Avian pathogenic escherichia coli attenuated strain and application thereof

Abstract

The invention discloses an avian pathogenic escherichia coli attenuated strain and application thereof, and belongs to the technical field of biology. The avian pathogenic escherichia coli attenuated strain APEC TW-XM delta yadA disclosed by the invention is a yadA gene deleted strain of a V-type secretion system, is relatively low in fatality rate, and can be used for remarkably reducing the bacterial load of blood and tissues of a tested animal and the content of inflammatory cytokines in serum. The APEC TW-XM delta yadA has the biological characteristics that the exercise capability and the biofilm forming capability are enhanced, and the cell adhesion capability and the macrophage phagocytosis resisting capability are weakened. The virulence of the strain is remarkably reduced, the strain has the potential possibility of being developed into a bacterial attenuated vaccine, and the strain is also beneficial to follow-up research and prevention and control on the pathogenic mechanism of avian pathogenic escherichia coli.

Description

TECHNICAL FIELD

[0001] The present application relates to an avian pathogenic Escherichia coli attenuated strain and its application, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Avian pathogenic Escherichia coli (APEC) belongs to extraintestinal pathogenic E. coli (ExPEC) and is a kind of facultative anaerobic gram-negative bacteria. APEC realizes colonization in the host body by adhesion and invasion, and infects different tissues and organs with the help of various virulence factors. APEC can cause local or systemic infection in poultry, and the clinical manifestations are various inflammations and septicemia. The intensive production of poultry and the rapid expansion of free-range system make poultry more frequently exposed to pathogens and stress factors, which increases the incidence of avian pathogenic Escherichia coli disease and brings huge economic losses to poultry farming. APEC and human ExPEC have many common serotypes, virulence factors and pathogenic mechanisms, which may lead to the spread of APEC to humans through infected poultry and their products, pose a potential threat to public health, and have the risk of zoonosis. In recent years, due to the continuous abuse of antibiotics, APEC has developed extensive drug resistance. Therefore, it is of great significance to strengthen the long-term monitoring and research of APEC for the development of poultry farming and public health safety.

[0003] In order to transport more proteins to the surface of bacteria, gram-negative bacteria have evolved various secretion systems (types I to XIII), and the type V secretion system (T5SS) is one of them. Since the T5SS output mechanism only relies on a polypeptide chain to transport through the outer membrane of bacteria, it does not consume ATP and auxiliary factors such as energy, so it is also called "self-transport system". T5SS-related proteins are widely distributed in APEC and participate in the infection and pathogenic process of APEC, and can significantly affect the adhesion and colonization ability of bacteria, which is an important virulence factor of bacteria.

[0004] Autotransporters (ATs) are the largest group of secreted and outer membrane proteins in Gram-negative bacteria, and trimeric autotransporter adhesins (TAAs) are important members of ATs. TAAs contain a variety of surface adhesins secreted by pathogenic Gram-negative bacteria, which play a key role in the pathogenic process and resistance to host immune response. As the most representative member of TAAs, YadA can effectively mediate the formation and motility of bacterial biofilm, promote bacterial adhesion, and enhance its ability to resist phagocytosis by macrophages, thereby significantly enhancing the pathogenicity of bacteria. Studies have shown that the virulence of Yersinia enterocolitica is closely related to its ability to adhere to collagen, fibronectin and laminin through YadA. In addition, this process can promote bacterial self-aggregation, and bacteria residing in biofilm communities can further enhance their pathogenicity, thereby posing a threat to public health safety. However, plague Yersinia also carries the yadA gene, but due to the influence of frame shift mutation, its TAA cannot be normally expressed. This shows that the function of YadA is not the same in different strains. Based on the influence of yadA gene on the biological characteristics and pathogenicity of APEC, the present application provides a weak strain of avian pathogenic Escherichia coli V-type secretion system, which aims to provide a new idea for understanding the pathogenic mechanism and prevention and control strategy of avian pathogenic Escherichia coli. SUMMARY

[0005] The purpose of the present application is to provide a weak strain of avian pathogenic Escherichia coli and its application.

[0006] Technical solution: The weak strain of avian pathogenic Escherichia coli provided by the present application is preserved in China Center for Type Culture Collection, and is named as Escherichia coli APEC TW-XMΔyadA. The preservation address is Wuhan, China, the preservation number is CCTCC NO: M 20251060, and the preservation date is May 14, 2025.

[0007] Further, the weak strain is obtained by knocking out the yadA gene of the APEC TW-XM strain by using λ-Red homologous recombination technology.

[0008] The application of the weak strain of avian pathogenic Escherichia coli in the preparation of avian pathogenic Escherichia coli attenuated vaccine.

[0009] Further, the dose of the weak strain of avian pathogenic Escherichia coli in the vaccine is 5x10 6 ~1x10 8 CFUs / 200 μL / animal.

[0010] Further, the animal is a mammal and a bird.

[0011] Further, the bird is a duck.

[0012] Further, the vaccine is an injection vaccine.

[0013] Further, the vaccine further contains a vaccine adjuvant.

[0014] The vaccine of the present application contains the above-mentioned avian pathogenic E. coli attenuated strain.

[0015] Further, the dose of the avian pathogenic E. coli attenuated strain in the vaccine is 5×10 6 ~ 1×10 8 CFUs / 200 μL / animal.

