CN111374094B - Method for establishing non-pregnant mouse uterine infection model caused by subcutaneous administration of donkey-derived salmonella abortus - Google Patents

Abstract

The invention belongs to the field of biotechnology, and in particular relates to a method for establishing a non-pregnant mouse uterus infection model caused by subcutaneously induced Salmonella abortus from donkeys. The equine-derived Salmonella abortus treatment group was raised in a conventional environment, and the backs of the mice were disinfected. The donkey-derived equine Salmonella abortus treatment group was subcutaneously inoculated with donkey-derived equine Salmonella abortus solution, and the blank control group was subcutaneously inoculated with PBS. The method improves the inoculation method, shortens the infection cycle, and reduces the experimental cost, and provides an animal model for further exploring the pathogenic mechanism and prevention and control technology of donkey-derived Salmonella abortus.

Description

Establishment of a non-pregnant mouse uterus infection model by subcutaneous Salmonella abortus from donkey origin

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for establishing a non-pregnant mouse uterus infection model caused by subcutaneous Salmonella abortus from donkey origin.

Background technique

Salmonella abortus is an important infectious disease causing abortion in equines during pregnancy. Studies have shown that non-vaginal uterine infection is one of the important ways of this pathogenic infection. With the development of China's donkey industry, the occurrence of donkey abortions caused by Salmonella abortus from donkeys has gradually increased, but the symptoms are different from those of Salmonella abortus infection in horses. As a result, breeding failure or gestational miscarriage may be caused, which requires the establishment of a non-pregnant animal model of Salmonella infection from donkeys for related research.

SUMMARY OF THE INVENTION

The invention provides a method for establishing a non-pregnant mouse uterus infection model caused by subcutaneous Salmonella abortus from donkey origin. Pathogenesis and prevention and control technology, animal models are provided.

To achieve the above object, the invention provides the following technical solutions:

A method for establishing a non-pregnant mouse uterus infection model by subcutaneous Salmonella abortus from donkey origin includes the following steps:

Clean-grade ICR non-pregnant mice were selected and divided into a blank treatment group and a donkey-derived equine Salmonella abortus treatment group. They were raised in a conventional environment and the backs of the mice were disinfected. The donkey-derived equine Salmonella abortus treatment group was subcutaneously inoculated with donkey-derived equine abortus Salmonella on the back. Bacterial liquid, blank control group was subcutaneously inoculated with PBS.

Further, said donkey-derived equine aborted Salmonella treatment components are donkey-derived equine aborted Salmonella treatment group I and donkey-derived equine aborted Salmonella treatment group II, respectively subcutaneously inoculated with 0.1 mL of 10 ⁹ cfu/mL donkey-derived equine aborted Salmonella solution and 0.1 mL 10 ¹¹ cfu/mL donkey-derived equine aborted Salmonella solution.

Further, after the inoculation, on the 8th day after the mice were infected, the mice were sacrificed by cervical dislocation.

Further, the mice are 8-10 weeks old.

Further, the mice were reared in a conventional environment for a week to acclimate, with free access to water and food, and the light and dark were alternated for 12 hours.

Further, the disinfection method is to dip a cotton swab in 75% alcohol to disinfect the back of the mouse.

Further, the preparation method of the donkey-derived equine aborted Salmonella liquid is as follows: take the frozen-preserved donkey-derived equine aborted Salmonella liquid, shake it well, inoculate it on LB solid medium, and place it in a 37°C constant temperature incubator for 15 hours, sterile. Pick a single colony of donkey-derived Salmonella abortus and inoculate it in LB liquid medium, culture at 120r/m, 37°C for 6h, take an appropriate amount of bacterial solution, 4000rpm, 5min, discard the supernatant, rinse the bacterial cells and homogeneous bacterial solution with PBS , adjusted to 10 ⁹ cfu/mL and 10 ¹¹ cfu/mL.

Further, on the 8th day after the mice were infected, the mice were enucleated and 200 μL of blood was collected into the anticoagulation tube, and the wall of the tube was flicked to facilitate the mixing of the anticoagulant and the blood, and then the hematological index was checked.

