CN111564219B - Method for establishing goat escherichia coli enteritis model and application - Google Patents
Method for establishing goat escherichia coli enteritis model and application Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for establishing a goat escherichia coli enteritis model and application thereof, and bacterial infection detection research and probiotics auxiliary treatment research are performed by using the animal model. The construction method of the model comprises the following steps: bacterial inoculation, probiotic (enterococcus faecium) intervention, hematological examination, intestinal pathology histology analysis and intestinal inflammation related gene expression analysis. The method has the advantages of simplicity, convenience, rapidness, high efficiency, economy and the like, and can be used for researching and providing a model for ruminant enteritis, screening probiotics, medicines and the like. The invention has great significance for researching the infection mechanism of the digestive system of ruminants and preventing and controlling treatment.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for establishing a goat escherichia coli enteritis model and application thereof.
Background
Colibacillosis is a common diarrhea disease of animals, and is also a common occurrence of ruminant goat breeding, and suffering from sheep often has symptoms of high body temperature, rapid respiration and heart rate, diarrhea and the like. Enterococcus faecium is used as one of feed microbial additives which are clearly allowed to be used by the agricultural department of China, and has the functions of regulating the balance of intestinal flora, inhibiting the adhesion and growth of pathogenic bacteria, reducing intestinal inflammation and the like. As ruminants belong to re-gastric animals, bacterial enteritis is a common cause of diarrhea diseases, and an infectious disease model similar to clinical morbidity is established, the method has important significance for researching pathogenesis, probiotics and drug screening. The existing experimental animals, namely mice and rabbits, belong to monogastric animals, the pathogenesis of the mice and the complex gastric animals are greatly different, and the pathogenic mechanism of enteritis models and pathogenic bacteria caused by virulence components or compounds is greatly different. The model is established to simulate clinical pathogenic microorganism infection and excite relevant immune reaction and pathological damage, modern molecular technology and classical pathological observation are used in the establishment of the model to demonstrate inflammation and damage, and probiotics are used for intervention treatment, so that the model and method are provided for relevant research in future.
Disclosure of Invention
The invention provides a method for establishing a goat escherichia coli enteritis model and application thereof, and the method has the advantages of simplicity, convenience, rapidness, high efficiency, economy and the like, and can be used for researching pathogenesis of enteritis caused by bacteria of ruminants, screening probiotics, medicines and the like.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for establishing a goat escherichia coli enteritis model comprises the following steps:
randomly dividing healthy adult female goats into 4 groups, namely a control group, a probiotic group, an escherichia coli group and a prevention group;
control group: normal saline is infused every day, and the period is 8 days continuously;
probiotic group: the probiotic suspension is infused daily for 4 days-4 to-1 of the test, and the test is continued for 4 days; the physiological saline is infused every day for 0 to 3 days of the test, and the test is continued for 4 days;
coli group: taking physiological saline every day for-4 to-1 days, and continuously taking for 4 days; e.coli suspension was infused daily for 4 days of the test;
preventive group: the probiotic suspension is infused daily for 4 days-4 to-1 of the test, and the test is continued for 4 days; e.coli suspension was infused daily for 4 days of the test;
and performing minimally invasive sampling on the 4d and the 8d of the test respectively, observing and evaluating pathological changes of the intestinal tract, and detecting the expression level of inflammatory genes related to the intestinal mucosa.
Further, the probiotics are enterococcus faecium.
Further, the preparation method of enterococcus faecium comprises the following steps: the frozen enterococcus faecium is streaked and inoculated on MRS solid culture medium, and is subjected to stationary culture at the constant temperature of 37 ℃ for 24 hours. After 2 times of plate streak activation, single colony is selected and inoculated in MRS liquid culture medium, after the culture is carried out at 37 ℃ and 120rpm to logarithmic phase, bacterial liquid is centrifuged, and is washed for 2 times by sterile normal saline, and the concentration is adjusted to 10 9 CFU/mL, cold storage at 4 ℃ for standby.
Further, the preparation method of the escherichia coli comprises the following steps: streaking and inoculating frozen escherichia coli on an LB solid culture medium, and standing and culturing at a constant temperature of 37 ℃ for 12 hours; after streaking activation by 2 times of flat plates, single colony is selected and inoculated in LB liquid culture medium, after culturing at 37 ℃ and 120rpm to logarithmic phase, bacterial liquid is centrifuged, washed 2 times by sterile normal saline, and the concentration is adjusted to 10 9 CFU/mL, cold storage at 4 ℃ for standby.
