chitosan-pectin composite film, the protection ability and slow-release performance of the active ingredients and probiotics in the gastrointestinal environment are significantly improved, and the efficacy persistence and intestinal microecological repair effect are enhanced.

[0017] The application realizes rapid relief of diarrhea emergency by setting a first oral administration dose of 20 g per head, and combines the continuous feeding mechanism of "mixing the remaining slow-release microcapsules into fermented highland barley feed at a ratio of 1:10", which not only ensures the rapid effect of the first dose, but also ensures the continuous supply of the drug and probiotics within the next 3 days, forming a stable intervention rhythm. The mechanism is suitable for the grazing management scene of yak pastures, and is simple to operate, low in cost and high in compliance. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only a part of the present application, and other drawings can also be obtained by those skilled in the art without creative effort based on these drawings.

[0019] Figure 1 The method flowchart of the embodiment of the present application is shown in the figure. Figure 2 The system flowchart of the embodiment of the present application is shown in the figure. DETAILED DESCRIPTION

[0020] The present application will be described in detail below in combination with the drawings and specific embodiments. It should be noted that in order to make the embodiments more detailed, the following embodiments are the best, preferred embodiments, and other alternative ways can also be used by those skilled in the art to implement them; and the drawings are only used to more specifically describe the embodiments, and are not intended to specifically limit the present application.

[0021] It should be noted that in the specification, "one embodiment", "embodiment", "exemplary embodiment", "some embodiments" and the like indicate that the described embodiments can include a specific feature, structure or property, but not necessarily every embodiment includes the specific feature, structure or property. In addition, when a specific feature, structure or property is described in combination with an embodiment, it should be within the knowledge of those skilled in the related art to realize such a feature, structure or property in combination with other embodiments (whether or not explicitly described).

[0022] Generally, the terms can be understood at least in part from the use in context. For example, depending at least in part on the context, the term "one or more" as used herein can be used to describe any feature, structure, or characteristic in the singular or can be used to describe combinations of features, structures, or characteristics, in the plural. In addition, the term "based on" can be understood as not necessarily intending to convey a set of exclusive factors, but can instead, depending at least in part on the context, allow for the existence of other factors not necessarily explicitly described.

[0023] As Figure 1As shown, a comprehensive treatment method for yak diarrhea based on Tibetan medicine compound includes the following steps: S1: Collect yak fecal samples, determine the type of diarrhea pathogen by multiplex PCR detection, and the pathogen type includes bacterial type, viral type, parasitic type and mixed infection type; S2: According to the determined pathogen type, the corresponding Tibetan medicine compound composition is retrieved from the pre-established Tibetan medicine compound database, and the Tibetan medicine compound composition is composed of Bo Longguazi, Xiaoye Dujuan, Yishoucao and Bangga in a specific ratio; S3: The obtained Tibetan medicine compound composition is extracted by low temperature dynamic extraction to obtain active extract, mixed with Lactobacillus plantarum CGMCC 1.555 freeze-dried powder, and prepared into slow-release microcapsules with a diameter of 1-2mm by using sodium alginate-chitosan double-phase embedding technology; S4: The diarrhea yak is first given 20g of slow-release microcapsules per head, and the remaining microcapsules are mixed with fermented highland barley feed at a ratio of 1:10 for continuous feeding for 3 days.

[0024] S1 includes: S11, collect fecal samples and pathogen nucleic acid extraction: select yak with obvious diarrhea symptoms, collect 50g of fresh fecal samples on site using sterile operation methods, immediately put into sampling tubes with RNA protectant, store at 4℃ and send to the laboratory within 12 hours. After arriving at the laboratory, extract pathogen DNA and RNA from yak fecal samples.

[0025] The extraction method specifically includes: weighing 0.3 g of uniform feces, adding 1 mL of CTAB lysis solution (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl, 0.1% β-mercaptoethanol, pH 8.0), incubating in a 65℃ water bath for 30 minutes, and inverting and mixing every 10 minutes during the incubation. Then add an equal volume of chloroform-isoamyl alcohol (24:1), mix vigorously for 1 minute, centrifuge at 12,000 rpm for 10 minutes, take the supernatant, precipitate the nucleic acid with isopropanol, wash with 70% ethanol, dissolve with TE buffer, and obtain the total nucleic acid extract.

