CN111374094B - Method for establishing non-pregnant mouse uterine infection model caused by subcutaneous administration of donkey-derived salmonella abortus - Google Patents
Method for establishing non-pregnant mouse uterine infection model caused by subcutaneous administration of donkey-derived salmonella abortus Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/12—Animals modified by administration of exogenous cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0337—Animal models for infectious diseases
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for establishing a model of non-gestation mouse uterine infection caused by subcutaneous administration of donkey-derived salmonella abortus equi, which comprises the following steps: selecting a clean ICR non-pregnant mouse, dividing the clean ICR non-pregnant mouse into a blank treatment group and a donkey-derived equine abortion salmonella treatment group, feeding the mouse in a conventional environment, sterilizing the back of the mouse, inoculating donkey-derived equine abortion salmonella bacterial liquid subcutaneously on the back of the donkey-derived equine abortion salmonella treatment group, and inoculating PBS subcutaneously on the blank control group. The method improves the inoculation mode, shortens the infection period, reduces the experiment cost, and provides an animal model for further researching the pathogenic mechanism and prevention and control technology of the donkey-derived equine abortion salmonella.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for establishing a model of non-gestation mouse uterine infection caused by subcutaneous administration of donkey-derived salmonella abortus equi.
Background
Salmonella abortus in equines is an important infectious disease that causes abortion in pregnancy in equine animals. Studies have shown that uterine infections of the non-vaginal route are one of the important routes of infection for this pathogen. With the development of the donkey industry in China, donkey abortion caused by donkey-derived salmonella abortus gradually increases, but symptoms of the donkey-derived salmonella abortus are different from those of infection caused by equine salmonella abortus to a certain extent, the specific mechanism of infection is unclear, whether non-gestational animal infection can be caused or not is judged, so that breeding failure or gestational abortion and the like are caused, and the donkey-derived salmonella infection non-gestational animal model needs to be established for relevant research.
Disclosure of Invention
The invention provides a method for establishing a model of non-pregnant mouse uterine infection caused by donkey-derived salmonella equine abortion subcutaneously, which improves an inoculation mode, shortens an infection period, reduces experiment cost, and provides an animal model for further researching a pathogenic mechanism and a prevention and control technology of donkey-derived salmonella equine abortion.
In order to achieve the purpose, the invention provides the following technical scheme:
the method for establishing the model of the subcutaneous non-pregnant mouse uterine infection caused by the donkey-derived salmonella abortus comprises the following steps:
selecting a clean ICR non-pregnant mouse, dividing the clean ICR non-pregnant mouse into a blank treatment group and a donkey-derived equine abortion salmonella treatment group, feeding the mouse in a conventional environment, sterilizing the back of the mouse, inoculating donkey-derived equine abortion salmonella bacterial liquid subcutaneously on the back of the donkey-derived equine abortion salmonella treatment group, and inoculating PBS subcutaneously on the blank control group.
Further, the donkey-derived salmonella equine abortus treatment component comprises a donkey-derived salmonella equine abortus treatment component I and a donkey-derived salmonella equine abortus treatment component II which are respectively inoculated with 0.1mL 10 mL of 109cfu/mL donkey source salmonella abortus liquid and 0.1mL 1011cfu/mL donkey source equine abortion salmonella liquid.
Further, after inoculation, the mice were sacrificed by cervical dislocation on day 8 after infection.
Further, the mice are 8-10 weeks old.
Further, the mice were housed in a conventional environment for one week, allowed free access to water and food, and light and dark alternated for 12 h.
Further, the disinfection method is that the back of the mouse is disinfected by dipping a cotton swab in 75% alcohol.
Further, the preparation method of the donkey-derived equine abortion salmonella liquid comprises the following steps: taking a frozen and preserved donkey-derived salmonella equine abortus bacterial liquid, fully shaking up, inoculating the liquid on an LB solid culture medium, culturing for 15h in a 37 ℃ constant temperature incubator, aseptically selecting a single bacterial colony of the donkey-derived salmonella equine abortus bacterial on the LB liquid culture medium, culturing for 6h at 120r/m and 37 ℃, taking an appropriate amount of bacterial liquid, rotating at 4000rpm for 5min, removing a supernatant, washing the bacterial body and the homogeneous bacterial liquid by PBS, and adjusting the bacterial liquid to 109cfu/mL and 1011cfu/mL。
Further, on the 8 th day after the infection of the mouse, the eyeball of the mouse is removed and blood is collected by 200 mu L into an anticoagulation tube, the tube wall is flicked, the anticoagulation agent and the blood are conveniently and uniformly mixed, and then the hematology index examination is carried out immediately.
