A strain of avian-derived Salmonella typhimurium ST0602 and its application

Abstract

The present invention discloses a strain of poultry-derived Salmonella typhimurium ST0602 and its application. The strain has been preserved, and the preservation unit is: China Center for Type Culture Collection; address: Wuhan University; preservation date: May 30, 2024; preservation number: CCTCC NO.M 20241100. The present invention also provides the application of the Salmonella typhimurium for constructing a poultry attenuated vaccine strain. Salmonella typhimurium ST0602 is sensitive to currently common antibacterial drugs and has good genetic stability, and is suitable for constructing a live attenuated vaccine strain of Salmonella typhimurium.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a strain of avian-origin Salmonella typhimurium ST0602 and application thereof in constructing an attenuated vaccine strain for avian use. Background Art

[0002] Salmonella typhimurium is a foodborne pathogen that is very widespread worldwide and is mainly transmitted through contaminated food. Salmonella typhimurium is an important cause of salmonellosis in humans and animals, characterized by a high mortality rate. According to incomplete statistics, there are more than 93.8 million cases of gastroenteritis and more than 155,000 related deaths caused by Salmonella typhimurium each year, which has brought huge public health and socioeconomic burdens to the world. In China, Salmonella typhimurium is one of the two dominant Salmonella serotypes that cause non-typhoidal Salmonella infections. Controlling Salmonella typhimurium infection in livestock and poultry can effectively block human food poisoning. At present, the control of Salmonella typhimurium in livestock and poultry in China usually uses antimicrobial drugs to reduce its harm, but there is confusion in the selection of drug types, routes of administration, methods, dosages and maintenance time, and there is a considerable degree of blindness. Abuse or waste is very common. At the same time, long-term and repeated use of drugs not only increases the cost of feeding, but also leads to a series of problems such as increased bacterial resistance and drug residues in livestock products. Facts have proved that relying solely on drug control is not a good way to prevent and control. In contrast, vaccine immunization does not have the problems of bacterial resistance and drug residues caused by antibiotics, and can produce lasting immunity, so it is more easily accepted by breeders and consumers.

[0003] Attenuated live vaccines are favored by researchers because of their strong immunogenicity, low immune dose and low price. The preparation of bacterial attenuated live vaccines mainly uses genetic engineering methods for attenuation, but the current clinically isolated Salmonella typhimurium is generally highly resistant, usually to ampicillin, chloramphenicol, sulfonamide, streptomycin and other drugs, and these drug resistances often conflict with the resistance tags brought by commonly used bacterial gene editing tools, thus limiting related research. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Salmonella typhimurium ST0602 that is sensitive to common antibacterial drugs, and conventional bacterial gene editing tools can be used to construct an attenuated vaccine strain.

[0005] The technical solution of the present invention is as follows:

[0006] On the one hand, the present invention relates to a Salmonella typhimurium ST0602, which is a strain with strong immunogenicity, good genetic stability and sensitivity to common antibacterial drugs obtained after years of repeated screening and verification from more than 400 strains of Salmonella typhimurium collected and preserved over the years. The strain can be used for the construction research of Salmonella typhimurium attenuated vaccine strains.

[0007] The Salmonella Typhimurium ST0602 of the present invention was sent to the China Center for Type Culture Collection (Wuhan University, Wuhan, China) for preservation on May 30, 2024. The name and identification characteristics are: Salmonella Typhimurium, strain number: ST0602, and preservation number: CCTCC NO.M 20241100.

[0008] The invention also provides the application of the Salmonella typhimurium in the research of constructing attenuated vaccine strains.

[0009] In a preferred embodiment, when constructing an attenuated vaccine strain of Salmonella typhimurium ST0602, bacterial gene editing tools containing resistance tags such as ampicillin, tetracycline, chloramphenicol, spectinomycin, neomycin, streptomycin, gentamicin and / or kanamycin can be used to construct a mutant strain of Salmonella typhimurium ST0602.

[0010] Preferably, Red homologous recombination system, RecET homologous recombination system, CRISPR gene editing system, Cre-lox recombination system, FLP-FRT recombination system, suicide plasmid vector and / or transposon vector bacterial gene editing tools are selected.

[0011] Another aspect of the present invention also relates to a method for constructing an attenuated vaccine strain of Salmonella Enteritidis, characterized in that the above-mentioned Salmonella Typhimurium ST0602 is used as a parent strain.

[0012] In a preferred embodiment, it is characterized in that the aroA gene of the parent strain is deleted or mutated.

[0013] The Salmonella typhimurium ST0602 provided by the present invention is sensitive to common antibacterial drugs, and various bacterial gene editing tools can be used to construct attenuated vaccine strains. In particular, the virulence can be significantly reduced by knocking out or mutating the aroA gene due to the editing tools. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1The results are the verification of the ST0602ΔaroA deletion mutant constructed in Example 2. In the figure: M is a DNA molecular weight standard Marker (100-2000 bp), lane 1 is ST0602, lane 2 is ST0602ΔaroA:cat, lane 3 is ST0602ΔaroA, and lane 4 is a negative control. DETAILED DESCRIPTION

[0015] The present invention is further described below in conjunction with the embodiments and drawings. The following description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention is within the protection scope of the present invention.