[0026] S12, multiplex PCR amplification reaction system construction and primer setting: On the basis of the extracted DNA and RNA, cDNA synthesis is performed on the RNA sample using a reverse transcription kit to ensure subsequent PCR amplification.

[0027] Three groups of PCR amplification reactions are constructed for the identification of bacterial type pathogens, viral type pathogens and parasitic type pathogens: Bacterial pathogen detection: use bacterial universal primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'; The length of the amplified target product was 1500 bp, and the PCR reaction system was 25 μL, which contained 2x PCR Master Mix, 0.4 μM of each primer, and 1 μL of template DNA.

[0028] Viral pathogen detection: rotavirus VP6 gene primers were used VP6-F: 5'-GACGGVGCRACTACATGGT-3'; VP6-R: 5'-GTCCAATTCATNCCTGGTGG-3'; The length of the amplified target product was 650 bp, and cDNA was used as a template, and the PCR reaction system was the same as above.

[0029] Parasitic pathogen detection: Cryptosporidium COWP gene primers were used.

[0030] COWP-F: 5'-TGAGTATCCGGTATTGATAG-3'; COWP-R: 5'-CACCACAAATGAAGTGCGAC-3'; The length of the amplified target product was 550 bp, and the template was extracted DNA.

[0031] The reaction conditions were as follows: pre-denaturation at 94°C for 3 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s; and final extension at 72°C for 5 min. The PCR products were detected by 2% agarose gel electrophoresis.

[0032] S13, detection result analysis and pathogen type determination: the PCR amplification products were detected by electrophoresis, and the pathogen type was determined by comparing the band position with the standard molecular weight Marker. The specific determination criteria were as follows: Only 1500 bp band appeared: determined as bacterial pathogen; Only 650 bp band appeared: determined as viral pathogen; Only 550 bp band appeared: determined as parasitic pathogen; If ≥2 bands appeared: determined as mixed infection pathogen.

[0033] For example, in one detection, two clear bands of 1500 bp and 650 bp were amplified from a fecal sample of a diarrheic yak, and combined with the specificity of the primers, it was determined that the yak was infected with both bacterial and viral pathogens, and therefore the diarrhea type was mixed infection.

[0034] S2 comprises: S21, compound calling rule matching: according to the determined diarrhea pathogen type, enter the pre-established Tibetan medicine compound database for compound combination query. Based on the theory of Tibetan medicine and historical clinical experience, the compound calling rules corresponding to different pathogen types are constructed, and the specific rules are as follows: Bacterial type, calling rule B: Pongouguazi: Small-leaf rhododendron: Wings first grass: Bangga = 5:3:1:1; Viral type, calling rule V: Pongouguazi: Small-leaf rhododendron: Wings first grass: Bangga = 3:4:2:1; Parasitic type, calling rule P: Pongouguazi: Small-leaf rhododendron: Wings first grass: Bangga = 4:2:1:2; Mixed infection type, calling rule M: Pongouguazi: Small-leaf rhododendron: Wings first grass: Bangga = 4:3:2:1; The compound calling rules are stored in the database in a structured format and can be quickly retrieved and matched by indexing the diarrhea pathogen type, ensuring the accuracy and consistency of subsequent medicinal material proportioning.

[0035] Example: If a yak is determined to be of viral diarrhea type by multiple PCR detection, the system automatically calls the compound calling rule V, and the corresponding proportioning is: Pongouguazi 3 parts, small-leaf rhododendron 4 parts, wings first grass 2 parts, and bangga 1 part.

[0036] S22, medicinal material extraction: according to the matched compound calling rule, the system instructs to retrieve the corresponding medicinal materials from the medicinal material storage associated with the Tibetan medicine compound database. The medicinal materials are traceable standardized dry products with batch identification, drying rate test report and pesticide residue test report.

[0037] During operation, an electronic balance is used for weighing, with a single reference mass of 100 g. After calculating the total amount, Pongouguazi, small-leaf rhododendron, wings first grass and bangga are taken in proportion. If 1 kg of viral type Tibetan medicine compound composition is required, 300 g of Pongouguazi, 400 g of small-leaf rhododendron, 200 g of wings first grass and 100 g of bangga are weighed respectively.

[0038] After the medicinal materials are taken, they are cleaned and impurities are removed by winnowing, and treated in an ozone sterilization cabinet for 30 minutes to ensure that the cleanliness and microbial control level of the medicinal materials meet the "Quality Standards for Chinese Herbal Medicines".