Furthermore, the volume of physiological saline, probiotic suspension or escherichia coli suspension infused by goats every day is 30mL; the concentration is 10 9 CFU/mL。
Further, the method for observing and evaluating the pathological changes of the intestinal tract comprises the following steps:
in 4d and 8d, jejunum and cecum tissues are taken, tissue sections are made, after HE staining, observation and photographing are carried out, and evaluation is carried out according to the following table;
scoring intestinal histopathological changes
Intestinal villi or epithelium is complete in 0 minutes, and tissue structure is normal
1 minute slight submucosal or lamina propria swelling separation
Moderate submucosal and/or lamina propria separation in 2 minutes, submucosal and/or myometrial edema
3-fold submucosal and/or lamina propria separation, submucosal and/or myometrial edema, local villus
Mao Tuola
4 divide intestinal villi disappear with necrosis of intestinal wall cells.
Further, fluorescent quantitative PCR is adopted to detect TLR4, IL-1 beta, IL-6, IL-8, TNF-alpha and beta-actin genes, the number of cycles undergone by the fluorescent signal reaches a threshold value is C t Value of 2 -ΔΔCt Pair C of method t The value is analyzed, beta-actin is taken as a reference gene, and the relative expression quantity of each gene is calculated, wherein the specific formula is as follows:
delta ct= (average Ct value of target gene of test group-average Ct value of reference gene of test group) - (average Ct value of target gene of control group-average Ct value of reference gene of control group).
Further, goat beta-actin, TLR4, IL-1β, IL-6, IL-8 and TNF- α were used with the following primers:
further, monitoring the goat body temperature, respiration and heart rate at-4, -2, 0, 2, 4, 6, 8, 10d of the test, respectively; collecting peripheral blood of goats, and performing white blood cell count examination; goat feces were collected and scored for diarrhea index.
The invention also provides application of the method for establishing the goat escherichia coli enteritis model in enteritis probiotics development.
The technical scheme provided by the invention realizes the following beneficial effects:
the patent designs a construction method for a goat infected by escherichia coli, and carries out bacterial infection detection research and probiotics auxiliary treatment research by using the animal model. The construction method of the model comprises the following steps: bacterial inoculation, probiotic (enterococcus faecium) intervention, hematological examination, intestinal pathology histology analysis and intestinal inflammation related gene expression analysis. The method has the advantages of simplicity, convenience, rapidness, high efficiency, economy and the like, and can be used for researching and providing a model for ruminant enteritis, screening probiotics, medicines and the like. The model is established, can reflect the body infection and defense process after clinical ruminant infection, and has great significance for researching the infection mechanism of the ruminant digestive system and preventing and controlling treatment.
The invention adopts the escherichia coli O55 to cause the escherichia coli diarrhea through drenching, confirms that the model is successfully constructed through detecting diarrhea index, pathological histology observation and inflammation related genes, and adopts enterococcus faecium probiotics for intervention, and confirms the auxiliary treatment effect of the probiotics in the index detection. The successful establishment of the model provides a ruminant infection model for clinical research of bacterial diarrhea of ruminants, particularly colibacillosis, and provides a foundation for development of enteritis probiotics.
Drawings
FIG. 1 is a diagram of an animal test protocol;
FIG. 2 shows the change in goat body temperature; and (3) injection: "x" indicates significant difference (p < 0.05) compared to control group, "x" indicates significant difference (p < 0.01) compared to control group; "#" indicates significant differences (p < 0.05) compared to the E.coli group, and "#" indicates very significant differences (p < 0.01) compared to the E.coli group;
FIG. 3 shows the variation of the respiratory rate of goats;
FIG. 4 is a change in goat heart rate;
FIG. 5 shows the change in the number of white blood cells in goat peripheral blood;
FIG. 6 is a goat jejunal tissue section 4d (HE); control group a (100×); enterococcus faecium group B (100×); coli group (100×); d preventive action group (100×); e control group (200X); f enterococcus faecium group (200×); group G E.coli (200X); h preventive action group (200×);
FIG. 7 is a goat jejunal tissue section 8d (HE); control group I (100×); j enterococcus faecium group (100×); k E.coli group (100X); l preventive action group (100×); m control group (200X); enterococcus faecium group (200×); o E.coli group (200X); p preventive action group (200×);
FIG. 8 is a goat jejunal pathology score;
FIG. 9 is a section of goat cecal tissue 4d (HE); control group a (100×); enterococcus faecium group B (100×); coli group (100×); d prophylaxis group (100×); e control group (200X); f enterococcus faecium group (200×); group G E.coli (200X); h preventive group (200×);
FIG. 10 is a section 8d (HE) of goat cecum tissue; control group I (100×); j enterococcus faecium group (100×); k E.coli group (100X); l preventive group (100×); m control group (200X); enterococcus faecium group (200×); o E.coli group (200X); p preventive group (200×);
FIG. 11 is a goat cecal pathology score;
FIG. 12 shows changes in the expression level of inflammatory genes in jejunum mucosa of goats;
FIG. 13 shows changes in the expression level of inflammatory genes in the cecum of goats.