Further, on the 8th day after the mice were infected, the mice were sacrificed by cervical dislocation, and the spleen and uterus samples were collected aseptically and inoculated into LB solid medium, placed in a 37°C constant temperature incubator for 12-15 hours, and then a single colony was picked. It was inoculated into XLD agar and placed in a constant temperature incubator at 37°C for 15-18 hours; a single colony to be tested was picked and identified by the MALDI Biotyper system.

Further, the whole genome DNA of Salmonella was extracted for PCT amplification, and MLST was performed on Salmonella. The 7 housekeeping genes used for MLST typing of Salmonella were aroC, dnaN, hisD, thrA, purE, hemD and sucA, housekeeping gene primers. The sequence is as follows:

The technical scheme provided by the present invention has achieved the following beneficial effects:

1. Less stress and easy operation: the present invention selects mice to inoculate bacteria subcutaneously on the back of the mice, the infection site is accurate and easy to operate, and the degree of stress to the mice is low. 2. Convenient feeding and low cost: the present invention selects low-cost, easy-to-buy ICR mice as experimental animals, and is raised in a conventional environment, which is convenient for management, and the experimental results conform to clinical practice. 3. Short infection period and high infection rate: After subcutaneous inoculation, only 8 days of infection period can cause bacterial infection and inflammation of the uterus of non-pregnant mice. 4. For the first time, the model of subcutaneous infection of uterus was established by Salmonella from donkey, and it was also confirmed that non-vaginal route was also one of the ways to cause uterine infection. 5. Provides an experimental animal model that replaces large animals as experimental animals, increasing the feasibility of animal experiments. 6. Significant clinical significance: The non-pregnant mouse model in the present invention can explore mechanisms such as abortion and breeding failure caused by uterine infection of donkey-derived Salmonella abortus, providing animal models for scientific research.

Description of drawings

Figure 1 shows the results of clinical symptoms and necropsy changes in mice;

Figure 2 shows the results of routine blood tests for mice;

Fig. 3 is Salmonella mass spectrum;

Figure 4 is the Biotyper matching result;

Fig. 5 is the PCR amplification diagram of Salmonella MLST housekeeping gene;

Fig. 6 is the comparison result of Salmonella ST type;

Figure 7 shows the results of bacterial load in mouse spleen and uterus;

Figure 8 is the histological change results of mouse spleen (HE);

Fig. 9 is the histological change result of mouse endometrial (HE);

Figure 10 shows the histological changes of mouse myometrium and adventitia (HE);

Figure 11 shows the results of qPCR detection of the expression of TLR4 and inflammatory factors in mouse uterus.

Note: Control control group; 10 ⁸ /S.Abortusequi 10 ⁸ /Salmonella abortus equine group; 10 ¹⁰ /S.Abortusequi 10 ¹⁰ /Salmonella abortus equine group

Detailed ways

1 material

1.1 Strains

Donkey-derived Salmonella abortus was isolated, identified and preserved by the Department of Veterinary Surgery, School of Veterinary Medicine, Yangzhou University.

1.2 Experimental animals

8-10-week-old clean-grade ICR non-pregnant mice were purchased from the Center for Comparative Medicine, Yangzhou University.

1.3 Main reagents

Tryptone and yeast extract, produced by OXOID company; xylose lysine deoxycholate (XLD) agar, produced by Hangzhou Microbial Reagent Co., Ltd.; agar powder, produced by Beijing Soleibo Technology Co., Ltd.; TIANampBacteria DNAKit and TIANGEN TRNzol Universal, produced by Tiangen Biochemical Technology Co., Ltd.; 2×EasyTaq PCR SuperMIX and DNA Marker, produced by Beijing Quanzhijin Biotechnology Co., Ltd.; Matrix Solution, produced by Bruker, Germany; HiScript Reverse Transcriptase Kit, Nanjing Manufactured by Novozymes Biotechnology Co., Ltd.