[0039] S23, mixing and grinding of medicinal materials to prepare compound composition: the treated seeds of Momordica balsamifera, Ledum palustre, and the above-mentioned medicinal materials are mixed and ground in a stainless steel high-speed grinder at a certain proportion, and then sieved through an 80-mesh sieve. The obtained product is a uniform and fine powder of the Tibetan medicine compound composition.

[0040] To ensure the powder dispersibility and uniformity, secondary mixing can be performed in a rotating mixing tank for 5 minutes. The obtained Tibetan medicine compound composition is used in the subsequent active extraction process.

[0041] Example: for 1 kg of compound composition powder prepared for viral pathogens, the average particle size is 185 μm, the water content is controlled at 6.3%, and the content of each main medicinal ingredient is verified by high-performance liquid chromatography fingerprinting to meet the set range.

[0042] S3 includes: S31, preparation of active extract: 100 g of the prepared powder of the Tibetan medicine compound composition is placed in a dynamic extraction tank with temperature control and inert gas interface, nitrogen (or argon) is introduced to form an inert gas protection environment, 900 mL of ethanol-water mixed solvent (i.e., 100 g of medicinal powder is added to 900 mL of ethanol-water mixed solution) is added, and 0.1% (v / v) of citric acid is added as an antioxidant stabilizer, and the initial pH is adjusted to 5.0-5.5.

[0043] The extraction is performed at a constant temperature of 45°C±2°C, and the extraction is performed for 3 times, each time for 30 minutes. The solvent is replaced and the inert gas is continuously filled to maintain the stability of the system between each extraction.

[0044] After the extraction is completed, the extract is filtered through a 0.22 μm microporous filter to remove residual insoluble powder, and the filtrate is collected to obtain the active extract. The obtained extract is light brown and transparent, with a pH of about 5.4, and is stored at 4°C in the dark for use.

[0045] The total flavonoid concentration in the extract is 0.46 mg / mL, the total polyphenol concentration is 0.85 mg / mL, and the extract contains characteristic components such as syringic acid from Momordica balsamifera and hesperidin from the winged grass, which are detected by HPLC.

[0046] S32, preparation of mixed slurry: under clean and sterile conditions, the above-mentioned active extract and Lactobacillus plantarum (Lactobacillus plantarum CGMCC 1.555) freeze-dried powder are mixed at a mass ratio of 2:1. Specifically, 200 g of the active extract is weighed and 100 g of Lactobacillus plantarum freeze-dried powder (viable bacterial count ≥1× CFU / g) is added, and then placed in a sterile conical flask.

[0047] Place the conical flask on a 25℃ shaker at a speed of 100 rpm for 30 minutes to ensure the lyophilized powder is evenly dispersed to form a uniform mixed slurry.

[0048] After actual mixing, the mixed slurry is light cream in color, with moderate viscosity, and the probiotics are evenly distributed without aggregation under microscopic observation. The pH value is maintained within the range of 5.2 ± 0.1, which is suitable for the subsequent embedding process.

[0049] S33, preparation of slow-release microcapsules: The mixed slurry is microencapsulated using a sodium alginate-chitosan two-phase embedding technique. The specific steps are as follows: Forming a sodium alginate gel core: Prepare a 3% (w / v) sodium alginate solution, load the mixed slurry into an electrostatic atomizer, and drop it into the sodium alginate solution at a constant flow rate of 0.5 mL / min through a fine capillary nozzle. After maintaining static conditions for 1 minute, the resulting droplets naturally settle into a cross-linking solution containing 2% (w / v) CaCl2. The droplets are cross-linked in this solution for 10 minutes to form a sodium alginate gel core with a certain strength, with a thickness controlled at 100-150 μm.

[0050] Chitosan coating of the outer layer: After the gel core is removed and gently rinsed to remove free calcium ions, it is transferred to a 0.8% (w / v) chitosan (molecular weight 80 kDa) acetic acid solution for outer layer coating. Maintain the coating reaction for 15 minutes, with slow magnetic stirring to ensure uniform coating. After coating is complete, rinse with deionized water for 3 times to remove the surface residual chitosan that has not been combined.

[0051] Freeze-drying: Place the coated microcapsules into a freeze dryer, pre-freeze at -40℃ for 2 hours, then start the vacuum drying program, and dry for 24 hours. Finally, smooth-surfaced, structurally stable slow-release microcapsules are obtained, with a diameter controlled within the range of 1-2 mm.