[0016] Example 1 Basic biological characteristics of Salmonella typhimurium ST0602

[0017] 1) Isolation, purification and staining microscopy

[0018] The strain of Salmonella typhimurium ST0602 was isolated from the liver of diseased goslings in Taizhou City, Jiangsu Province in February 2006. The specific isolation method is: the liver of goslings was collected aseptically and added to SC selective enrichment broth for overnight culture at 37°C, then inoculated with xylose lysine deoxycholate agar for overnight culture at 37°C, and the colonies with metallic black centers were picked and inoculated into LB agar medium for culture at 37°C for 24 hours. The pure cultured bacteria were examined by Gram staining and then observed under an optical microscope, showing elongated rods with slightly rounded ends.

[0019] 2) Biochemical identification

[0020] Colonies were picked from the LB agar plate for biochemical identification tests. Microbiological identification tubes were purchased from Hangzhou Microbiological Reagent Co., Ltd. and were operated according to the instructions. Results All biochemical indicators of the strain (see Table 1) were consistent with the biochemical characteristics of Salmonella.

[0021] Table 1. Biochemical identification results of ST0602

[0022] Biochemical indicators <![CDATA[H 2 S]]> Indigo Urea KCN Lysine Mannitol Sorbitol ONPG Test results + - - - + + + -

[0023] 3) Serotype identification

[0024] Pick colonies from the LB agar plate and place them in saline. Use turbidimetry to adjust the bacterial concentration to 1.0 × 10 per ml. 10CFU, the method of slide agglutination is used to perform serological typing of the strain. The specific operation method is as follows: take a clean slide, use a micropipette to draw 25μl of Salmonella positive serum (purchased from S&A Laboratory in Thailand) and drop it on the slide, draw another 25μl of saline as a control, and then draw 25μl of bacterial solution and add it to the serum and saline respectively, and mix them thoroughly. Gently shake the slide and determine the result after 2 minutes. The test results show that the antigenic formula of the Salmonella is O4,12:Hi;1,2 According to the antigenic formula, the Salmonella is determined to be Salmonella Typhimurium.

[0025] Table 2. ST0602 serotype identification results

[0026]

[0027]

[0028] Note: “+” represents agglutination, and “-” represents no agglutination.

[0029] 4) Drug resistance test

[0030] The broth dilution method combined with the disk diffusion method was used to test the sensitivity of ST0602 to common drugs, and the results were determined according to international bacterial resistance detection standards such as the Clinical and Laboratory Standards Institute (CLSI), US National Antimicrobial Resistance Monitoring System (NARMS) and European Committee on Antimicrobial Susceptibility Testing (EUCAST). As shown in Table 3, ST0602 was sensitive to the 20 drugs tested (ampicillin, cefazolin, cefotaxime, ceftriaxone, ceftazidime, aztreonam, imipenem, streptomycin, gentamicin, kanamycin, amikacin, tetracycline, nalidixic acid, ciprofloxacin, sulfamethoxazole, trimethoprim, co-trimoxazole, chloramphenicol, polymyxin B, and rifampicin).

[0031] Table 3. ST0602 drug resistance test results

[0032]

[0033]

[0034] 5) Genetic stability test of ST0602

[0035] After ST0602 freeze-dried bacteria were resuscitated and propagated in TSB broth, they were continuously subcultured overnight on TSA agar medium without selection. 50 colonies were randomly selected from the agar plate every 10 generations, and the variation of colony type was observed under a solid dissecting microscope. The total number of generations was 100. As shown in Table 4, ST0602 did not undergo colony type variation after 30 generations of subculture, and the variation rate was less than or equal to 10% after 60 consecutive subcultures, so it has good genetic stability.

[0036] Table 4. ST0602 genetic stability test results

[0037] Generation Number Detection of colony count Number of mutant colonies Mutation rate (%) 10th Generation 50 0 0 20th Generation 50 0 0 30th Generation 50 0 0 40s 50 2 4 50th Generation 50 3 6 60s 50 5 10 70s 50 9 18 80s 50 11 22 90s 50 13 26 100th Generation 50 17 34

[0038] Example 2 Construction of aroA gene deletion mutant of ST0602 using Red homologous recombination system

[0039] Primers were designed based on the upstream and downstream sequences of the aroA gene in the ST0602 genome sequence. Primers ST-ΔaroA-F / ST-ΔaroA-R were used to identify the size of the fragments before and after the aroA gene deletion, and primers CAT-ΔaroA-F / CAT-ΔaroA-R were used to amplify the chloramphenicol resistance gene, as shown in Table 5.