Further, on the 8 th day after the infection of the mice, the mice are killed by removing necks, spleen and uterus samples of the mice are aseptically collected and inoculated to an LB solid culture medium, the mice are placed in a constant-temperature incubator at 37 ℃ for culture for 12-15h, then a single colony is selected and inoculated to XLD agar, and the mice are placed in the constant-temperature incubator at 37 ℃ for culture for 15-18 h; a single colony to be detected is picked up and identified by a MALDI Biotyper system of bacteria.
Further, extracting the whole genome DNA of the salmonella for PCT amplification, and carrying out MLST typing on the salmonella, wherein 7 housekeeping genes for MLST typing of the salmonella are aroC, dnaN, hisD, thrA, purE, hemD and sucA, and the housekeeping gene primer sequences are as follows:
the technical scheme provided by the invention realizes the following beneficial effects:
1. small stress and simple and convenient operation: the invention selects the subcutaneous inoculation of bacteria on the back of the mouse, the infected part is accurate and easy to operate, and the stress degree to the mouse is low. 2. Convenient feeding and low cost: the invention selects ICR mice with low cost and easy purchase as experimental animals, and the ICR mice are raised in a conventional environment, so that the management is convenient, and the experimental result is fit for clinical practice. 3. Short infection period and high infection rate: after subcutaneous inoculation, only 8 days of infection period is needed to cause bacterial infection and inflammation of uterus of non-pregnant mice. 4. The donkey-derived salmonella is used for establishing a subcutaneous route infected uterus model for the first time, and the non-vaginal route is also proved to be one of the routes for causing the uterus infection. 5. Provides an experimental animal model replacing a large animal as an experimental animal, and increases the feasibility of animal experiments. 6. The clinical significance is great: the non-pregnant mouse model in the invention can explore abortion, mating failure and other mechanisms caused by uterine infection of donkey-derived equine abortion salmonella, and provides an animal model for scientific research.
Drawings
FIG. 1 shows the results of clinical symptoms and necropsy changes in mice;
FIG. 2 is a conventional result of mouse blood;
FIG. 3 is a Salmonella mass spectrum;
FIG. 4 is Biotyper match results;
FIG. 5 is a diagram of PCR amplification of Salmonella MLST housekeeping genes;
FIG. 6 shows the result of ST-type alignment of Salmonella;
FIG. 7 is the mouse spleen and uterus bacterial load results;
FIG. 8 shows the results of histological changes in mouse spleen (HE);
FIG. 9 shows the results of histological changes (HE) of mouse endometrium;
FIG. 10 shows the histological change results (HE) of the myometrium and adventitia of a mouse;
FIG. 11 shows the results of expression of TLR4 and inflammatory factors in the uterus of mice detected by qPCR.
Note: control group; 108/S.Abortusequi 108The Salmonella abortus group; 1010/S.Abortusequi 1010Salmonella abortus group
Detailed Description
1 Material
1.1 strains
The equine abortion salmonella of donkey origin is isolated, identified and preserved by veterinary surgery research and development room of veterinary college of beast medical college of Yangzhou university.
1.2 test animals
8-10 week old clean grade ICR non-pregnant mice, purchased from the Yangzhou university center for comparative medicine.
1.3 Primary reagents
Tryptone and yeast extract, produced by OXOID corporation; xylose Lysine Deoxycholate (XLD) agar, produced by Hangzhou microbial agents, Inc.; agar powder, produced by Beijing Soilebao technologies, Inc.; TIANAmp Bacteria DNAkit and TIANGEN TRNzol Universal, produced by Tiangen Biochemical technology Ltd; 2 × EasyTaq PCR SuperMIX and DNA Marker, produced by Beijing Quanjin Biotechnology Ltd; a matrix solution produced by bruke, germany; HiScript Reverse Transcriptase kit, produced by Nanjing Novozam Biotech, Inc.
1.4 Main Instrument
PCR instrument, Gel Doc XR + Gel imaging system, fluorescence quantitative PCR instrument, manufactured by BIO-RAD of America; merrill BC-2800 animal blood corpuscle analyzer, produced by Shenzhen Merrill biomedical electronics, Inc.; an ultraviolet-visible spectrophotometer manufactured by Shanghai spectrometer Co., Ltd; an ultraviolet spectrophotometer manufactured by Thermo corporation of usa; MALDI Biotyper microorganism identification system, produced by Bruker, Germany; a biomicroscope (model number Olympus CX22) manufactured by Olympus corporation of japan; a multi-purpose table refrigerated centrifuge manufactured by eppendorf corporation, germany; a full-temperature culture shaking table and an electric heating constant-temperature air blast drying box which are produced by Shanghai sperm macro experimental equipment Limited company; paraffin microtomes, manufactured by Leica, Germany.