[0016] Advantages: Compared with the prior art, the present application has the following remarkable advantages: the avian pathogenic E. coli attenuated strain APEC TW-XMΔyadA of the present application is a V-type secretion system yadA gene deletion strain, which has a lower mortality rate, can significantly reduce the bacterial load in the blood and tissues of the test animals and the content of inflammatory cytokines in the serum. APEC TW-XMΔyadA has the biological characteristics of enhanced motility and biofilm formation ability, and weakened adhesion to cells and resistance to phagocytosis by macrophages. The virulence of the strain of the present application is significantly reduced, which has the potential to be developed into a bacterial attenuated vaccine, and is also helpful for subsequent research and prevention and control of the pathogenic mechanism of avian pathogenic E. coli. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 A diagram for identifying APEC TW-XMΔyadA gene deletion strain (M: Trans2K plus II marker; 1: amplification product of wild strain; 2: amplification product of deletion strain);

[0018] Figure 2 APEC TW-XM, APEC TW-XMΔyadA and APEC TW-XMΔyadA / PyadA bacterial growth curves;

[0019] Figure 3 APEC TW-XM, APEC TW-XMΔyadA and APEC TW-XMΔyadA / PyadA bacterial motility (A) and percentage of motility ring diameter formed in semi-solid medium (B);

[0020] Figure 4 APEC TW-XM, APEC TW-XMΔyadA and APEC TW-XMΔyadA / PyadA bacterial biofilm formation

[0021] Figure 5 Adhesion of APEC TW-XM, APEC TW-XMΔyadA and APEC TW-XMΔyadA / PyadA bacteria to human brain microvascular endothelial cells (hBMEC) and human cervical cancer cells (HeLa) cells;

[0022] Figure 6 Phagocytosis of APEC TW-XM, APEC TW-XMΔyadA and APEC TW-XMΔyadA / PyadA bacteria against mouse monocyte macrophage leukemia cells (RAW264.7), mouse immortalized bone marrow-derived macrophages (iBMDM) and chicken macrophages (HD11);

[0023] Figure 7 Survival curve of APEC TW-XM, APEC TW-XMΔyadA and APEC TW-XMΔyadA / PyadA bacteria in mice;

[0024] Figure 8 Bacterial load determination of APEC TW-XM, APEC TW-XMΔyadA and APEC TW-XMΔyadA / yadA in mice blood;

[0025] Figure 9 Bacterial load determination of APEC TW-XM, APEC TW-XMΔyadA and APEC TW-XMΔyadA / PyadA in mice tissues;

[0026] Figure 10 Changes in the content of inflammatory cytokines and S100A8, S100A9 in the serum of APEC TW-XM, APEC TW-XMΔyadA and APEC TW-XMΔyadA / PyadA in mice;

[0027] Figure 11 Bacterial load determination of APEC TW-XM, APEC TW-XMΔyadA and APEC TW-XMΔyadA / PyadA in duckling tissues;

[0028] Figure 12 Changes in the content of inflammatory cytokines in the serum of APEC TW-XM, APEC TW-XMΔyadA and APEC TW-XMΔyadA / PyadA in ducklings. DETAILED DESCRIPTION

[0029] The technical solutions of the present application will be further described below in conjunction with the accompanying drawings.

[0030] Example 1 Construction of APEC TW-XM Ayada gene deletion strain and determination of growth curve

[0031] The present disclosure includes the use of lambda-Red homologous recombination technology to edit the APEC TW-XM gene to obtain the deletion strain APEC TW-XM Ayada.

[0032] 1. Primer design

[0033] According to the sequence of yadA in the whole genome of APEC TW-XM published on NCBI (NCBI Reference Sequence: NZ_CP025328.1), the upstream and downstream primer sequences of yadA were designed, as shown in Table 1. Among them, P1 and P2 are yadA gene identification primers, and P3 and P4 are homologous recombination targeting fragment amplification primers; the upstream and downstream homologous arms of the amplification primers are located inside the upstream and downstream homologous arms of the identification primers; the underlined sequences in the amplification primers are the complementary sequences on both sides of the chloramphenicol resistance gene cat.

[0034] Table 1 Primer sequences for constructing yadA gene deletion strain

[0035]

[0036] 2. Preparation and recovery of fusion PCR product

[0037] A single colony of DH5a containing pKD3 plasmid (Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000 Jun 6; 97(12): 6640-5. doi: 10.1073 / pnas.120163297. PMID: 10829079; PMCID: PMC18686.) was inoculated into LB medium, centrifuged after overnight growth, and the supernatant was discarded after washing the bacterial pellet with an equal volume of ultrapure water and centrifuging again. The bacterial pellet was resuspended with 200 μL of ultrapure water, boiled in a water bath for 10 min, centrifuged, and the supernatant was collected to obtain the pKD3 template. PCR amplification was performed using the pKD3 plasmid (GenBank: AY048742.1) as the template. The amplification system was as follows: 5 μL of pKD3 plasmid template, 0.75 μL of rTaq enzyme, 5 μL of 10x buffer, 5 μL of dNTPs, 1 μL of each of the amplification primers P3 and P4, and 32.25 μL of ultrapure water. The amplification program is shown in Table 2. The PCR product (yadA-cat gene fusion product) was recovered using an agarose gel kit, and the DNA concentration was determined. The recovered product was stored at -20°C after determining the DNA recovery concentration using a NanoDrop 2000 micro-nucleic acid detector.

[0038] Table 2 Fusion PCR amplification program

[0039]

[0040]

[0041] 3. Preparation of APEC TW-XM competent cells and transformation of pKD46 plasmid

[0042] APEC TW-XM competent cells were prepared by a conventional method, and the pKD46 plasmid (GenBank: AY048746.1) was electroporated into the APEC TW-XM strain (O2:K1:H7, kindly provided by Professor Lu Chengping of Nanjing Agricultural University) with a capacitance of 25 μF, a resistance of 200 Ω, and a voltage of 1.8 kV. Amp R and Cm R resistance plates were used for screening, and APEC TW-XM pKD46 positive clones were obtained after incubation at 30°C for 16 h. The yadA-cat gene fusion product was electroporated into APEC TW-XM pKD46 competent cells, and Amp R and CmR APEC TW-XMΔyadA::cat + pKD46.

