| With the rapid development of pig breeding industry in China,the frequency of pig bacterial diseases is getting higher and higher,and the local epidemic,if it can not be controlled in time,it is easy to cause the spread and aggravation of the disease.Escherichia coli is an important intestinal pathogen,which can lead to changes in intestinal flora of piglets,causing severe diarrhea and even death.Especially the piglets before and after weaning often cause great economic losses to the pig industry.There are many serum types of E.coli,and antibiotics are mostly used in clinical treatment,but long-term use of antibiotics will affect the intestinal symbiotic microflora,lead to intestinal flora imbalance,and directly affect the health of piglets.and long-term use of antibiotics will lead to excessive drug residues in animal products,which is harmful to human health.Traditional Chinese medicine has the characteristics of antibacterial,no drug residue and little side effects.Fermented traditional Chinese medicine is on the basis of traditional Chinese medicine inheritance,combined with modern advanced research means of innovative technology,can regulate the balance of intestinal flora,enhance the activity of traditional Chinese medicine,and improve body immunity.In recent years,it has been widely used in animal husbandry because of its green,natural,fast efficacy and other characteristics.Therefore,it has become a research focus to find a better compound prescription of fermented traditional Chinese medicine as a substitute for the treatment of colibacillosis in piglets.In this study,in order to find a fermented compound prescription of traditional Chinese medicine with effective,good therapeutic effect and stable intestinal flora,34 traditional Chinese medicines were selected from the single traditional Chinese medicine with the main functions of heat-clearing and detoxification,astringent intestine and diarrhea,astringent and astringent,and relieving pain.the bacteriostatic test in vitro was carried out,and then combined with the theory of Chinese veterinary medicine to establish the compound.After the establishment of the compound prescription,preliminary screening was carried out through animal experiments,and the final traditional Chinese medicine compound prescription was fermented with Lactobacillus rhamnoides,Bacillus subtilis,Lactobacillus plantarum and Bacillus coagulans respectively.E.coli isolated from the samples collected from Jilin Province were modeled to explore the therapeutic effect of traditional Chinese medicine fermentation compound on piglet E.coli,so as to provide reference for the research and development of traditional Chinese medicine fermentation to prevent and cure the disease.The contents and results of the study are as follows:1.Isolation and identification of pathogenic E.coli.The anal swab samples of diarrhea piglets were collected from a farm around Changchun.The bacteria were identified by Mc Conkey Agar medium,E.coli chromogenic medium,microscope examination and biochemical test,and then further detected by PCR.The results showed that the size of the amplified band was about 1500 bp and the sequence analysis was Escherichia coli.A strain of pathogenic E.coli E-3 was screened by animal pathogenicity test.2.The establishment of compound prescription of traditional Chinese medicine and the construction of mouse diarrhea model.Fifty Kunming mice of about 20 g were randomly divided into 5 groups.The experimental groups were injected with E-3 intraperitoneally with concentrations of 1×10~8cfu/m L,2×10~8cfu/m L,3×10~8cfu/m L and 4×10~8cfu/m L respectively,and the control group was injected with normal saline.The results showed that 3×10~8cfu/m L was the best concentration for injection.By consulting the literature,34 traditional Chinese medicines were selected from the single traditional Chinese medicine with the main functions of heat-clearing and detoxification,astringent intestine and diarrhea,astringent astringency,pain relief and so on.The effective components of 34traditional Chinese medicines were extracted by water extraction and alcohol extraction,and the concentration of traditional Chinese medicine was adjusted to 1g/m L.The drug sensitivity test was carried out by Agar drilling method,and 11traditional Chinese medicines with good bacteriostatic effect were selected.The combined drug sensitivity test of the 11 traditional Chinese medicines was carried out.According to the results of combined drug sensitivity,combined with the theory of Chinese veterinary medicine,seven groups of compound prescriptions of traditional Chinese medicine were established,and the results of bacteriostatic comparison with western medicine in vitro were used to select the best compound prescriptions of three groups.3.Screening of compound prescription of traditional Chinese medicine.Fifty Kunming mice