[0040] Table 5. aroA gene mutation primer sequences

[0041]

[0042]

[0043] Note: The underlined part is a 50bp homologous sequence

[0044] Using CAT-ΔaroA-F / CAT-ΔaroA-R as primers and pKD3 plasmid as template PCR, DNA fragments with homology to the upstream and downstream sequences of the aroA gene at both ends and the chloramphenicol resistance gene CAT in the middle were amplified, and the products after PCR amplification were cut and recovered. ST0602 prepared competent cells were mixed with pKD46 plasmid and then electrotransformed, and the transformed bacterial solution was coated on an LB plate containing ampicillin resistance, and the transformants were screened by overnight culture. The positive single colony was pure cultured to construct the ST0602-pKD46 strain. The ST0602-pKD46 strain was added to LB broth containing L-arabinose for culture, and further made into competent cells, mixed with the aroA targeting fragment, and then electrotransformed, and the transformants were screened on an LB plate containing chloramphenicol. The transformants were cultured at 42°C to induce the loss of the auxiliary plasmid pKD46. The strains with the loss of the auxiliary plasmid pKD46 were screened according to the ampicillin antibiotic test, thereby obtaining a deletion mutant of ST0602ΔaroA:cat that only contained chloramphenicol resistance. The pCP20 plasmid carrying the FLP recombinase was mixed with the ST0602ΔaroA:cat competent cells and then electrotransformed. The transformants were screened on LB plates containing ampicillin and chloramphenicol resistance, and then transferred to non-resistant LB plates for culture at 42°C to detect the loss of the auxiliary plasmid pCP20 and CAT resistance genes, thereby obtaining a non-resistant gene mutant strain named ST0602ΔaroA.

[0045] The genomic DNA of ST0602ΔaroA:CAT and ST0602ΔaroA was extracted, and the wild-type DNA was used as a control. The ST-ΔaroA-F / ST-ΔaroA-R primers in Table 5 were used for PCR amplification and identification. The gel electrophoresis results showed that the aroA gene deletion mutation was successful. After the chloramphenicol resistance gene replaced the target gene, the aroA sequence size was about 1384 bp. After the chloramphenicol resistance plate identified the elimination of the resistance gene, the size of the target gene was about 370 bp, and the size of the wild-type amplification was about 1654 bp ( Figure 1 ).

[0046] Example 3 LD50 determination of ST0602 wild-type strain and ST0602 ΔaroA mutant strain

[0047] The wild-type ST0602 strain and the ΔaroA mutant ST0602 strain were enriched in TSB broth and injected intramuscularly into 2-day-old SPF chicks. The mortality was observed for 15 days and the median lethal dose (LD50) was calculated using the Koch method. As shown in Table 6, the LD50 of the wild-type ST0602 strain is 10 6.82 CFU, the LD50 after the deletion of aroA gene is 10 7.98 CFU, the virulence decreased by dozens of times.

[0048] Table 6. LD50 test results of ST0602 wild-type strain and ST0602ΔaroA mutant strain

[0049]

[0050]

[0051] Example 4 Determination of the immune efficacy of Salmonella typhimurium ST0602ΔaroA attenuated live vaccine

[0052] To determine the immune efficacy of ST0602ΔaroA live vaccine, 40 7-day-old SPF chicks were divided into 4 groups, with 10 chicks in each group. Each group was intramuscularly injected with 6.32×10 6 CFU, 6.32×10 5 CFU, 6.32×10 4 CFU of ST0602ΔaroA and PBS (blank control group). Two weeks after immunization, the wild strain of ST0602 was used for challenge and the survival and death of each group were observed. As shown in Table 7, SPF chickens immunized with 6.32×10 5 After receiving ST0602ΔaroA live vaccine at doses of 100% or above, the immune protection rate reached 100%, indicating that ST0602ΔaroA attenuated live vaccine has good immunogenicity.

[0053] Table 7. Results of determination of immune efficacy of ST0602ΔaroA attenuated live vaccine

[0054] Immunization dose (CFU / mouse) Survival Survival rate <![CDATA[6.32×10 6 ]]> 10 100％ <![CDATA[6.32×10 5 ]]> 10 100％ <![CDATA[6.32×10 4 ]]> 7 70％ PBS control 1 10％

[0055] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A strain of avian Salmonella Typhimurium ST0602, characterized in that: The strain has been deposited in the China Center for Type Culture Collection; Address: Wuhan University; Deposit date: May 30, 2024; Deposit number: CCTCC NO.M 20241100.

2. Use of the Salmonella typhimurium ST0602 according to claim 1 in constructing an attenuated vaccine strain for poultry.

3. The use of Salmonella typhimurium ST0602 according to claim 2 in constructing an attenuated vaccine strain for poultry, characterized in that: Bacterial gene editing tools containing ampicillin, tetracycline, chloramphenicol, gentamicin or kanamycin resistance tags were used to construct mutant strains of Salmonella typhimurium ST0602.

4. The use according to claim 3, characterized in that: Red homologous recombination system, RecET homologous recombination system, CRISPR gene editing system, Cre-lox recombination system, FLP-FRT recombination system, suicide plasmid vector or transposon vector are selected as bacterial gene editing tools.

5. A method for constructing a Salmonella typhimurium attenuated vaccine strain, characterized in that: The Salmonella typhimurium ST0602 of claim 1 is used as a parent strain; the aroA gene of the parent strain is deleted or mutated.

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