A strain of poultry-derived Salmonella enteritidis SE0730 and its application

Abstract

The present invention discloses a strain of poultry-derived Salmonella enteritidis SE0730 and its application. The strain has been deposited at the China Center for Type Culture Collection; address: Wuhan University; deposit date: May 30, 2024; deposit number: CCTCC NO.M 20241099. The present invention also provides an application of the Salmonella enteritidis for constructing a poultry attenuated vaccine strain. The Salmonella enteritidis SE0730 is sensitive to currently common antibacterial drugs and has good genetic stability, and is suitable for constructing a Salmonella enteritidis attenuated vaccine strain.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a strain of avian-origin Salmonella Enteritidis SE0730 and an application thereof in constructing an attenuated vaccine strain for avian use. Background Art

[0002] Salmonella enteritidis is one of the pathogens that are non-host specific and invasive, and its hosts include humans and various animals. Salmonella enteritidis can not only cause the disease and death of livestock and poultry, causing serious economic losses, but also the contaminated livestock and poultry products can be carriers of Salmonella enteritidis, further endangering human health. Studies have shown that the reason why Salmonella enteritidis is widely spread in poultry products is that after the chicken flock is purified from pullorum, Salmonella enteritidis will replace the original ecological colonization site of Salmonella pullorum, thereby causing widespread spread. At present, acute gastroenteritis (food poisoning) caused by Salmonella enteritidis in humans has become an important issue of international public health.

[0003] Controlling the infection of Enteritidis Salmonella in chickens can effectively prevent food poisoning in humans. Antimicrobial drugs are usually used to control Enteritidis Salmonella in chickens to reduce its harm, but the type of drug, route of administration, method, dosage and maintenance time must be carefully selected. At the same time, long-term and repeated use of drugs not only increases the cost of breeding, but also leads to a series of problems such as increased bacterial resistance and drug residues in poultry products. Facts have proved that relying solely on drug control is not a good way to prevent and control Enteritidis Salmonella. 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 farmers and consumers.

[0004] 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 Enteritidis Salmonella 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

[0005] The purpose of the present invention is to provide a strain of Salmonella Enteritidis SE0730 that is sensitive to common antibacterial drugs, and conventional bacterial gene editing tools can be used to construct an attenuated vaccine strain.

[0006] The technical solution of the present invention is as follows:

[0007] On the one hand, the present invention relates to a Salmonella Enteritidis SE0730, which is a strain with strong immunogenicity, good genetic stability and sensitivity to common antibacterial drugs obtained after repeated screening and verification for many years from more than 1,300 strains of Salmonella Enteritidis collected and preserved over the years. The strain can be used for the construction research of attenuated vaccine strains of Salmonella Enteritidis.

[0008] The Salmonella Enteritidis SE0730 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 Enteritidis, strain number: SE0730, and preservation number: CCTCC NO.M 20241099.

[0009] The invention also provides the application of the enteritidis salmonella in the research of constructing attenuated vaccine strains.

[0010] In a preferred embodiment, when constructing an attenuated vaccine strain of Salmonella Enteritidis SE0730, a bacterial gene editing tool 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 Enteritidis SE0730.

[0011] 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.

[0012] 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 Enteritidis SE0730 is used as a parent strain.

[0013] In a preferred embodiment, it is characterized in that the aroA gene of the parent strain is deleted or mutated.

[0014] The Enteritidis Salmonella SE0730 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

[0015] Figure 1The results are the verification of the SE0730ΔaroA deletion mutant constructed in Example 2. In the figure: M is a DNA molecular weight standard Marker (100-2000 bp), lane 1 is SE0730, lane 2 is SE0730ΔaroA:cat, lane 3 is SE0730ΔaroA, and lane 4 is a negative control. DETAILED DESCRIPTION

[0016] 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.

[0017] Example 1 Basic biological characteristics of Salmonella enteritidis SE0730

[0018] 1) Isolation, purification and staining microscopy

[0019] The enteritidis Salmonella SE0730 strain was isolated from chicken embryos in Yangzhou City, Jiangsu Province in October 2007. The specific isolation method is: aseptically collect chicken samples and add them to SC selective enrichment broth for overnight culture at 37°C, then inoculate xylose lysine deoxycholate agar for overnight culture at 37°C, pick the colonies with metallic black centers, inoculate them in LB agar medium, and culture them 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.

[0020] 2) Biochemical identification

[0021] 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.

[0022] Table 1. SE0730 biochemical identification results

[0023] Biochemical indicators <![CDATA[H2S]]> Indigo Urea KCN Lysine Mannitol Sorbitol ONPG Test results + - - - + + + -

[0024] 3) Serotype identification

[0025] 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 absorb 25μl of Salmonella positive serum (purchased from S&A Laboratory in Thailand) and drop it on the slide, and absorb 25μl of saline as a control, and then absorb 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 antigen formula of the Salmonella is O9,12:Hg,m. According to the antigen formula, the Salmonella is determined to be Salmonella Enteritidis.