[0052] In actual preparation, the slow-release microcapsules have a particle size distribution range of 1.12-1.87 mm, a drug loading rate of about 72.6%, and a probiotic embedding efficiency of 89.3%. The scanning electron microscope image shows that the shell is evenly coated without cracks, with good slow-release performance and gastric juice resistance.

[0053] S41, first-time administration of slow-release microcapsules: The prepared slow-release microcapsules are directly administered to the diarrheic yak through an oral syringe at a dose of 20 g / head.

[0054] Before operation, confirm that the slow-release microcapsules have a particle size of 1-2 mm, the particles are complete, and the surface is free of cracking, mold, etc. The utensil is a reusable veterinary oral syringe made of plastic or silicone, which is used to suspend the slow-release microcapsules in saline to form a slurry for administration, ensuring smooth passage through the syringe catheter into the esophagus, preventing regurgitation or aspiration.

[0055] Example: If the number of yaks with diarrhea to be treated is 30, then: Total amount of sustained-release microcapsules to be administered = 20 g / head × 30 heads = 600 g.

[0056] The administration should be performed on an empty stomach before feeding, and the patient should be fasted for 2 hours afterward to ensure that the microcapsules are effectively adsorbed and transported through the stomach to the small intestine, thereby improving bioavailability.

[0057] S42, Sustained-release microcapsules mixed with fermented highland barley feed: The secondary dosing mechanism is initiated 8 hours after oral administration. The remaining sustained-release microcapsule dosage is calculated using the following formula: Remaining sustained-release microcapsules = Total amount of sustained-release microcapsules prepared - 20g / head × Number of yaks treated; Assuming that step S3 produces 2000 g of sustained-release microcapsules, and the treatment involves 30 yaks, then: Remaining sustained-release microcapsules = 2000 − 20 × 30 = 1400g; The remaining slow-release microcapsules were mixed into the fermented barley feed at a mass ratio of 1:10. The fermented barley feed was obtained by mixing medicinal residue, barley straw, and yeast in a ratio of 10:85:5 and anaerobic fermenting at 25°C for 72 hours, and has good palatability and microecological regulation ability.

[0058] The mixing method involves using a horizontal feed mixer at low speed for 5 minutes to ensure uniform dispersion of the slow-release microcapsules and avoid localized accumulation that could lead to uneven feeding. After mixing, the uniformity of the slow-release microcapsule distribution in the sample is tested, and the coefficient of variation (CV) should be ≤10%.

[0059] S43, continuous free-feeding: The mixed feed containing microcapsules is evenly placed in a clean feed trough for the yaks with diarrhea to eat freely for 3 consecutive days.

[0060] The daily feeding amount should be set according to body weight: for yaks with an average weight of 300–400 kg, the daily intake of mixed feed should be about 2.5–3 kg, ensuring that the intake of slow-release microcapsules is maintained within the range of about 15–18 g / head per day to achieve the purpose of maintaining the therapeutic dose.

[0061] In actual feeding, fecal samples taken on the 1st and 3rd days after oral administration were subjected to quantitative analysis of viable bacteria and high performance liquid chromatography analysis. The results showed that the level of intestinal lactic acid bacteria was significantly increased (p<0.01), and the active ingredient, melon seed extract, remained stable, indicating that the microcapsules have a good controlled release effect.

[0062] like Figure 2 As shown, a comprehensive treatment system for yak diarrhea based on Tibetan medicine compound is used to implement the aforementioned comprehensive treatment method for yak diarrhea based on Tibetan medicine compound, and includes the following modules: The sample collection and detection module is used for collecting yak dung samples, extracting pathogen DNA and RNA, and combining multiple PCR detection to identify bacterial type, viral type, parasitic type and mixed infection type pathogen types; The compound matching and combination module is used for calling preset compound calling rules in the Tibetan medicine compound database according to the detection results, extracting Bolenguazi, Xiaoye Rhododendron, Yishoucao and Bangga, crushing and mixing them in proportion, and generating a powder-shaped Tibetan medicine compound composition; The active extraction and microcapsule preparation module is used for extracting Tibetan medicine active ingredients with a buffer solution and mixing them with Lactobacillus plantarum freeze-dried powder, and preparing a sustained-release microcapsule with a diameter of 1-2 mm through a sodium alginate-chitosan two-phase embedding technology. The gavage and feeding module is used for gavaging the yak with the sustained-release microcapsule at a dose of 20 g per head, and then continuously feeding the yak with fermented highland barley feed mixed with the remaining sustained-release microcapsule at a mass ratio of 1:10 for 3 days, so as to realize continuous treatment intervention.