Detailed Description
Example 1
1 Material
1.1 test animals
Healthy adult female goats were selected and weighing 30+ -5 kg. Is provided by a large-scale goat farm in Yangzhou city.
1.2 species
Enterococcus faecium (laboratory preservation); coli O55 (Sigma, usa);
1.3 major instrumentation
Animal blood cell analyzer (BC-2800, shenzhen Michael biomedical electronics Co., ltd., china); full temperature culture shaking table (QYC-200, shanghai Xinmiao medical instruments manufacturing Co., ltd., china); ultra clean bench (su zhou purification equipment limited, china); refrigerator (BCD-246 WD, tin-free pine cooler limited, china); electrothermal constant temperature blast drying oven (DWP-9272, shanghai macrolaboratory equipment Co., ltd., china); biological microscope (Olympus CX22, japan); rotary microtomes (RM 2016, leica, germany); a tabletop refrigerated Centrifuge (Centrifuge 5810r, eppendorf, germany); ultraviolet spectrophotometers (Thermo corporation, usa); refrigerated centrifuges (Eppendorf, germany); ultra low temperature refrigerator (Eppendorf, germany); PCR instrument (BioRad corporation, usa); fluorescent quantitative PCR instrument (BioRad corporation, usa); autoclave (TOMY corporation, japan) other instruments: surgical knife, forceps, surgical scissors, hemostatic forceps, suture needle, absorbable suture, trephine, etc.
2 test method
2.1 preparation of live bacteria preparation
Preparation of E.coli: the frozen escherichia coli is streaked and inoculated on LB solid medium, and is subjected to stationary culture at a constant temperature of 37 ℃ for 12 hours. After streaking activation by 2 times of flat plates, single colony is selected and inoculated in LB liquid culture medium, after culturing at 37 ℃ and 120rpm to logarithmic phase, bacterial liquid is centrifuged, washed 2 times by sterile normal saline, and the concentration is adjusted to 10 9 CFU/mL, cold storage at 4 ℃ for standby.
Preparation of enterococcus faecium: the frozen enterococcus faecium is streaked and inoculated on MRS solid culture medium, and is subjected to stationary culture at the constant temperature of 37 ℃ for 24 hours. After 2 times of plate streak activation, single colony is selected and inoculated in MRS liquid culture medium, after the culture is carried out at 37 ℃ and 120rpm to logarithmic phase, bacterial liquid is centrifuged, and is washed for 2 times by sterile normal saline, and the concentration is adjusted to 10 9 CFU/mL, cold storage at 4 ℃ for standby.
2.2 grouping and treatment of test animals
Healthy adult female goats were randomly divided into 4 groups, control, enterococcus faecium, escherichia coli and prophylaxis.
Control group: 30mL of physiological saline was infused daily for 8 days.
Enterococcus faecium group: the test was performed for-4 to-1 days, 30mL (10 9 CFU/mL) enterococcus faecium suspension, 4 days in succession. 30mL of physiological saline was infused daily for 4 days of the test, followed by 4 days.
Coli group: for-4 to-1 days, 30mL of physiological saline is infused daily for 4 days. 30mL (10) was drenched daily for 0 to 3 days of the test 9 CFU/mL) of escherichia coli suspension, 4 days in succession.
Preventive group: the test was performed for-4 to-1 days, 30mL (10 9 CFU/mL) enterococcus faecium suspension, 4 days in succession. 30mL (10) was drenched daily for 0 to 3 days of the test 9 CFU/mL) of escherichia coli suspension, 4 days in succession.
Minimally invasive sampling was performed at trial 4d and 8d, respectively, as shown in fig. 1:
the following operations were performed at-4, -2, 0, 2, 4, 6, 8, 10d of the test, respectively: (1) monitoring the goat's body temperature, respiration and heart rate; (2) Collecting peripheral blood of goats, and performing white blood cell count examination; (3) collecting goat feces for diarrhea index scoring.