[0043] 4. Identification of the primary recombinant strain and elimination of pKD46 plasmid

[0044] APEC TW-XMΔyadA::cat + pKD46 positive clones were inoculated into LB liquid medium containing Amp R and Cm R for culture, and template DNA was prepared. The reaction system was 1 μL of template, 12.5 μL of Green Taq mix, 1 μL of each of gene identification primers P1 and P2, and 10.5 μL of ultrapure water. PCR amplification was used to identify positive clones, and the amplification procedure is shown in Table 3. The pKD46 plasmid is a temperature-sensitive plasmid, and the correct APEC TW-XMΔyadA::cat + pKD46 single colonies were continuously passaged in a 42°C incubator, and the temperature-sensitive characteristics were used to eliminate the pKD46 plasmid. Positive strains sensitive to Amp and not sensitive to Cm were screened and named APEC TW-XMΔyadA::cat.

[0045] Table 3 PCR amplification procedure for identification of the recombinant strain

[0046]

[0047] 5. Secondary recombination and elimination of pCP20 plasmid

[0048] APEC TW-XMΔyadA::cat competent cells were prepared by the same method as described above, and the pCP20 plasmid (GenBank: HB393402) was electroporated into the APEC TW-XMΔyadA::cat competent cells. Positive clones were screened on ampicillin-resistant plates and named APEC TW-XMΔyadA::cat + pCP20. A single APEC TW-XMΔyadA::cat + pCP20 positive clone was picked and continuously passaged in a 42°C incubator. The pCP20 plasmid fully expressed the FLP recombinase to eliminate the cat gene and was gradually eliminated during the high-temperature passage process. The secondary recombinant strain was identified by PCR, and the amplification system and procedure were the same as above. The identification results are shown in Table 4. The secondary recombinant strain was named APEC TW-XMΔyadA, which is the avian pathogenic Escherichia coli V-type secretion system yadA gene deletion strain described in the present application. Figure 1

[0049] ​A small amount of bacteria liquid was picked from the bacteria stock and streaked on LB plate, and incubated at 37°C for 12h to form white, well-defined, slightly convex, and uniform-sized colonies. A single colony was picked and inoculated into appropriate amount of LB liquid medium, and incubated at 37°C, 200rpm in a shaker. The bacteria liquid in the logarithmic growth phase was the fresh bacteria liquid. The bacteria number was 5x10 600nm CFUs / mL when the OD 8 =1.

[0050] 6. Construction and identification of yadA gene complementation strain

[0051] Expression primers P5 and P6 were designed. The sequence of P5 is shown in SEQ ID NO. 5, and the sequence of P6 is shown in SEQ ID NO. 6. P5: 5'-CGCGCTAGCATGAAAACTGTAAACGTAGCTT-3'; P6: 5'-GACGTCGACT TAGAACGAATATCCTACGCC-3'. The yadA gene amplified by PCR was cloned into pBR322 plasmid (TaKaRa, Code No. 3050) using APEC TW-XM genomic DNA as the template and P5 and P6 as primers, to obtain recombinant plasmid pBR322-yadA (Nhe I-Sal I). The recombinant plasmid was transformed into the deletion strain APEC TW-XMΔyadA to obtain the complementation strain APEC TW-XMΔyadA / PyadA.

[0052] 7. Detection of growth curve

[0053] An appropriate amount of bacteria liquid was taken from the bacteria stock of APEC TW-XM, APEC TW-XMΔyadA (ΔyadA), and APEC TW-XMΔyadA / PyadA (PyadA), and streaked on the corresponding resistant LB medium. After overnight incubation at 37°C, a single colony was picked and inoculated into the corresponding resistant LB liquid medium, and incubated for 12h. The culture was transferred into fresh LB liquid medium at a ratio of 1:100, and the transfer was repeated for three generations. When the OD 600 =1.0, 50μL of the corresponding bacteria liquid was taken and inoculated into 5mL of fresh LB liquid medium, and incubated for 12h. The OD 600 value of the corresponding bacteria liquid was measured at an interval of 1h during the incubation, and the value was recorded. The results showed that there was no significant difference in the growth rate of APEC TW-XM, APEC TW-XMΔyadA, and APEC TW-XMΔyadA / PyadA strains in each growth period Figure 2 , indicating that the deletion of yadA gene did not affect the growth of APEC TW-XM.

[0054] Example 2 Biological characteristics of APEC TW-XM AyadA gene deletion strain

[0055] 1. Motility test

[0056] APEC TW-XM, APEC TW-XM AyadA (AyadA) and APEC TW-XM AyadA / PyadA (AyadA / PyadA) were cultured in liquid medium to OD 600 = 1.0; 1500 x g centrifugation for 5 min, discard the supernatant, PBS wash the bacterial pellet. Take 0.5 μL of bacterial liquid drop on the surface of ordinary semi-solid medium (semi-solid medium formula is Tryptone 1%; NaCl 0.25% and Agar 0.25%); place the semi-solid medium vertically in a 37°C bacterial incubator, observe the size of the motility ring formed by each strain after 36h of culture, measure the radius of the motility ring and take pictures. Record the obtained test data and organize, use GraphPad Prism 9.0 to plot and perform one-way ANOVA (p value < 0.05 is considered significant, p value ≥ 0.05 is considered not significant). The results show that Figure 3 ), APEC TW-XM, APEC TW-XM AyadA (AyadA) and APEC TW-XM AyadA / PyadA (AyadA / PyadA) strains can form obvious motility rings on semi-solid medium. Compared with APEC TW-XM, the motility ring formed by APEC TW-XM AyadA is significantly larger than APEC TW-XM. The motility ring formed by APEC TW-XM AyadA / PyadA is reduced, but it is still larger than APEC TW-XM. Compared with APEC TW-XM, the motility ring formed by APEC TW-XM AyadA is 2.01 times that of APEC TW-XM, with significant difference (p < 0.001); the motility ring formed by APEC TW-XM AyadA / PyadA is 1.35 times that of APEC TW-XM (p > 0.05). It shows that the deletion of yadA leads to the significant enhancement of the motility of APEC TW-XM (p < 0.001).