1.4 Main instruments

PCR instrument, Gel Doc XR+ gel imaging system, fluorescence quantitative PCR instrument, produced by BIO-RAD, USA; Mindray BC-2800 animal blood cell analyzer, produced by Shenzhen Mindray Biomedical Electronics Co., Ltd.; UV-Vis Spectrophotometer, Produced by Shanghai Spectrum Instrument Co., Ltd.; UV spectrophotometer, produced by Thermo Company of the United States; MALDI Biotyper Microbial Identification System, produced by Bruker Company of Germany; Biological microscope (model Olympus CX22), produced by Olympus Company of Japan; machine, produced by German Eppendorf company; full-temperature culture shaker, electric heating constant temperature blast drying oven, produced by Shanghai Jinghong Experimental Equipment Co., Ltd.; paraffin microtome, produced by German Leica company.

2 methods

2.1 Recovery of strains

Take the cryopreserved bacterial liquid, shake it well, inoculate it on LB solid medium, and place it in a constant temperature incubator at 37°C for 15h.

2.2 Preparation of bacterial solution

Aseptically pick a single colony of donkey-derived Salmonella abortus (hereinafter referred to as Salmonella abortus), inoculate it in LB liquid medium, cultivate at 120r/m, 37°C for 6h, take an appropriate amount of bacterial liquid, 4000rpm, 5min, discard the supernatant , PBS washed the cells and the homogenized bacterial solution, and adjusted them to 10 ⁹ cfu/mL and 10 ¹¹ cfu/mL.

2.3 Animal grouping and infection

18 ICR mice were randomly divided into 3 groups, namely blank treatment group, Salmonella abortus treatment group I and II, 6 mice in each group, reared in conventional environment, acclimatized for one week, freely drinking water and food, light and dark alternated for 12 hours . A cotton swab was dipped in 75% alcohol to disinfect the back of the mice, and 0.1 mL of bacterial solution (10 ⁹ cfu/mL and 10 ¹¹ cfu/mL) was subcutaneously inoculated in Salmonella abortus treatment groups I and II, respectively, and 0.1 mL of PBS was subcutaneously inoculated in the blank control group. , continuously observed until the 8th day, the mice were necropsied.

2.4 Routine blood test

On the 8th day after the mice were infected, the mice were enucleated and 200 μL of blood was collected into the anticoagulation tube, and the wall of the tube was flicked to facilitate the mixing of the anticoagulant and the blood, and then the hematological index was checked.

2.5 Bacterial isolation, culture and identification

2.5.1 Bacterial isolation and culture

On the 8th day after the mice were infected, the mice were sacrificed by cervical dislocation, the spleen and uterus samples were collected aseptically and inoculated into LB solid medium, placed in a constant temperature incubator at 37°C for 12-15 hours, and then a single colony was picked and inoculated into XLD agar , placed in a constant temperature incubator at 37°C for 15-18h.

2.5.2 MALDI Biotyper System Identification of Bacteria

Pick a single colony to be tested, spread it on the MALDI target plate, and dry naturally; add 1 μL of 70% formic acid to cover the colony coating and dry naturally; then add 1 μL of matrix solution to cover and dry naturally. The target plate is placed in the mass spectrometer for detection. FlexControl and MALDI Biotyper RTC collect specific maps, and perform bacterial identification by matching with the reference maps of the Biotyper database. When the Bruker Biotyper score is ≥ 2.0, the result is considered to be highly credible.

2.5.3 MLST typing of Salmonella

2.5.3.1 Salmonella DNA extraction

Pick a single colony of Salmonella and inoculate it in LB liquid medium. After culturing for 18-24 hours at 37°C and 120r/m, take the prepared bacterial culture solution as a sample and extract the whole genome DNA of Salmonella according to the instructions of the TIANamp Bacteria DNA Kit. Make PCR template.

2.5.3.2 Primer Design and Synthesis

The seven housekeeping genes used for MLST typing of Salmonella are aroC, dnaN, hisD, thrA, purE, hemD and sucA. The primer sequences of the housekeeping genes are shown in Table 1.