Surgical sampling analysis was performed at trial 4d and 8d, respectively, with the following operations: (1) observation and evaluation of pathological changes of intestinal tracts; (2) detection of the expression level of inflammatory genes related to intestinal mucosa.
2.3 determination of physiological index and diarrhea index
2.3.1 body temperature, respiration and heart rate
The respiratory rate was measured by observing the number of times the chest and abdomen of each group of goats fluctuated, and the body temperature and heart rate of goats were measured using a thermometer and a stethoscope.
2.3.2 peripheral blood white blood cell count
Each group of goats was collected by jugular vein and placed in an anticoagulant tube with peripheral blood white cell count examination using an animal blood cell analyzer.
2.3.3 diarrhea index
Fresh manure from goats was collected and the manure status of goats from each group was recorded. Feces were graded with reference to bristol fecal sorting and goat physiological properties, and the scoring criteria are shown in table 1.
Diarrhea index = sum of stool scores/total goat head count for each group of trial. Diarrhea index of 2 or more is considered to occur.
Table 1 diarrhea index scoring criteria
2.4 pathological histology of intestinal tract
At 4d and 8d, jejunal and cecal tissues were taken, tissue sections were prepared, HE stained, observed and photographed, and pathology scored with reference to the Nadler scoring criteria (see table 2) and statistically analyzed.
TABLE 2Nadler intestinal histopathological scoring
2.5 fluorescent quantitative PCR detection of inflammatory Gene expression
Corresponding primers were designed based on the mRNA sequences of goat beta-actin, TLR4, IL-1β, IL-6, IL-8 and TNF- α, and the primer information is shown in Table 3:
TABLE 3 primer sequences of target genes for experiments
Detection of TLR4, IL-1 beta, IL-6, IL-8, TNF-alpha and beta-actin genes by fluorescent quantitative PCR with cycle number C when the fluorescent signal reaches the threshold t Value of 2 -ΔΔCt Pair C of method t The value is analyzed, beta-actin is taken as a reference gene, and the relative expression quantity of each gene is calculated, wherein the specific formula is as follows:
delta ct= (average Ct value of objective gene of test set-average Ct value of reference gene of test set) - (average Ct value of objective gene of control set-average Ct value of reference gene of control set)
2.6 data analysis
The experimental data were statistically analyzed using SPSS 17.0. Data are expressed as Mean ± standard error (Mean ± SEM). Data analysis was performed using One-way ANOVA and Dunnett's method was used for pairwise comparison. p <0.05 indicates that the difference is significant, and p <0.01 indicates that the difference is extremely significant.
3 results
3.1 goat physiological index and diarrhea index determination
3.1.1 goat body temperature
As shown in FIG. 2, the goat body temperature of both the control group and the enterococcus faecium group did not change significantly. On day 2 of the experiment, the body temperature of the E.coli group increased extremely significantly (p < 0.01) compared to the control group; the prophylaxis group body temperature was significantly lower than that of the E.coli group (p < 0.05). On days 4 and 6 of the trial, the body temperature was very significantly increased (p < 0.01) in both the E.coli and prophylactic groups compared to the control group.
3.1.2 goat respiratory rate
As shown in FIG. 3, the respiratory rate of goats in the control group and the enterococcus faecium group did not change significantly. On day 2 of the trial, the frequency of group aspiration was prevented from increasing (p < 0.05) compared to the control group. On day 4 of the experiment, the respiratory rate of the E.coli group was increased (p < 0.05) compared to the control group.
3.1.3 goat heart rate
As shown in FIG. 4, the goat heart rate of both the control group and the enterococcus faecium group did not significantly change. On day 4 of the trial, the heart rate was significantly increased in the E.coli group (p < 0.01) compared to the control group, and significantly increased in the prophylaxis group (p < 0.05).
3.1.4 goat peripheral blood leukocyte count
As shown in FIG. 5, the peripheral blood leukocyte counts of goats in the control group and enterococcus faecium group were not significantly changed. On day 2 of the experiment, the white blood cell count of the E.coli group appeared to be extremely significantly elevated (p < 0.01) compared to the control group; and the prophylaxis group was significantly lower than the E.coli group (p < 0.05). On day 4 of the experiment, a significant increase in E.coli group white blood cell count (p < 0.05) still occurred compared to the control group
3.1.5 goat diarrhea index
As shown in Table 4, neither the control nor the enterococcus faecium group goats developed diarrhea. On day 2 of the test, the stool score was significantly elevated (p < 0.01) in the e.coli group compared to the control group, diarrhea occurred; compared to the E.coli group, the fecal scores were reduced in the prophylaxis group (p < 0.05) and no diarrhea occurred. Diarrhea occurred in both the E.coli and prophylactic groups on day 4 of the trial. On days 6 and 8 of the trial, the fecal scores were extremely significantly elevated (p < 0.01) in the escherichia coli group compared to the control group, still diarrhea; compared to the E.coli group, the stool score was reduced in the prophylaxis group (p < 0.05) and no diarrhea occurred.