[0063] The present application covers any substitution, modification, equivalent method and scheme made on the essence and scope of the present application. In order to make the public have a thorough understanding of the present application, specific details are described in the following preferred embodiments of the present application, and the present application can also be fully understood without the description of these details to those skilled in the art. In addition, in order to avoid unnecessary confusion to the essence of the present application, well-known methods, processes, procedures, elements and circuits are not described in detail.

[0064] The above is only the preferred embodiment of the present application, and it should be pointed out that for ordinary skilled in the art, without departing from the principle of the present application, a number of improvements and refinements can be made, and these improvements and refinements should be considered as the protection scope of the present application.

Claims

1. A comprehensive treatment method for yak diarrhea based on Tibetan medicine compound, characterized in that, The method comprises the following steps: S1: Collecting yak dung samples, and determining the types of diarrhea pathogens by multiple PCR detection, wherein the types of diarrhea pathogens include bacterial type, viral type, parasitic type and mixed infection type; S2: According to the determined type of diarrhea pathogens, the corresponding Tibetan medicine compound composition is retrieved from the pre-established Tibetan medicine compound database, wherein the Tibetan medicine compound composition is composed of Bo Long Gua Zi, Xiao Ye Du Quan, Yi Shou Cao and Bang Ga in a specific proportion; S3: The obtained active extract of the Tibetan medicine compound composition is obtained by low-temperature dynamic extraction, mixed with Lactobacillus plantarum CGMCC 1.555 freeze-dried powder, and prepared into slow-release microcapsules with a diameter of 1-2mm by using sodium alginate-chitosan double-phase embedding technology; S4: The diarrhea yak is first fed with 20g of the prepared slow-release microcapsules per head, and then the remaining microcapsules are mixed into fermented highland barley feed at a ratio of 1:10 for continuous feeding for 3 days.

2. The yak diarrhea comprehensive treatment method based on Tibetan medicine compound according to claim 1, characterized in that, The S1 comprises: S11: Collecting yak dung samples, and simultaneously extracting pathogen DNA and RNA in the yak dung samples by using the CTAB method; S12: Performing multiple PCR detection based on the extracted pathogen DNA and RNA, and generating multiple PCR detection results; S13: Analyzing the multiple PCR detection results, and determining the type of diarrhea pathogens according to the analysis results.

3. The yak diarrhea comprehensive treatment method based on Tibetan medicine compound according to claim 2, characterized in that, The multiple PCR detection comprises: Detecting bacterial pathogens by using bacterial universal primers; Detecting viral pathogens by using rotavirus gene primers; Detecting parasitic pathogens by using cryptosporidium gene primers.

4. The yak diarrhea comprehensive treatment method based on Tibetan medicine compound according to claim 3, characterized in that, The determination of the type of diarrhea pathogens according to the analysis results comprises: If only the bacterial universal primers amplify a 1500bp band, it is determined that the type of diarrhea pathogens is bacterial type; If only the rotavirus primers amplify a 650bp band, it is determined that the type of diarrhea pathogens is viral type; If only the cryptosporidium primers amplify a 550bp band, it is determined that the type of diarrhea pathogens is parasitic type; If ≥2 target bands are amplified at the same time, it is determined that the type of diarrhea pathogens is mixed infection type.

5. The yak diarrhea comprehensive treatment method based on Tibetan medicine compound according to claim 4, characterized in that, The S2 comprises: S21: According to the determined type of diarrhea pathogens, the pre-established compound calling rule is matched in the pre-established Tibetan medicine compound database; S22: According to the matched compound calling rule, the corresponding quality ratio of Bo Long Gua Zi, Xiao Ye Du Quan, Yi Shou Cao and Bang Ga is extracted from the medicinal material storage of the Tibetan medicine compound database; S23: The extracted Bo Long Gua Zi, Xiao Ye Du Quan, Yi Shou Cao and Bang Ga are mixed, crushed and passed through an 80-mesh sieve to generate a powder-like Tibetan medicine compound composition.