Table 1 MLST housekeeping gene primer sequences

2.5.3.3 PCR reaction program

The total PCR reaction system is 25 μL: 12.5 μL of 2×EasyTaq PCR SuperMIX, 1 μL of upstream and downstream primers (10 μM), 2 μL of template DNA, and supplemented with RNase-free water to 25 μL. The PCR reaction program was as follows: pre-denaturation temperature 94 °C, time 3 min; denaturation temperature 94 °C, time 30 s; annealing temperature 55 °C, time 30 s; extension temperature 72 °C, time 1 min; 30 cycles, and a final extension at 72 °C for 5 min. After the reaction, 5 μL of the PCR reaction stock solution was taken and electrophoresed on a 1% agarose gel at a voltage of 120 V for 30 min, and the results were observed on a Gel Doc XR+ gel imaging system. The PCR positive reaction product was sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing, and MEGA5.0 was used to edit and process the sequence results. The obtained sequences were uploaded to the MLST website to obtain the allele number of each strain, and then determine each strain. The ST type.

2.6 Determination of tissue bacterial load by plate colony counting

Mice were sacrificed by cervical dislocation on the 8th day after infection. Part of the spleen and uterine tissues of the mice were collected, weighed, added with an appropriate amount of PBS, and ground into tissue homogenate with a mortar. 10 μL of bacterial solution with appropriate dilution concentration was inoculated on LB solid medium, each gradient was repeated three times, the average value was taken, and the bacteria were cultured at a constant temperature of 37 °C for 15-18 h, counted, and calculated according to the formula. The formula for calculating the bacterial load of the tissue is:

n: the number of colonies on the plate; m: the weight of the homogenized tissue (unit: g); r: the dilution ratio; the results are expressed in the form of logarithm (log ₁₀ ).

2.7 Pathological observation

The spleen and uterus of the mice were collected, fixed in 10% formalin solution, sliced, and stained with HE for histopathological observation.

2.8 qPCR to detect the expression of TLR4 and inflammatory factors

2.8.1 Sample Collection

The mouse uterine tissue was collected into cryopreservation tubes, quick-frozen in liquid nitrogen, and transferred to a -80°C refrigerator for storage.

2.8.2 Uterine tissue RNA extraction and reverse transcription

According to the instructions of TIANGEN TRNzol Universal and HiScript Reverse Transcriptase kits, RNA was extracted from uterine tissue and then reverse transcribed into cDNA, which was used as qPCR template.

2.8.3 Primer Design and Synthesis

According to the primer sequences reported in the reference, a total of 9 qPCR primers were designed and amplified, which were synthesized by Nanjing Qingke Biotechnology Co., Ltd. The primer sequences are shown in Table 2.

Table 2 Target gene primer sequences

2.8.4 qPCR reaction program

The total qPCR reaction system is 10 μL: 5 μL of SYBR qPCR Master MIX, 1 μL of upstream and downstream primers (10 μM), 2 μL of template cDNA, and supplemented with RNase-free water to 10 μL. The PCR reaction program was 95°C, 30s; 95°C, 5s, 60°C, 30s followed by 40 cycles; 95°C, 15s; 60°C, 1 min; 95°C, 15s and then stored at 37°C.

2.8.5 Data Analysis

The 2- ^△△Ct method was used to calculate the expression of each gene, and the data were statistically analyzed by One-Way ANOVA in SPSS software, and the results were expressed in the form of Mean±SEM. p<0.05 means significant difference, p<0.01 means extremely significant difference.

3 results

3.1 Clinical symptoms and necropsy changes

Compared with the blank control group, mice in Salmonella abortus equi-treated group I and II had obvious hair disorganization (Fig. 1A). Mice in Salmonella abortus equi-treated group II had severe appetite loss and were reluctant to walk. On the 5th day of infection, some The mice died; on the 8th day of infection, the necropsy showed that the spleen enlargement and hemorrhage were obvious in the Salmonella abortus-treated group I and II (Fig. 1B), and the length of the uterine horn was significantly shortened (Fig. 1C).

3.2 Blood routine results

As shown in Figure 2A and B, the number of white blood cells and neutrophils in the blood of the mice treated with Salmonella abortus equi I and II were significantly increased compared with the blank control group (p<0.05), and exceeded the range of physiological normal values. ; The number of leukocytes and neutrophils in the blood of mice in treatment group II were higher than those in treatment group I, but the difference was not statistically significant (p>0.05); The number of lymphocytes in the blood of the mice increased, but there was no statistical difference, and it did not show a dose-dependence.