Table 4 goat faeces score for each group
3.2 goat intestinal tract histopathological changes
3.2.1 goat jejunal pathological histology observations
As shown in fig. 6 and 7, the jejunum villus of the goats of the control group and the enterococcus faecium group is complete, has clear structure and has no obvious lesions. On test day 4, compared with the control group, the jejunum villus of the escherichia coli group is blunt, atrophic lodging and oedema of the mucous membrane layer; compared with the coliform group, the intestinal villus morphology of the prevention group is better, but the intestinal villus has a small amount of hyperemia phenomenon. On day 8 of the test, the jejunum villus of the E.coli group was still atrophic and the mucosal layer was edematous compared to the control group; the jejunum villus structure of the prevention group is complete, and the shape is better than that of the escherichia coli group.
3.3.2 goat jejunal pathology scoring
As shown in fig. 8, on day 4 of the trial, jejunal pathology scores were significantly elevated (p < 0.01) in both the e.coli group and the prophylaxis group compared to the control group; the prophylaxis group scores were significantly lower than the e.coli group (p < 0.05). On day 8 of the trial, the escherichia coli group pathology score was still very significantly elevated compared to the control group (p < 0.01); the prophylaxis group scores were very significantly reduced compared to the e.coli group (p < 0.01).
3.3.5 goats cecal pathological histology observations
As shown in fig. 9 and 10, the cecum mucosal layer and submucosa structures of the goats in the control group and the enterococcus faecium group are clear and complete, and the crypt morphology and arrangement are normal. On day 4 of the test, the E.coli group and the prophylaxis group showed mild edema of the mucosal layer, accompanied by inflammatory cell infiltration, compared to the control group. On day 8 of the test, there was still mild oedema in the mucosal layer of the E.coli group.
3.3.6 goat cecal pathology score
As shown in fig. 11, there was no significant change in cecal pathology scores for both the control group and the enterococcus faecium group goats. On day 4 of the trial, both the e.coli group and the prophylaxis group had a very significant increase in disease score (p < 0.01) compared to the control group. On day 8 of the trial, the e.coli group score was very high (p < 0.01) compared to the control group, and the prophylaxis group score was very significantly lower than the e.coli group (p < 0.01).
3.5 changes in goat intestinal mucosal inflammatory Gene expression
3.5.1 changes in goat jejunal mucosal inflammatory Gene expression
As shown in fig. 12, the expression of TLR4 and each inflammatory gene was not significantly changed in the jejunal mucosa of goats in the control group and enterococcus faecium group. On day 4 of the experiment, the expression of TLR4, IL-1 beta, IL-6, IL-8 and TNF-alpha genes was extremely significantly increased (p < 0.01) in the E.coli group compared to the control group; the prophylactic group had significantly elevated IL-6 and TNF-alpha gene expression (p < 0.05). Compared with the escherichia coli group, the expression level of each inflammatory gene in the prevention group TLR4 is significantly reduced (p < 0.05). On day 8 of the experiment, the expression of TLR4, IL-1. Beta., IL-6 and IL-8 genes was still significantly elevated in the E.coli group (p < 0.05) compared to the control group. Compared to the E.coli group, the prophylactic group showed significantly reduced TLR4, IL-1. Beta. And IL-6 gene expression (p < 0.05).
3.5.2 changes in goat cecal mucosal inflammatory Gene expression
As shown in FIG. 13, the expression of the cecum mucosa inflammatory factor gene of the goats in the control group and the enterococcus faecium group is not changed obviously. On day 4 of the experiment, the expression of TLR4, IL-1 beta, IL-6, IL-8, TNF-alpha genes was significantly increased (p < 0.05) in the E.coli and prophylaxis groups compared to the control group; compared with the E.coli group, the prevention group TLR4, IL-6, IL-8 and TNF-alpha gene expression were significantly reduced (p < 0.05). Day 8 of the experiment, the E.coli group had significantly elevated TLR4, IL-1. Beta., IL-6, IL-8 and TNF-. Alpha.gene expression (p < 0.05) compared to the control group; the prophylactic group had significantly elevated IL-8 gene expression (p < 0.05). The prophylactic groups TLR4, IL-1 beta, IL-6, IL-8 and TNF-alpha gene expression were significantly reduced (p < 0.05) compared to the e.coli group.