[0057] 2. Biofilm formation test

[0058] The biofilm induction liquid medium formula is: (A) Tryptone 3.0 g, yeast extract 1.5 g, NaCl 0.75 g, dissolved in ultrapure water 150 mL, cooled to room temperature after high pressure; (B) K2HPO4 2.1 g, KH2PO4 0.9 g, FeSO4 2.745 mg, (NH4)2SO4 0.6 g, MgSO4 0.615 g, thiamine hydrochloride 0.6 g, dissolved and filtered through a 0.22 μm microporous filter, then mixed with A, and ensure that it is prepared and used immediately. Clean glass test tubes are used for biofilm formation capacity detection. Activate the bacteria at a ratio of 1:100 in the biofilm induction liquid medium. Incubate vertically in a 30°C bacterial incubator for 36 h; carefully discard the culture medium and gently rinse with ultrapure water for 5 times; add 5 mL of 1% crystal violet solution filtered through a 0.22 μm microporous filter, and stand at room temperature for 30 min; carefully discard the crystal violet solution and gently rinse with ultrapure water; observe the biofilm ring formed by each strain, place it on a white board and take a photo for record. Quantitative detection of biofilm formation is carried out in a 96-well culture plate. Activate the bacteria at a ratio of 1:100 in the biofilm induction liquid medium; add the bacterial solution to the 96-well plate, 150 μL / well, set 6 parallel holes for each strain, and incubate in a 30°C bacterial incubator for 36 h; carefully discard the culture medium and gently rinse with ultrapure water for 5 times; add 1% crystal violet solution filtered through a 0.22 μm microporous filter, 150 μL / well; stand at room temperature for 30 min; carefully discard the crystal violet solution and gently rinse with ultrapure water; add 95% ethanol solution to dissolve the crystal violet, and after the crystal violet is fully dissolved, read the OD 600 value with the help of a full-wavelength multifunctional enzyme label instrument and record it. In addition, the strain initially detected yadA has a cold-adapted property, and different culture temperatures (16°C and 42°C) are selected for comparative study of biofilm formation capacity. The results show that Figure 4), APEC TW-XM, APEC TW-XM AyadA (AyadA) and APEC TW-XM AyadA / PyadA (PyadA) strains could form ring-shaped biofilm in the biofilm induction liquid medium at 30°C. Compared with APEC TW-XM, APEC TW-XM AyadA formed biofilm ring with darker staining and more biofilm formation, indicating that deletion of yadA promoted the formation of APEC TW-XM biofilm. At 30°C, the expression of APEC TW-XM AyadA biofilm increased to 114% of APEC TW-XM, with a significant difference (p<0.001); the expression of APEC TW-XM AyadA / PyadA biofilm was similar to that of APEC TW-XM (p>0.05). The quantitative results of biofilm were consistent with the trend of biofilm qualitative test, indicating that deletion of yadA promoted the formation of APEC TW-XM biofilm.

[0059] Different culture temperatures (16°C and 42°C) were selected for testing to determine the effect of temperature on the biofilm formation ability of APEC TW-XM. Under the conditions of 16°C and 42°C, APEC TW-XM, APEC TW-XM AyadA and APEC TW-XM AyadA / PyadA strains could form ring-shaped biofilm in the biofilm induction liquid medium. This result was consistent with the biofilm qualitative detection results at 30°C. At 16°C, the expression of APEC TW-XM AyadA biofilm increased to 133% of APEC TW-XM, with a significant difference (p<0.001); at 42°C, the expression of APEC TW-XM AyadA biofilm increased to 122% of APEC TW-XM, with a significant difference (p<0.05). This result was consistent with the biofilm quantitative detection results at 30°C. The above results showed that in the culture environment of 16°C, 30°C and 42°C, the ability of AyadA strain to form biofilm was stronger than that of APEC TW-XM strain, and temperature was not the main factor affecting the formation of bacterial biofilm.

[0060] 3. Bacterial adhesion test

[0061] Considering the zoonotic risk of APEC and the shortage of avian cell lines, human brain microvascular endothelial cells (hBMEC) and human cervical cancer cells (HeLa) were used for cross-validation. After recovering the cells, they were passaged for three generations to restore the cell viability. When the monolayer of hBMEC and HeLa cells in the T-25 cell bottle were fully grown, 0.25% trypsin was added for digestion; the supernatant was discarded after centrifugation; the cell pellet was resuspended with fresh cell culture medium; the cell suspension was aspirated and added to a 96-well plate at 100 μL / well; the 96-well plate was placed in a cell incubator at 37°C with a CO2 content of 6% for overnight culture; when the monolayer of hBMEC and HeLa cells fully grew on the bottom wall of the 96-well plate, the cell culture medium was discarded and the cells were washed with PBS for 3 times. The bacterial sample was pretreated as in Example 1, and after washing the bacterial pellet with sterile PBS, 1 mL of DMEM medium without serum was added to resuspend the bacterial pellet. After the bacteria and cells were prepared, the bacteria were added to the cells at a ratio of MOI = 100 (5 replicates for each strain), 100 μL / well, and the 96-well plate was gently tapped on the side wall to ensure that the bacteria and cells were in full contact; the 96-well plate was placed in a bacterial incubator at 37°C for 1 h; the cell culture medium and bacterial liquid mixture were discarded, and the cells were washed with PBS for 3 times to remove the bacteria that failed to adhere successfully; 100 μL / well of autoclaved 1% Triton X-100 lysis solution was added; the plate was placed in a bacterial incubator at 37°C for 30 min for lysis; the lysis solution in the wells was gently blown with a pipette and diluted by 10 times gradient; 10 μL of the diluted solution was added to LB medium, and the LB medium was placed in a bacterial incubator at 37°C for 12 h; the single colonies were counted and recorded. The data analysis and plotting were the same as in the biofilm formation test. The results showed that Figure 5 ), compared with APEC TW-XM, the ability of APEC TW-XMΔyadA to adhere to hBMEC cells decreased to 70% of that of APEC TW-XM, with a significant difference (p < 0.05); the ability of APEC TW-XMΔyadA / PyadA to adhere to hBMEC cells recovered to 97% of that of APEC TW-XM (p > 0.05); in HeLa cells, compared with APEC TW-XM, the ability of APEC TW-XMΔyadA to adhere to HeLa cells decreased to 54% of that of APEC TW-XM, with a significant difference (p < 0.05); the ability of APEC TW-XMΔyadA / PyadA to adhere to HeLa cells recovered to 80% of that of APEC TW-XM (p > 0.05). The above results show that the deletion of yadA can inhibit the adhesion of bacteria to hBMEC and HeLa cells.