6. The Tibetan medicine compound-based comprehensive treatment method for yak diarrhea according to claim 5, characterized in that, The compound calling rule is specifically: If the type of diarrhea pathogens is bacterial type, the calling rule B is Bo Long Gua Zi:Xiao Ye Du Quan:Yi Shou Cao:Bang Ga=5:3:1:1; If the type of diarrhea pathogens is viral type, the calling rule V is Bo Long Gua Zi:Xiao Ye Du Quan:Yi Shou Cao:Bang Ga=3:4:2:1; If the type of diarrhea pathogens is parasitic type, the calling rule P is Bo Long Gua Zi:Xiao Ye Du Quan:Yi Shou Cao:Bang Ga=4:2:1:2; If the type of diarrhea pathogens is mixed infection type, the calling rule M is Bo Long Gua Zi:Xiao Ye Du Quan:Yi Shou Cao:Bang Ga=4:3:2:

1.

7. The Tibetan medicine compound-based comprehensive treatment method for yak diarrhea according to claim 6, characterized in that, The S3 comprises: S31: placing the powdered Tibetan compound composition prepared in S2 into a dynamic extraction tank, adding 10 times the mass of pH 5.5 citric acid-disodium hydrogen phosphate buffer, stirring at 200 rpm at 40°C for 2 hours, filtering through a 0.22 μm filter membrane to obtain an active extract; S32: mixing the active extract with the Lactobacillus plantarum freeze-dried powder at a mass ratio of 2:1, uniformly oscillating at 25°C in a sterile environment for 30 minutes to form a uniform mixed slurry; S33: treating the mixed slurry by using a sodium alginate-chitosan double-phase embedding technology to generate slow-release microcapsules.

8. The yak diarrhea comprehensive treatment method based on Tibetan medicine compound according to claim 7, characterized in that, The treatment of the mixed slurry by using the sodium alginate-chitosan double-phase embedding technology specifically comprises: dropping the mixed slurry into a 3% sodium alginate solution to form droplets at a flow rate of 0.5 mL / min through an electrostatic atomizer; collecting the droplets in a 2% CaCl2 solution for solidification for 10 minutes to form sodium alginate gel cores; transferring the gel cores to a 0.8% chitosan acetic acid solution for coating for 15 minutes, washing with deionized water, and then freeze-drying to obtain slow-release microcapsules with a diameter of 1-2 mm.

9. The Tibetan medicine compound-based comprehensive treatment method for yak diarrhea according to claim 8, characterized in that, The S4 comprises: S41: orally administering the prepared slow-release microcapsules to the diarrheic yaks at a dose of 20 g per head using a oral syringe; S42: after 8 hours of administration, uniformly mixing the remaining slow-release microcapsules into fermented barley feed at a mass ratio of 1:10 to form a mixed feed containing microcapsules; The remaining slow-release microcapsules = total amount of prepared slow-release microcapsules - 20 g per head x number of treated yaks; S43: placing the mixed feed containing microcapsules in the feeding trough for the diarrheic yaks to freely eat, and continuously feeding for 3 days.

10. A Tibetan medicine compound-based yaks diarrhea comprehensive treatment system for implementing the Tibetan medicine compound-based yaks diarrhea comprehensive treatment method according to any one of claims 1-9, characterized in that, The following modules are included: a sample collection and detection module for collecting yak fecal samples, extracting pathogen DNA and RNA, and detecting and identifying bacterial, viral, parasitic, and mixed infection types of pathogens by multiplex PCR; a compound matching and combination module for calling preset compound calling rules in a Tibetan compound database based on the detection results, extracting Boangle Guazi, Small-leaf Rhododendron, Wings Head Grass, and Bangga, and mixing them in proportion to generate a powdered Tibetan compound composition; an active extraction and microcapsule preparation module for extracting active ingredients of Tibetan medicine by buffer and mixing with Lactobacillus plantarum freeze-dried powder, and preparing slow-release microcapsules with a diameter of 1-2 mm by using a sodium alginate-chitosan double-phase embedding technology; an administration and feeding module for first orally administering slow-release microcapsules to yaks at a dose of 20 g per head, and then uniformly mixing the remaining slow-release microcapsules into fermented barley feed at a mass ratio of 1:10 for continuous feeding for 3 days to achieve continuous treatment intervention.

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