Claims (5)
1. The method for establishing the goat escherichia coli enteritis model is characterized by comprising the following steps of:
randomly dividing healthy adult female goats into 4 groups, namely a control group, an enterococcus faecium group, an escherichia coli group and a prevention group;
control group: normal saline is infused every day, and the period is 8 days continuously;
enterococcus faecium group: the enterococcus faecium suspension is infused daily for 4 days-4 to-1 days of the test, and the test is continued for 4 days; the physiological saline is infused every day for 0 to 3 days of the test, and the test is continued for 4 days;
coli group: taking physiological saline every day for-4 to-1 days, and continuously taking for 4 days; e.coli suspension was infused daily for 4 days of the test;
preventive group: the enterococcus faecium suspension is infused daily for 4 days-4 to-1 days of the test, and the test is continued for 4 days; e.coli suspension was infused daily for 4 days of the test;
performing minimally invasive sampling on the 4d and 8d of the test respectively, observing and evaluating pathological changes of the intestinal tract, and detecting the expression level of inflammatory genes related to the intestinal mucosa;
the method for observing and evaluating the pathological changes of the intestinal tract comprises the following steps:
in 4d and 8d, jejunum and cecum tissues are taken, tissue sections are made, after HE staining, observation and photographing are carried out, and evaluation is carried out according to the following table;
scoring intestinal histopathological changes
Intestinal villi or epithelium is complete in 0 minutes, and tissue structure is normal
1 minute slight submucosal or lamina propria swelling separation
Moderate submucosal and/or lamina propria separation in 2 minutes, submucosal and/or myometrial edema
3-component severe submucosal and/or lamina propria separation, submucosal and/or myometrial edema, local villus shedding
4 intestinal villus disappearance with intestinal wall cell necrosis
Detection of TLR4, IL-1 beta, IL-6, IL-8, TNF-alpha and beta-actin genes by fluorescent quantitative PCR with cycle number C when the fluorescent signal reaches the threshold t Value of 2 -ΔΔCt Pair C of method t The value is analyzed, beta-actin is taken as a reference gene, and the relative expression quantity of each gene is calculated, wherein the specific formula is as follows:
delta ct= (average Ct value of target gene of test group-average Ct value of reference gene of test group) - (average Ct value of target gene of control group-average Ct value of reference gene of control group);
the primers used for goat beta-actin, TLR4, IL-1β, IL-6, IL-8 and TNF- α were as follows:
monitoring the body temperature, respiration and heart rate of the goat at-4, -2, 0, 2, 4, 6, 8, 10d of the test, respectively; collecting peripheral blood of goats, and performing white blood cell count examination; goat feces were collected and scored for diarrhea index.
2. The method for establishing a goat colibacillosis model according to claim 1, wherein the method for preparing enterococcus faecium is as follows: streaking and inoculating frozen enterococcus faecium on MRS solid culture medium, and standing at 37 ℃ for culturing for 24h; after 2 times of plate streak activation, single colony is selected and inoculated in MRS liquid culture medium, after the culture is carried out at 37 ℃ and 120rpm to logarithmic phase, bacterial liquid is centrifuged, and is washed for 2 times by sterile normal saline, and the concentration is adjusted to 10 9 CFU/mL, cold storage at 4 ℃ for standby.
3. The method for establishing a goat colibacillosis model according to claim 1, wherein the preparation method of the escherichia coli is as follows: streaking and inoculating frozen escherichia coli on an LB solid culture medium, and standing and culturing at a constant temperature of 37 ℃ for 12 hours; after streaking activation by 2 times of flat plates, single colony is selected and inoculated in LB liquid culture medium, after culturing at 37 ℃ and 120rpm to logarithmic phase, bacterial liquid is centrifuged, washed 2 times by sterile normal saline, and the concentration is adjusted to 10 9 CFU/mL, cold storage at 4 ℃ for standby.
4. The method for establishing the goat escherichia coli enteritis model according to claim 1, wherein the volume of physiological saline, probiotic suspension or escherichia coli suspension infused by the goat every day is 30mL; the concentration is 10 9 CFU/mL。
5. The application of the method for establishing the goat escherichia coli enteritis model according to any one of claims 1-4 in the development of enteritis probiotics.
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