[0026] Table 2. SE0730 serotype identification results

[0027]

[0028]

[0029] Note: “+” represents agglutination, and “-” represents no agglutination.

[0030] 4) Drug resistance test

[0031] The broth dilution method combined with the disk diffusion method was used to test the sensitivity of SE0730 to common drugs, and the results were determined according to the 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). The results are shown in Table 3, which shows that SE0730 is 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).

[0032] Table 3. SE0730 drug resistance test results

[0033]

[0034]

[0035] 5) Genetic stability test of SE0730

[0036] After the freeze-dried strain of SE0730 was revived and propagated in TSB broth, it was 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, the variation rate of SE0730 within 50 generations was less than 10%, and the variation rate after 80 generations was also less than 20%, so it has good genetic stability.

[0037] Table 4. SE0730 genetic stability test results

[0038] 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 1 2 40s 50 1 2 50th Generation 50 4 8 60s 50 6 12 70s 50 5 10 80s 50 9 18 90s 50 15 30 100th Generation 50 19 38

[0039] Example 2 Construction of aroA gene deletion mutant of SE0730 using Red homologous recombination system

[0040] Primers were designed based on the upstream and downstream sequences of the aroA gene in the SE0730 genome sequence. Primers SE-ΔaroA-F / SE-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.

[0041] Table 5. Primer sequences for aroA gene mutation

[0042]

[0043]

[0044] Note: The underlined part is a 50bp homologous sequence

[0045] 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. SE0730 prepared competent cells were mixed with pKD46 plasmid and then electrotransformed, and the transformed bacterial solution was spread on an LB plate containing ampicillin resistance, and the transformants were screened overnight. The positive single colony was pure cultured to construct the SE0730-pKD46 strain. The SE0730-pKD46 strain was added to LB broth containing L-arabinose for culture, and further made into competent cells, mixed with the aroA target 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 loss strains of the auxiliary plasmid pKD46 were screened according to the ampicillin antibiotic test, thereby obtaining a deletion mutant strain of SE0730ΔaroA:cat containing only chloramphenicol resistance. The pCP20 plasmid carrying the FLP recombinase was mixed with the SE0730ΔaroA: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 SE0730ΔaroA.

[0046] The genomic DNA of SE0730ΔaroA:CAT and SE0730ΔaroA was extracted, and the wild-type DNA was used as a control. The SE-ΔaroA-F / SE-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 1217 bp. After the chloramphenicol resistance plate identified the elimination of the resistance gene, the size of the target gene was about 203 bp, and the size of the wild-type amplification was about 1487 bp ( Figure 1 ).

[0047] Example 3 LD50 determination of SE0730 wild-type strain and SE0730ΔaroA mutant strain

[0048] The wild-type SE0730 strain and the ΔaroA mutant SE0730 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 SE0730 strain was 10 6.78 CFU, the LD50 after the deletion of aroA gene is 10 8.77 CFU, the virulence decreased by about 100 times.

[0049] Table 6. LD50 test results of SE0730 wild-type strain and SE0730ΔaroA mutant strain

[0050]

[0051]

[0052] Example 4 Determination of the immune efficacy of Salmonella Enteritidis SE0730ΔaroA attenuated live vaccine

[0053] To determine the immune efficacy of SE0730ΔaroA live vaccine, 40 7-day-old SPF chicks were divided into 4 groups, with 10 chicks in each group. Each group was injected intramuscularly with 7.67×10 6 CFU, 7.67×10 5 CFU, 7.67×10 4 CFU of SE0730ΔaroA and PBS (blank control group). Two weeks after immunization, the chickens were challenged with a lethal dose of SE0730 wild strain and the survival and death rates of each group were observed. As shown in Table 7, 7.67×10 5 After receiving SE0730ΔaroA live vaccine at doses above 100%, the immune protection rate reached 100%, indicating that SE0730ΔaroA attenuated live vaccine has good immunogenicity.

[0054] Table 7. Results of determination of immune efficacy of SE0730ΔaroA attenuated live vaccine

[0055] Immunization dose (CFU / mouse) Survival Survival rate <![CDATA[7.67×10 6 ]]> 10 100％ <![CDATA[7.67×10 5 ]]> 10 100％ <![CDATA[7.67×10 4 ]]> 6 60％ PBS control 0 0

[0056] 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 poultry-derived Salmonella Enteritidis ( Salmonella enteritidis )SE0730, characterized in that: It is deposited in China Center for Type Culture Collection; Address: Wuhan University; Deposit date: May 30, 2024; Deposit number: CCTCCNO.M 20241099.

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