2 method
2.1 recovery of the Strain
And taking the frozen and preserved bacterium liquid, fully shaking up, inoculating the bacterium liquid on an LB solid culture medium, and placing the bacterium liquid in a constant-temperature incubator at 37 ℃ for culturing for 15 h.
2.2 preparation of bacterial liquid
Aseptically picking single colony of donkey-derived Salmonella abortus (hereinafter abbreviated as Salmonella abortus) and inoculating to LB liquid culture medium, culturing at 37 deg.C at 120r/m for 6 hr, taking appropriate amount of bacterial liquid, culturing at 4000rpm for 5min, discarding supernatant, washing thallus and homogeneous bacterial liquid with PBS, and adjusting to 109cfu/mL and 1011cfu/mL。
2.3 animal grouping and infection
The 18 ICR mice were randomly divided into 3 groups, i.e., blank treatment group, Salmonella abortus treatment group I and II, and 6 mice in each group, were bred in a conventional environment, adapted for one week, and had free access to water and food, and irradiated with lightAlternating with a dark 12 h. The back of the mouse was sterilized by dipping a cotton swab in 75% alcohol, and 0.1mL of the bacterial solution (10 mL) was subcutaneously inoculated into each of the Salmonella abortus-treated groups I and II9cfu/mL and 1011cfu/mL), the blank group was inoculated subcutaneously with 0.1mLPBS, and after inoculation, the 8 th d, dissected mice were observed continuously.
2.4 routine examination of blood
On the 8 th day after the infection of the mouse, the eyeball of the mouse is removed and blood is collected by 200 mu L into an anticoagulation tube, the tube wall is flicked, the anticoagulation agent and the blood are conveniently and uniformly mixed, and the hematology index examination is immediately carried out.
2.5 isolation, culture and identification of bacteria
2.5.1 isolation and culture of bacteria
On day 8 after the infection of the mice, the mice were killed by decapitation, spleen and uterus samples were aseptically collected and inoculated on LB solid medium, cultured in a 37 ℃ constant temperature incubator for 12-15h, and then a single colony was picked and inoculated on XLD agar, cultured in a 37 ℃ constant temperature incubator for 15-18 h.
2.5.2 MALDI Biotypeer System identification of bacteria
Selecting a single colony to be detected, coating the colony on a MALDI target plate, and naturally drying; adding 1 mu L of 70% formic acid to cover the colony coating, and naturally drying; then, 1. mu.L of the matrix solution was added to cover the reaction mixture, and the mixture was naturally dried. The target plate was placed in a mass spectrometer for detection. Flex Control and MALDI Biotyper RTC collect specificity atlas, through matching with Biotyper database reference atlas, carry on the bacterium identification, when Bruker Biotyper score ≥ 2.0, think the result is high credibility.
2.5.3 MLST typing of Salmonella
2.5.3.1 Salmonella DNA extraction
Selecting a single salmonella colony, inoculating the single salmonella colony in an LB liquid culture medium, culturing at 37 ℃ and 120r/m for 18-24h, taking the prepared bacterial culture solution as a sample, extracting the whole genome DNA of the salmonella according to the instruction of a TIAnmp Bacteria DNA Kit, and using the whole genome DNA as a PCR template.
2.5.3.2 primer design and Synthesis
The 7 housekeeping genes used for typing of Salmonella MLST were aroC, dnaN, hisD, thrA, purE, hemD and sucA, and the housekeeping gene primer sequences are shown in Table 1.
TABLE 1 MLST housekeeping Gene primer sequences
2.5.3.3PCR reaction procedure
The total PCR reaction was 25 μ L: 2 × EasyTaq PCR SuperMIX 12.5. mu.L, upstream and downstream primers (10. mu.M) each 1. mu.L, template DNA 2. mu.L, supplemented with RNase-free water to 25. mu.L. The PCR reaction program is that the pre-denaturation temperature is 94 ℃ and the time is 3 min; the denaturation temperature is 94 ℃ and the time is 30 s; the annealing temperature is 55 ℃ and the annealing time is 30 s; the extension temperature is 72 ℃ and the time is 1 min; 30 cycles, and finally an extension at 72 ℃ for 5 min. After the reaction is finished, 5 μ L of PCR reaction stock solution is subjected to electrophoresis in 1% agarose Gel at a voltage of 120V for 30min, and the result is observed in a Gel Doc XR + Gel imaging system. And (3) sending the PCR positive reaction product to Nanjing engine biotechnology limited for sequencing, editing sequence results by using MEGA5.0, uploading each obtained sequence to an MLST website, obtaining the number of each strain allele, and further determining the ST type of each strain.