[0062] 4. Bacterial phagocytosis test

[0063] Considering the potential zoonotic risks associated with APEC, mouse mononuclear macrophage leukemia cells (RAW264.7), immortalized mouse bone marrow-derived macrophages (iBMDM), and chicken macrophages (HD11) were used in the experiments. The pretreatment methods for bacterial and cell samples were the same as for the 'bacterial adhesion assay'. After both bacteria and cells were prepared, the bacterial culture was added to 48-well plates containing cells at a ratio of MOI=100 (5 replicates per strain), 150 μL / well. The sidewalls of the 48-well plates were gently tapped to ensure sufficient contact between bacteria and cells. The 48-well plates were incubated at 37°C for 1 hour. The cell culture medium and bacterial culture mixture were discarded, and the plates were washed three times with PBS. 150 μL / well of serum-free DMEM containing gentamicin (200 μg / mL) was added, and the plates were incubated at 37°C for 1 hour to kill extracellular bacteria. The mixture was discarded, and the plates were washed three times with PBS. 1% Triton X-rays were added after autoclaving. X-100 lysis buffer, 150 μL / well; incubate at 37°C for 30 min for complete lysis; gently pipette the lysis buffer from the wells and serially dilute 10-fold; add 10 μL of the dilution to LB medium and incubate at 37°C for 12 h, count and record single colonies. Data analysis and graphing are the same as in the 'Biofilm Formation Assay'. Results show ( Figure 6 In RAW264.7 cells, the relative phagocytic rate of APEC TW-XMΔyadA was 166%; in iBMDM cells, it was 137%; and in HD11 cells, it was 124%. All these rates were significantly higher than those of the APEC TW-XM strain (p<0.05). This indicates that the absence of yadA in bacteria leads to massive phagocytosis by macrophages, weakening their resistance to phagocytosis.

[0064] Example 3: Study on the pathogenicity of the APEC TW-XMΔyadA gene deletion strain in mice.

[0065] 1. Establish a mouse infection model

[0066] Three-week-old BALB / c mice were purchased from the Comparative Medicine Center of Yangzhou University. The ambient temperature was around 25℃, and the mice were given 12 hours of light and 12 hours of darkness per day, along with a regular diet and water.

[0067] (I) Infection method via gavage: Fifteen 3-week-old BALB / c mice were randomly divided into 5 groups of 3 mice each. The infection dose of APECTW-XM strain was set at 1×10⁻⁶. 8 CFUs / 500μL / each; 5×10 8 CFUs / 500μL / each; 1×10 9 CFUs / 500μL / ; 2.5×109 CFUs / 500μL / only and 5x10 9 CFUs / 500μL / only. The mice were fasted and deprived of water 12 h before gavage and were gavaged with 5% NaHCO3 solution 30 min before infection to neutralize stomach acid; the mice were observed for clinical symptoms and death within 72 h after gavage. The results showed that the mice in different dose infection groups did not show any clinical symptoms within 72 h after infection, indicating that the APEC TW-XM strain failed to infect mice by oral gavage.

[0068] (ii) Intraperitoneal infection method: 16 three-week-old BALB / c mice were randomly divided into four groups, including APEC TW-XM strain infection groups (infection doses were 5x10 7 CFUs / 100μL / only, 1x10 8 CFUs / 100μL / only, 2.5x10 8 CFUs / 100μL / only) and a control group, with 4 mice in each group. The control group of mice was intraperitoneally injected with an equal amount of autoclaved PBS. The mice were observed for clinical symptoms and death after intraperitoneal injection. The results showed that the mice in the control group did not show obvious clinical symptoms; all mice in the 2.5x10 8 CFUs / 100μL / only infection group died within 8 h after infection and no obvious clinical symptoms were observed; the mice in the 5x10 7 CFUs / 100μL / only infection group showed mild clinical symptoms after infection; the mice in the 1x10 8 CFUs / 100μL / only infection group died within 24 h after infection and showed obvious clinical symptoms. At the beginning of the course, the APEC TW-XM group of mice were depressed, had no appetite, had secretions at the corners of the eyes, had messy fur, had damp and hard perianal fur, and were contaminated with loose feces; at the point of death, the mice showed neurological symptoms such as convulsions and opisthotonos. In summary, the subsequent mouse infection test used intraperitoneal injection, and the infection dose was 1x10 8 CFUs / 100μL / only.