3.3.1 Bacterial isolation and culture results

There was no bacterial growth in the spleen and uterus samples of the mice in the blank control group; the bacteria isolated from the spleen and uterus of the mice in the Salmonella abortus treatment group I and II were round and transparent colonies on LB solid medium and on XLD agar plates. Colonies are round pink and negative for hydrogen sulfide and lactose.

3.3.2 MALDI Biotyper system identification results of bacteria

According to the matching results between Flex Control and MALDI Biotyper RTC collection-specific maps and the Biotyper database, the isolated bacteria were identified as Salmonella. The mass spectrum and Biotyper matching results are shown in Figures 3, 4 and Table 3.

Table 3 Biotyper matching results

3.3.3 MLST molecular typing results

As shown in Figures 5, 6 and Table 4, the isolated bacteria can amplify 7 housekeeping genes of Salmonella, and the ST type is Salmonella abortus ST251.

Table 4 Salmonella ST type comparison results

ST aroC dnaN hemD hisD purE sucA thrA ST251 93 4 18 89 5 96 87

3.4 Bacterial load results

The spleen and uterus of mice in each group were counted for Salmonella equivarius on the 8th day of infection, and the bacterial loads in the spleen and uterus were shown in Figure 7. No Salmonella abortus was detected in the spleen and uterus of the mice in the blank control group; the bacterial loads in the spleen and uterus of the mice in the Salmonella abortus treatment group II were higher than those in the Salmonella abortus treatment group I, showing a dose-dependent manner.

3.5 Pathological observations

3.5.1 Pathological observation results of spleen

As can be seen from Figure 8A and B, the spleen structure of the mice in the blank control group was complete, the boundary between the white pulp and the red pulp was clear, and there were abnormal pathological changes. In the spleen of Salmonella abortus equine-treated group I and II mice, the proliferation of white pulp to red pulp was obvious, the structure of periarterial lymphatic sheath was changed, the area was enlarged, and the area of lymphocyte aggregation was enlarged, red blood cells in red pulp were significantly increased, and spleen hemorrhage (Fig. 8C). , D, E and F).

3.5.2 Endometrial pathological observation results

It can be seen from Figures 9A and B that the endometrium (ie, the epithelium and the stromal layer) of the mice in the blank control group has a complete structure and no abnormal pathological changes. As shown in Figure 9C, D, E and F, the structure of the uterine epithelium and stromal layer of the mice in Salmonella abortus equina-treated group I and II was intact; neutrophil infiltration appeared in the uterine glands in the stromal layer; Neutrophil infiltration was significantly more than that in treatment group Ⅰ.

3.5.3 Pathological observation results of myometrium and adventitia

It can be seen from Figure 10A and B that the structure of the myometrium and adventitia layer of the mice in the blank control group was complete, with a small amount of neutrophil infiltration, and no abnormal pathological changes. As shown in Figure 10C, D, E and F, the structure of the myometrium and adventitia of the mice in Salmonella abortus equina-treated group I and II were intact; Neutrophil infiltration was significantly increased in the membrane layer.

3.6 The expression results of TLR4 and inflammatory factors detected by qPCR

As shown in Figure 11A, compared with the blank control group, the expression level of TLR4 gene was significantly increased (p<0.01) after infection with different concentrations of Salmonella abortus in a concentration-dependent manner. From Figure 11B, C, D, E, F and G, it can be seen that the pro-inflammatory factor IL-1β, chemokine (IL-6 and IL-8), Th1-type cytokine (TNF-α) in Salmonella abortus treatment groups I and II -α and IFN-γ) and Th2-type cytokine (IL-10) gene expression levels were significantly higher than those in the blank group (p<0.05), but not in a concentration-dependent manner. For example, IFN in Salmonella abortus treatment group II The level of -γ gene was significantly lower than that of treatment group I (p<0.05). Compared with the blank control group, the expression level of COX-2 gene increased in a dose-dependent manner after S. abortus infection (Fig. 11H), and the high-concentration S. abortus treatment group II was significantly different from the blank treatment group (p<0.01).

The technology of the present invention adopts the common animal model of Salmonella infection from donkeys, namely ICR non-pregnant mice, to establish an animal infection model. Methods Detection methods: An animal model of donkey-derived Salmonella infection in non-pregnant mice was established. The successful establishment of this model improves the inoculation method, shortens the infection cycle, and reduces the experimental cost. It provides an animal model for further exploration of the pathogenic mechanism and prevention and control technology of Salmonella abortus from donkeys.