2.6 plate colony count determination of tissue bacterial load
The method comprises the following steps of taking a mouse out of the neck and killing the mouse at the 8 th day after infection, collecting partial spleen and uterus tissues of the mouse, weighing, adding a proper amount of PBS (phosphate buffer solution), grinding the tissues by a mortar until the tissues are homogenized, diluting the tissue homogenate by 10-fold gradient, selecting 10 mu L of bacterial liquid with proper dilution concentration, inoculating the bacterial liquid on an LB (LB) solid culture medium, repeating the steps for three times for each gradient, taking an average value, carrying out static culture at the constant temperature of 37 ℃ for 15-18h, counting, calculating according to a formula, wherein the formula of the tissue bacterial load calculation is shown as
n is the number of plate colonies; m is the weight of the homogenate (unit: g); r: dilution times; the results are in log (log)10) And (4) form representation.
2.7 pathological observations
Spleen and uterus of mice were collected and fixed in 10% formalin solution, and sections were prepared and stained by HE for histopathological observation.
2.8 qPCR detection of TLR4 and inflammatory factor expression
2.8.1 sample Collection
Collecting mouse uterus tissue to a freezing tube, quickly freezing with liquid nitrogen, and storing in a refrigerator at-80 deg.C.
2.8.2 uterine tissue RNA extraction and reverse transcription
RNA in uterine tissue was extracted and then Reverse transcribed into cDNA according to the TIANGEN TRNzol Universal and HiScript Reverse Transcriptase kit instructions and used as a qPCR template.
2.8.3 primer design and Synthesis
According to the primer sequences reported in the reference literature, 9 kinds of qPCR primers are designed and amplified in total and synthesized by Nanjing Ongjingki Biotech Co., Ltd, and the primer sequences are shown in Table 2.
TABLE 2 primer sequences for genes of interest
2.8.4 qPCR reaction procedure
The qPCR reaction was 10 μ L overall: SYBR qPCR Master MIX 5. mu.L, upstream and downstream primers (10. mu.M) 1. mu.L each, template cDNA 2. mu.L, and RNase-free water supplemented to 10. mu.L. The PCR reaction program is 95 ℃ for 30 s; circulating for 40 times at 95 deg.C, 5s, 60 deg.C and 30 s; 95 ℃ for 15 s; 60 deg.C for 1 min; storing at 95 deg.C for 15s and 37 deg.C.
2.8.5 data analysis
By using 2-△△CtThe expression quantity of each gene is calculated by the method, the data is subjected to statistical analysis by adopting One-Way ANOVA in SPSS software, and the result is expressed in the form of Mean +/-SEM. p is a radical of<0.05 indicates significant difference, p<0.01 indicates that the difference is extremely significant.
3 results
3.1 clinical symptoms and Caesarean Change results
Compared to the placebo group, both mice in the Salmonella abortus-treated groups I and II showed significant hairiness disorder (FIG. 1A); the appetite of mice in the equine abortion salmonella treatment group II is seriously weakened, the mice do not want to walk, the mice are infected with the disease at the 5 th day, and the individual mice die; at 8d, the splenomegaly and bleeding were evident in mice I and II treated with Salmonella abortus horse (FIG. 1B), and the uterine horn length was significantly shortened (FIG. 1C)
3.2 blood routine results
As shown in FIGS. 2A and B, the numbers of leukocytes and neutrophils in the blood of mice in the Salmonella abortus-treated groups I and II were significantly increased (p <0.05) compared with the control group, and exceeded the physiological normal range; the number of leucocytes and neutrophils in the blood of the mice in the treatment group II is higher than that of the mice in the treatment group I, but the statistical difference is not significant (p is more than 0.05); the mice in groups I and II treated with Salmonella abortus in equine showed elevated numbers of lymphocytes in blood compared to the control group without statistical differences and showed no dose dependence.