[0069] (iii) Median lethal dose (LD 50 ) test: 80 three-week-old BALB / c mice were randomly divided into four groups, namely APEC TW-XM (group A), APEC TW-XMΔyadA (group B), APEC TW-XMΔyadA / PyadA (group C) and a control group (group D). Groups A to C each had a total of 25 mice; each group was further divided into 5 subgroups (corresponding to the 5 infection doses set), with 5 mice in each subgroup; group D had 5 mice. The infection doses were set at 1x10 5 CFUs / 100μL / only; 1x10 6CFUs / 100μL / unit; 1×10 7 CFUs / 100μL / unit; 1×10 8 CFUs / 100μL / each and 1×10 9 CFUs / 100μL / mouse. Mice in each group were infected via intraperitoneal injection, while control mice were injected intraperitoneally with an equal volume of autoclaved PBS. The number of mice that died within 7 days of infection was recorded, and the LD50 of each strain was calculated using the modified Kohl's method. 50 The results showed that the median lethal dose (LD50) of APEC TW-XM was 5.01 × 10⁻⁶. 6 The median lethal dose (LD50) of CFUs, APEC TW-XMΔyadA, is 2 × 10⁻⁶. 8 CFUs; The median lethal dose (LD50) of APEC TW-XMΔyadA / PyadA is 3.16 × 10⁻⁶. 8 CFUs. The results showed that, compared with APEC TW-XM, APEC TW-XM had a higher median lethal dose of ΔyadA and a lower lethality, indicating that the pathogenicity of the strain decreased after the deletion of yadA.

[0070] 2. Mouse survival curve

[0071] Thirty 3-week-old BALB / c mice were randomly divided into three groups: APEC TW-XM group (WT), APEC TW-XMΔyadA group (ΔyadA), and APEC TW-XMΔyadA / PyadA group (PyadA), with 10 mice in each group. The infection dose was set at 1×10⁻⁶. 8 CFUs / 100μL / mouse; mice in each group were infected via intraperitoneal injection; the number of mice that died within 24 hours of infection was recorded and the survival rate of each group was calculated. The results showed ( Figure 7 Within 24 hours of infection, all mice in the APEC TW-XM group developed the disease and died, with a survival rate of 0%; the survival rate in the APEC TW-XMΔyadA group was 100%; and the survival rate in the APEC TW-XMΔyadA / PyadA group was 60%. This indicates that the pathogenicity of the strain was significantly reduced after the yadA deletion.

[0072] 3. Determination of bacterial load in mouse blood

[0073] Eight 3-week-old BALB / c mice were randomly divided into two groups: the APEC TW-XM group (WT) and the APEC TW-XMΔyadA group (ΔyadA), with four mice in each group. The infection dose was set at 1×10⁻⁶. 8CFUs / 100 μL / mouse; infection method was intraperitoneal injection; within 10 h after infection, blood of mice was collected every hour, 10 μL of the diluted solution was added on LB medium after dilution by gradient ratio, and single colony was counted after incubation at 37 °C for 12 h. The results showed that Figure 8 ), APEC TW-XM strain continuously proliferated after entering the blood of mice, showing an upward trend; while the bacterial load in the blood of APEC TW-XMΔyadA group mice decreased significantly within 1 h after infection, and remained at a low level within 9 h after infection, and no death occurred.

[0074] 4. Bacterial load determination of mouse tissues

[0075] 14 three-week-old BALB / c mice were randomly divided into four groups, APEC TW-XM (group A), APEC TW-XMΔyadA (group B), APEC TW-XMΔyadA / PyadA (group C) and control group (group D). Groups A to C, 4 each; group D, 2. The infection dose was set to 1×10 8 CFUs / 100 μL / mouse; infection method was intraperitoneal injection; the control group was injected with high-pressure sterilized PBS, 100 μL / mouse. 12 h after infection, the mice were euthanized and dissected; the tissues and organs of mice (heart, liver, spleen, lung, kidney and brain) were obtained, ground after weighing, and diluted by gradient ratio of 10 times; 10 μL of the diluted solution was added on LB medium, and single colony was counted after incubation at 37 °C for 12 h. The results showed that Figure 9 ), compared with APEC TW-XM infection group, the bacterial load of each tissue of APEC TW-XMΔyadA infection group mice decreased significantly; it was proved that deletion of yadA could reduce the colonization and survival of APEC TW-XM in mouse tissues.

[0076] 5. ELISA detection of the content of inflammatory cytokines and S100A8, S100A9 in mouse serum

[0077] The test procedure is the same as that of 'Bacterial load determination of mouse tissues'. Blood samples were collected from mice in each group and left at room temperature for 30 min. The serum was collected by centrifugation and used for determination of IL-1β (pro-inflammatory cytokine, mainly produced by activated macrophages, involved in inflammatory response and immune response), IL-10 (anti-inflammatory cytokine, involved in immune regulation), IL-8 (chemotactic factor, capable of chemotaxis of neutrophils to the infection site), IL-6 (multifunctional cytokine, involved in immune regulation, inflammatory response, metabolism and tissue repair), TNF-α (tumor necrosis factor, involved in systemic inflammation), IFN-γ (interferon, capable of anti-virus and activation of macrophages) and S100A8 and S100A9 (important markers for reflecting the degree of inflammation in the clinic). The ELISA kit used for detection was purchased from Wuhan Genemei Biotechnology Co., Ltd. The subsequent detection was performed according to the operation steps of the kit instruction: dilute the standard and add sample; add enzyme-labeled reagent; seal the plate with sealing film and incubate in a 37°C incubator for 30 min; dilute the concentrated washing solution with distilled water; take the plate out of the 37°C incubator, carefully remove the sealing film; discard the liquid and shake dry; add enough washing solution to each well, stand for 30 s, then discard, repeat this step 5 times, and then tap dry. Add 50 μL of color developing agent A to each well, then add 50 μL of color developing agent B; mix gently, develop color at 37°C for 10 min in the dark; add 50 μL of stop solution to each well to stop the reaction; set the blank well to zero, and read the OD 450 values with a full-wavelength multifunctional enzyme marker and record them; record and organize the obtained data; draw a standard curve; substitute the read OD 450 values into the linear regression equation of the standard curve to calculate the actual concentration of the sample. Data analysis and plotting are the same as in Example 2. The results show that Figure 10 the contents of IL-1β, IL-10, IL-8, IL-6, TNF-α and IFN-γ in the APEC TW-XMΔyadA group were decreased compared with those in the APEC TW-XM group. The results show that the inflammatory response caused by APEC TW-XMΔyadA is weaker than that caused by the APEC TW-XM strain, and yadA may exacerbate tissue damage by specifically regulating the pro-inflammatory / anti-inflammatory balance. The content of S100A8 in the serum of mice in the APEC TW-XMΔyadA group was 115 pg / mL (p<0.05), and the content of S100A9 was 1,630 pg / mL (p<0.001), which was decreased and had a significant difference compared with the APEC TW-XM group. This indicates that the degree of inflammation caused by APEC TW-XMΔyadA is weaker than that caused by the APEC TW-XM strain.