Claims (8)

1. the establishment method of the non-pregnant mouse uterus infection model caused by subcutaneous Salmonella abortus from donkey origin, is characterized in that, comprises the following steps: Clean-grade ICR non-pregnant mice were selected and divided into a blank treatment group and a donkey-derived equine Salmonella abortus treatment group. They were raised in a conventional environment and the backs of the mice were disinfected. The donkey-derived equine Salmonella abortus treatment group was subcutaneously inoculated with donkey-derived equine abortus Salmonella on the back. Bacterial liquid, blank control group was subcutaneously inoculated with PBS; The donkey-derived equine abortus Salmonella treatment components are donkey-derived equine abortus treatment group I and donkey-derived equine aborted Salmonella treatment group II, respectively subcutaneously inoculated with 0.1 mL 10 ⁹ cfu/mL donkey-derived equine abortus Salmonella liquid and 0.1 mL 10 ¹¹ . cfu/mL donkey-derived equine abortion Salmonella solution; the mice were 8-10 weeks old. 2. the establishment method of the non-pregnant mouse uterus infection model caused by subcutaneous Salmonella abortus from donkey origin according to claim 1, it is characterized in that, after inoculation, in the 8th day after mouse infection, the mouse is killed by cervical dislocation . 3. the establishment method of the non-pregnant stage mouse uterus infection model caused by subcutaneous Salmonella abortus of donkey origin according to claim 1, it is characterized in that, described mice are reared in conventional environment to adapt to one week, freely drink water and eat, Light and dark alternated for 12h. 4. the establishment method of the non-pregnant mouse uterus infection model caused by subcutaneous Salmonella abortus from donkey origin according to claim 1, it is characterized in that, described disinfection method is that cotton swab is dipped in 75% alcohol to disinfect mouse back. 5. the establishment method of the non-pregnant stage mouse uterus infection model caused by subcutaneous Salmonella abortus from donkey origin according to claim 1, it is characterized in that, described donkey origin equine abortion Salmonella liquid preparation method is: get the donkey that is cryopreserved The source horse aborted Salmonella liquid, shaken well, inoculated on LB solid medium, placed in a constant temperature incubator at 37°C for 15 hours, and then aseptically picked a single colony of donkey-derived horse aborted Salmonella and inoculated into LB liquid medium, 120r/ m, incubate at 37°C for 6 hours, take an appropriate amount of bacterial liquid, 4000 rpm, 5 min, discard the supernatant, rinse the bacterial cells and homogeneous bacterial liquid with PBS, and adjust them to 10 ⁹ cfu/mL and 10 ¹¹ cfu/mL. 6. The method for establishing a non-pregnant mouse uterus infection model caused by subcutaneous Salmonella abortus from donkey origin according to claim 1, is characterized in that, on the 8th day after the mouse infection, the mouse is enucleated and 200 μL of blood is collected to the point of resistance. In the coagulation tube, flick the tube wall to facilitate the mixing of the anticoagulant and the blood, and then perform the hematological index examination. 7. the establishment method of the non-pregnant stage mouse uterus infection model caused by subcutaneous Salmonella abortus of donkey origin according to claim 1, it is characterized in that, in the 8th day after the mouse infection, the mice were killed by de-cervical, sterile The spleen and uterus samples were collected and inoculated into LB solid medium, placed in a 37°C constant temperature incubator for 12-15 hours, and then picked a single colony and inoculated into XLD agar, placed in a 37°C constant temperature incubator for 15-18 hours; Colonies were detected and the bacteria were identified by the MALDI Biotyper system. 8. the establishment method of the non-pregnant stage mouse uterus infection model caused by subcutaneous Salmonella abortus of donkey origin according to claim 7, it is characterized in that, the whole genome DNA of extracting Salmonella carries out PCT amplification, and Salmonella is carried out MLST typing , the 7 housekeeping genes used for Salmonella MLST typing are aroC, dnaN, hisD, thrA, purE, hemD and sucA. The housekeeping gene primer sequences are as follows:

Images