3.3.1 bacterial isolation and culture results
Spleen and uterus samples from placebo mice had no bacterial growth; salmonella abortus treatment groups I and II the bacteria isolated from the spleen and uterus appeared as round, transparent colonies on LB solid medium and as round pink, hydrogen sulfide and lactose negative colonies on XLD agar plates.
3.3.2 characterization of bacteria by MALDI Biotyper System
According to the results of matching the specific spectrum acquired by Flex Control and MALDI Biotyper RTC with the Biotyper database, the separated bacteria is identified as salmonella, and the mass spectrum and the Biotyper matching results are shown in FIGS. 3 and 4 and Table 3.
TABLE 3 Biotyper matching results
3.3.3 MLST molecular typing results
As shown in FIGS. 5, 6 and Table 4, the isolated bacterium amplified 7 housekeeping genes of Salmonella, and ST type is Salmonella abortus ST 251.
TABLE 4 Salmonella ST-type alignment
| ST | aroC | dnaN | hemD | hisD | purE | | thrA |
| ST251 | |||||||
| 93 | 4 | 18 | 89 | 5 | 96 | 87 |
3.4 bacterial load results
Spleen and uterus of each group of mice were counted on day 8 of infection for Salmonella abortus in horses, and spleen and uterus bacterial loads are shown in FIG. 7. No salmonella abortus equi was detected in spleen and uterus of placebo mice; mice in Salmonella abortus group II had higher spleen and uterus bacterial loads than those in Salmonella abortus group I, indicating dose dependence.
3.5 pathological observations
3.5.1 observations of spleen pathology
As can be seen from FIGS. 8A and B, the spleen structure of the mice in the blank control group is intact, the white marrow and the red marrow are clearly demarcated, and abnormal pathological changes occur. In the spleen of mice in the Salmonella abortus-treated groups I and II, there were marked proliferation of white marrow into red marrow, structural changes in periarterial lymph sheath, increased area and enlarged lymphocyte accumulation zone, marked increase in red blood cells in red marrow, and spleen bleeding (FIGS. 8C, D, E and F).
3.5.2 Observation of endometrial Pathology
As can be seen from FIGS. 9A and B, the endometrium (i.e., epithelial and stromal layers) of the mice in the placebo group was structurally intact and showed no abnormal pathological changes. As shown in FIGS. 9C, D, E and F, the uterine epithelium and matrix layer of mice in groups I and II treated with Salmonella abortus equi were structurally intact; neutrophil infiltration of the uterine gland within the stromal layer; treatment group ii neutrophil infiltration in the uterine ducts was significantly greater than treatment group i.
3.5.3 Observation of myometrium and adventitial pathology
As can be seen from FIGS. 10A and B, the structure of the myometrium and adventitia layer of the mice in the placebo group was intact, and there was little neutrophil infiltration and no abnormal pathological changes. As shown in FIGS. 10C, D, E and F, the myometrium and adventitia structures of mice of the Salmonella abortus-treated groups I and II were intact; the mice of treatment group I had significantly increased neutrophil infiltration in the myometrium and adventitial layers compared to treatment group II.
3.6 expression results of TLR4 and inflammatory factor detected by qPCR
As shown in fig. 11A, TLR4 gene expression levels were significantly elevated (p <0.01) and concentration-dependent after infection with different concentrations of salmonella abortus equi compared to the blank control group. As can be seen from FIGS. 11B, C, D, E, F and G, the gene expression levels of the proinflammatory factors IL-1 β, chemokines (IL-6 and IL-8), Th1 type cytokines (TNF-. alpha.and IFN-. gamma.), and Th2 type cytokine (IL-10) in the Salmonella abortus-treated groups I and II were significantly increased (p <0.05) compared to the blank group, but were not concentration-dependent, e.g., the IFN-. gamma.gene level in the Salmonella abortus-treated group II was significantly lower than that in the treated group I (p < 0.05). Compared with the blank control group, the COX-2 gene expression level after the infection of the salmonella abortus equi shows dose-dependent increase (figure 11H), and the high-concentration salmonella abortus equi treatment group II has a significant difference (p <0.01) compared with the blank treatment group.
The invention adopts an animal model common to the infection of the donkey-derived salmonella, namely an ICR non-pregnant mouse, to establish an animal infection model, and establishes the animal model of the donkey-derived salmonella infected non-pregnant mouse by advanced detection methods such as blood routine, flat plate counting of tissue bacterial load, histopathology, related inflammatory gene expression and the like. The successful establishment of the model improves the inoculation mode, shortens the infection period, reduces the experiment cost, and provides an animal model for further researching the pathogenic mechanism and prevention and control technology of the donkey-derived equine abortion salmonella.