[0078] Example 4 Study on the pathogenicity of APEC TW-XMΔyadA gene deletion strain to ducklings

[0079] 1. Establishing the duckling infection model

[0080] The original ducklings (7-day-old, purchased from Yangzhou Jiangsu Gaoyou Duck Development Group Co., Ltd., and fed with non-antibiotic feed) were selected as the infection objects, and the strains for infecting the ducklings were APEC TW-XM, APEC TW-XMΔyadA, and APEC TW-XMΔyadA / PyadA. The ducklings were grouped according to the research purposes; three infection doses were set, which were 5×10 8 CFUs / 200 μL / duckling, 1×10 8 CFUs / 200 μL / duckling, and 5×10 6 CFUs / 200 μL / duckling; each group had 6 ducklings, and the infection method was intraperitoneal injection; and the appropriate infection dose was screened to establish the duckling infection model. The results showed that all the ducklings in the 5×10 8 CFUs / 200 μL / duckling dose group died within 12 h after infection (including the APEC TW-XM, APEC TW-XMΔyadA, and APEC TW-XMΔyadA / PyadA infection groups). The ducklings in the 1×10 8 CFUs / 200 μL / duckling dose group showed obvious clinical symptoms such as lethargy, unstable standing or paralysis, dull response to external stimuli, depression, diarrhea, and feces adhering to the cloaca within 20 h after infection, and the ducklings in the APEC TW-XM group died successively. The ducklings in the APEC TW-XMΔyadA group showed milder clinical symptoms. In the 5×10 6 CFUs / 200 μL / duckling dose group, all the ducklings showed only mild clinical symptoms within 72 h after infection, and the number of dead individuals was extremely small. According to the test results of establishing infection models with different infection doses, 1×10 8 CFUs / 200 μL / duckling and 5×10 6 CFUs / 200 μL / duckling were selected as the effective infection doses.

[0081] 2. Bacterial load determination of duckling tissues

[0082] Combined with the test of establishing infection models with different infection doses, two infection doses were selected for tissue bacterial load detection.

[0083] (I) 1×10 8 CFUs / 200 μL / duckling dose: 35 7-day-old ducklings were divided into four groups, which were APEC TW-XM (group A), APEC TW-XMΔyadA (group B), APEC TW-XMΔyadA / PyadA (group C), and the control group (group D). Each of groups A to C had 10 ducklings, and group D had 5 ducklings. The infection dose was 1×10 8CFUs / 200μL / duckling; infection method: intraperitoneal injection; control group: injection of physiological saline, 200μL / duckling. Ducklings were euthanized and necropsies performed within 20 hours post-infection; various organs (heart, liver, spleen, kidney, pancreas, and brain) were collected, weighed, ground, and serially diluted 10-fold; 10μL of each dilution was added to LB agar and incubated at 37℃ for 12 hours. Single colonies were counted and recorded. Results showed ( Figure 11 Compared with the APEC TW-XM group, the bacterial load in various tissues of ducklings in the APEC TW-XMΔyadA group was significantly reduced, indicating that the absence of yadA can reduce the colonization and survival of APEC TW-XM in duckling tissues.

[0084] (ii) 5×10 6 CFUs / 200μL / duckling infection dose: 54 7-day-old ducklings were divided into 3 groups: APEC TW-XM (WT), APEC TW-XMΔyadA (ΔyadA), and APEC TW-XMΔyadA / PyadA (PyadA); 18 ducklings in each group; infection dose was 5 × 10⁻⁶ CFUs / 200μL / duckling. 6 CFUs / 200μL / duckling; infection method: intraperitoneal injection. The infection time was divided into 6 time points (24h, 36h, 48h, 60h, 72h, and 84h post-infection). At each specified time point, 3 ducklings in each group were euthanized and necropsy performed. Various organs (heart, liver, spleen, kidney, pancreas, and brain) were collected, weighed, ground, and serially diluted 10-fold. 10μL of each dilution was added to LB agar and incubated at 37℃ for 12h. Single colonies were counted and recorded. Results showed ( Figure 11 Compared to the APEC TW-XM group, the APEC TW-XMΔyadA group showed decreased bacterial loads in the heart and pancreas at all six time points; however, bacterial loads in the liver, spleen, kidneys, and brain increased at the fourth time point (60 hours) post-infection. In the APEC TW-XMΔyadA group, bacterial loads were only observed in the heart between 36 and 48 hours, while in the APEC TW-XMΔyadA / PyadA group, bacterial loads were only observed in the heart and pancreas between 48 and 60 hours. This may be because the infectious dose was lower, the bacterial count was lower in the early stages of infection, and plate counting had a lower limit of detection, resulting in undetectable bacterial loads at some time points. Combining the tissue bacterial load results from the two infectious doses, it is concluded that the absence of yadA reduces the colonization and survival of APEC TW-XM in duckling tissues.