of about 20 g were randomly divided into 5 groups:group 1 of traditional Chinese medicine compound,group 2 of traditional Chinese medicine compound,group 3 of traditional Chinese medicine compound,bacterial attack control group and healthy control group.Except for the healthy control group,the other four groups were intraperitoneally injected with E.coli 0.5 m L with the concentration of 3×10~8 cfu/m L.After the onset of symptoms,the mice in the traditional Chinese medicine group were treated with 0.2 m L for 7 days,and the feed intake,weight,cure rate and mortality were recorded every day.The blood samples of mice in each group were collected before treatment and 3 days,5 days and 7 days during treatment,respectively,and the immune indexes were determined,and the middle segments of duodenum and ileum were taken to make pathological sections and observe the pathological changes.The results showed that the average weight gain and feed intake of the two groups were higher than those of the other four groups,the difference was significant(P<0.05),and the cure rate reached 100%.The contents of IL-2,IL-6,IL-8 and TNF-αin serum of mice in the second group of traditional Chinese medicine compound prescription were significantly different from those in the other four groups.The results of pathological sections showed that the intestinal gland structure of the mice in the second group of traditional Chinese medicine compound was relatively complete,and the repair of intestinal villi was obvious,and the degree of repair of intestinal villi in the second group was higher than that in the first group and the third group.According to the comprehensive results,the therapeutic effect of the second group of traditional Chinese medicine compound is better than that of the first group and the third group of traditional Chinese medicine compound,the therapeutic effect of the first group of traditional Chinese medicine compound is better than that of the third group of traditional Chinese medicine compound,and the therapeutic effect of the third group of traditional Chinese medicine compound is poor.4.Study on the therapeutic effect of traditional Chinese medicine compound products fermented by probiotics on mice infected with E.coli.Sixty Kunming mice of about 20 g were randomly divided into 6 groups:Bacillus subtilis fermentation group,Lactobacillus plantarum fermentation group,Lactobacillus rhamnosus fermentation group,Bacillus coagulans fermentation group,challenge control group and healthy control group.Except for the healthy control group,the other five groups were intraperitoneally injected with E.coli 0.5 m L with the concentration of 3×10~8cfu/m L.After the onset of symptoms,the mice in the fermentation group were given the drug 0.2 m L for 7 days,and the feed intake,body weight,cure rate and mortality were recorded every day.The blood samples of mice in each group were collected before treatment and 3 days,5 days and 7days during treatment,respectively,and the immune indexes were determined,and the middle segments of duodenum and ileum were taken to make pathological sections and observe the pathological changes.The results showed that the average weight gain of mice in Lactobacillus rhamnosus fermentation group was significantly different from that in challenge control group and healthy control group(P<0.05).The feed intake of mice was higher than that of the other five groups,which was significantly different from that of the other four groups except the healthy control group(P<0.05),and the cure rate reached 100%.The contents of IL-2,IL-6,IL-8 and TNF-αin serum of mice fermented by Lactobacillus rhamnosus were significantly different from those of the other five groups.The results of pathological sections showed that the intestinal gland structure of Lactobacillus rhamnosus fermentation group was intact,the intestinal villi recovered better,the intestinal mucosa was continuous and clear,and there was no inflammatory cell infiltration,which was better than other groups.According to the results,the four groups of fermentation compound can effectively treat Escherichia coli diarrhea in mice,but the therapeutic effect of Lactobacillus rhamnosus fermentation compound is the best.To sum up,a strain of pathogenic E.coli was successfully isolated from the anal swab sample of piglets,and the mouse diarrhea model was successfully established.According to a series of results of drug sensitivity and incompatibility of traditional Chinese medicine,three groups of compound prescriptions of traditional Chinese medicine were established and screened.The results of various indexes showed that the therapeutic effect of compound II of traditional Chinese medicine was good,and the therapeutic effect was enhanced after probiotic fermentation,among which the therapeutic effect was the best after fermentation by Lactobacillus rhamnosus. |