Claims (8)
1. The method for establishing the model of the subcutaneous non-pregnant mouse uterine infection caused by the donkey-derived salmonella abortus is characterized by comprising the following steps:
selecting a clean ICR non-pregnant mouse, dividing the clean ICR non-pregnant mouse into a blank treatment group and a donkey-derived equine abortion salmonella treatment group, feeding the mouse in a conventional environment, sterilizing the back of the mouse, inoculating donkey-derived equine abortion salmonella bacterial liquid subcutaneously on the back of the donkey-derived equine abortion salmonella treatment group, and inoculating PBS subcutaneously on a blank control group;
the donkey-derived salmonella equine abortus treatment component comprises a donkey-derived salmonella equine abortus treatment component I and a donkey-derived salmonella equine abortus treatment component II which are respectively inoculated with 0.1mL 10 mL of 109cfu/mL donkey source salmonella abortus liquid and 0.1mL 1011cfu/mL donkey source equine abortion salmonella liquid; the mice are 8-10 weeks old.
2. The method for modeling a non-pregnant mouse uterine infection caused by Salmonella abortus of Equus of claim 1, wherein the mice are killed by cervical dislocation at 8 days after infection of the mice after inoculation.
3. The method for establishing the model of the subcutaneous non-pregnant mouse uterine infection with Salmonella abortus of Equus asinensis according to claim 1, wherein the mouse is raised in a conventional environment for one week, is freely drunk and eaten, and is alternately lighted and dark for 12 hours.
4. The method for establishing the model of the subcutaneous infection of the uterus of the non-pregnant mice caused by the salmonella equi-source equine abortion in claim 1, wherein the sterilization method is to dip a cotton swab with 75% alcohol to sterilize the backs of the mice.
5. The method for establishing the model of the subcutaneous non-pregnant mouse uterine infection caused by the donkey-derived salmonella equine abortus according to claim 1, wherein the preparation method of the donkey-derived salmonella equine abortus liquid comprises the following steps: taking a frozen and preserved donkey-derived salmonella equine abortus bacterial liquid, fully shaking up, inoculating the liquid on an LB solid culture medium, culturing for 15h in a 37 ℃ constant temperature incubator, then aseptically selecting a single bacterial colony of the donkey-derived salmonella equine abortus, inoculating the single bacterial colony on an LB liquid culture medium, culturing for 6h at 120r/m and 37 ℃, taking an appropriate amount of bacterial liquid, rotating at 4000rpm for 5min, discarding supernatant, washing the bacterial body and the homogeneous bacterial liquid with PBS, and adjusting the bacterial liquid to 10 DEG C9cfu/mL and 1011cfu/mL。
6. The method for establishing the model of the subcutaneous non-pregnant mouse uterine infection with Salmonella abortus of Equus asinus according to claim 1, wherein 200 μ L of blood is collected from the mouse eyeball and transferred to an anticoagulation tube, and the tube wall is flicked to facilitate the mixing of the anticoagulation agent and the blood on the 8 th day after the mouse infection, and then the hematological index examination is performed.
7. The method for establishing the model of the subcutaneous non-gestational mouse uterine infection by the Equus equi-abortion salmonella as claimed in claim 1, wherein the mice are killed by cervical dislocation at 8 days after the infection, spleen and uterus samples are aseptically collected and inoculated to LB solid medium, the culture is carried out in a constant temperature incubator at 37 ℃ for 12-15h, then single colonies are picked and inoculated to XLD agar, and the culture is carried out in the constant temperature incubator at 37 ℃ for 15-18 h; a single colony to be detected is picked up and identified by a MALDI Biotyper system of bacteria.
8. The method for constructing the model of the subcutaneous non-pregnant mouse uterine infection with Salmonella abortus of Equus as claimed in claim 7, wherein the whole genome DNA of Salmonella is extracted for PCT amplification, and MLST typing is performed on Salmonella, and the 7 housekeeping genes used for MLST typing of Salmonella are aroC, dnaN, hisD, thrA, purE, hemD and sucA, and the housekeeping gene primer sequences are as follows:
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| CN110511889A (en) * | 2019-08-20 | 2019-11-29 | 山东省农业科学院畜牧兽医研究所 | Salmonella AD19 plants of one plant of donkey source and its application |
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| CN110511889A (en) * | 2019-08-20 | 2019-11-29 | 山东省农业科学院畜牧兽医研究所 | Salmonella AD19 plants of one plant of donkey source and its application |
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