[0085] 3. ELISA detection of inflammatory cytokine levels in duckling serum

[0086] The blood of each group of ducklings in the 'bacterial load determination of duckling tissues' was collected (1 x 10 8 CFUs / 200 μL / infectious dose group and 5 x 10 6 CFUs / 200 μL / infectious dose group, wherein 5 x 10 6 CFUs / 200 μL / infectious dose group combined with the results of the tissue bacterial load test to set two detection times (36 h and 72 h after infection), and placed at room temperature for 30 min; centrifugal collection of duckling serum; detection of IL-1β, IL-10 and TNF-α content in serum of ducklings in different infection groups. Refer to the operation steps in the instruction manual of the ELISA kit of Nanjing Bestbio Technology Co., Ltd. The basic steps are: dilute the standard and add sample; add enzyme-labeled reagent; after sealing the plate with sealing film, incubate in a 37°C incubator for 30 min; dilute the concentrated washing solution with distilled water; take the plate out of the 37°C incubator, carefully remove the sealing film; discard the liquid and spin dry; add enough washing solution to each well, stand for 30 s, then discard, repeat this step 5 times, then tap dry. Add color developing agent A 50 μL to each well, then add color developing agent B 50 μL; gently shake to mix, develop color at 37°C for 10 min in the dark; add stop solution 50 μL to each well to stop the reaction; zero the blank well, read the OD 450 value with a full-wavelength multifunctional enzyme label meter and record; record and organize the obtained data; draw a standard curve; substitute the read OD 450 value into the linear regression equation of the standard curve to calculate the actual concentration of the sample. Data analysis and plotting are the same as in Example 2.

[0087] The results show that Figure 12 , (i) 1 x 10 8 CFUs / 200 μL / infectious dose: compared with APEC TW-XM, the IL-1β, IL-10 and TNF-α contents of the APEC TW-XM ΔyadA group decreased; among them, IL-10 and TNF-α had significant differences (p < 0.05), indicating that yadA affects the regulation of inflammatory cytokines. (ii) 5 x 10 6CFUs / 200 μL / infectious dose: At 36 h post-infection, the contents of inflammatory cytokines IL-1β, IL-10 and TNF-α in the serum of the APEC TW-XMΔyadA group were lower than those of the APEC TW-XM, with no significant difference; at 72 h post-infection, the contents of inflammatory cytokines IL-1β and IL-10 in the serum of the APEC TW-XMΔyadA group were lower than those of the APEC TW-XM, and the content of TNF-α was higher than that of the APEC TW-XM. This may be because at 72 h post-infection, the immune regulation of the duck body is imbalanced, the pro-inflammatory response is enhanced, and the anti-inflammatory regulation is weakened. Combined with the results of the above two infectious doses, it shows that the inflammatory response caused by APEC TW-XMΔyadA is weaker than that of the APEC TW-XM strain.

[0088] According to the results of the above examples, the avian pathogenic E. coli attenuated strain described in the present application is a V-type secretion system yadA gene deletion strain, which is characterized in that it has similar morphological, cultural and biochemical identification characteristics to the APEC TW-XM strain; has enhanced biological characteristics such as motility, biofilm formation ability, and weakened adhesion to cells and resistance to phagocytosis by macrophages. The avian pathogenic E. coli attenuated strain described in the present application has significantly reduced virulence, and has the potential to be developed into a bacterial attenuated vaccine. Further, the avian pathogenic E. coli V-type secretion system yadA gene deletion strain APEC TW-XMΔyadA provided in the present example has the characteristics of low lethality, and significant reduction in bacterial load in blood and tissues and the content of inflammatory cytokines in serum of test animals, and can be applied to the preparation of vaccines for the prevention and control of avian pathogenic E. coli disease.

[0089] The above examples are only used to describe the basic principles of the present application, the main characteristics and application of the avian pathogenic E. coli attenuated strain APEC TW-XMΔyadA.

Claims

1. An avian pathogenic E. coli attenuated strain, characterized in that, The avian pathogenic Escherichia coli attenuated strain is deposited in China Center for Type Culture Collection, named as Escherichia coli APEC TW-XMΔyadA, and is located in Wuhan, China, with a preservation number of CCTCC NO: M 20251060 and a preservation date of May 14, 2025.

2. The avian pathogenic E. coli attenuated strain according to claim 1, characterized in that, The attenuated strain is obtained by knocking out the yadA gene of the APEC TW-XM strain by using a λ-Red homologous recombination technique.

3. The avian pathogenic Escherichia coli attenuated strain of claim 1 is used in the preparation of an avian pathogenic Escherichia coli attenuated vaccine.

4. Use according to claim 3, characterized in that, The dose of the avian pathogenic E. coli attenuated strain in the vaccine is 5 x 10 6 ~ 1 x 10 8 CFUs / 200 μL / animal.

5. Use according to claim 4, characterized in that, The animals include mammals and birds.

6. Use according to claim 5, characterized in that, The birds are ducks.

7. Use according to claim 3, characterized in that, The vaccine is an injection vaccine.

8. Use according to claim 3, characterized in that, The vaccine further contains a vaccine adjuvant.

9. A vaccine comprising a polynucleotide of claim 1. The vaccine contains the avian pathogenic Escherichia coli attenuated strain of claim 1.

10. The vaccine according to claim 9, characterized in that the dose of the avian pathogenic E. coli attenuated strain in the vaccine is 5 x 10 6 ~ 1 x 10 8 CFUs / 200 μL / animal.

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