A strain of Salmonella pullorum SP0936 from poultry and its application

Abstract

The present invention discloses a strain of Salmonella pullorum SP0936 of poultry origin 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 20241098. The present invention also provides the application of the Salmonella pullorum for constructing a poultry attenuated vaccine strain. Salmonella pullorum SP0936 is sensitive to 20 common antibacterial drugs and has good genetic stability, and is suitable for constructing a Salmonella pullorum attenuated vaccine strain.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a strain of avian-origin Salmonella pullorum SP0936 and an application thereof in constructing an attenuated vaccine strain for avian use. Background Art

[0002] Pullorum is a systemic infectious disease caused by Salmonella pullorum that poses a serious threat to the poultry industry. Salmonella pullorum usually causes acute systemic disease in chicks. Although it rarely causes major clinical disease in adult chickens, it is closely associated with weight loss, diarrhea, and reproductive tract inflammation. Although pullorum is currently well controlled in commercial chicken flocks in developed Western countries, it remains a major problem that threatens the healthy development of the poultry industry in most developing countries, including China.

[0003] At present, the control of pullorum in commercial chickens in China usually uses antimicrobial drugs to reduce its harm, but there is confusion in the selection of drug types, administration routes, 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 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 antimicrobial drug control is not a good way to prevent and treat pullorum. 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.

[0004] Attenuated live vaccines are favored by researchers because of their strong immunogenicity, low immune dose and low price. At present, the preparation of bacterial attenuated live vaccines mainly adopts genetic engineering methods for attenuation. However, the drug resistance of clinically isolated Salmonella pullorum is generally enhanced, especially for some conventional veterinary drugs such as ampicillin, chloramphenicol, sulfadiazine, streptomycin, etc. When bacteria develop resistance to these drugs, they cannot be screened using the resistance tags brought by bacterial gene editing tools, which limits related research. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Salmonella pullorum SP0936 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 pullorum SP0936, which is a strain with strong immunogenicity, good genetic stability and sensitivity to conventional antibacterial drugs obtained after repeated screening and verification for many years from more than 500 strains of Salmonella pullorum collected and preserved over the years. The strain can be used for the construction research of attenuated vaccine strains of Salmonella pullorum.

[0008] The Salmonella Pullorum SP0936 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 Pullorum, strain number: SP0936, and preservation number: CCTCC NO.M 20241098.

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

[0010] In a preferred embodiment, when constructing an attenuated vaccine strain of Salmonella pullorum SP0936, 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 pullorum SP0936.

[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 a Salmonella pullorum attenuated vaccine strain, characterized in that: the Salmonella pullorum SP0936 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 Salmonella pullorum SP0936 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 SP0936ΔaroA deletion mutant constructed in Example 2. In the figure: M is a DNA molecular weight standard Marker (100-2000 bp), lane 1 is SP0936, lane 2 is SP0936ΔaroA:cat, lane 3 is SP0936ΔaroA, 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 pullorum SP0936

[0018] 1) Isolation, purification and staining microscopy

[0019] The SP0936 strain of Salmonella pullorum was isolated from the lungs of dead chicks in Yangzhou City, Jiangsu Province in November 2009. The specific isolation method is: the lungs of dead chicks are collected aseptically and added to SC selective enrichment broth, and cultured overnight at 37°C. Then, xylose lysine deoxycholate agar is inoculated and cultured overnight at 37°C, and small colorless and transparent colonies are picked and inoculated into LB agar medium, and cultured at 37°C for 24 hours. The pure cultured bacteria are subjected to Gram staining microscopy 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. Biochemical identification results of SP0936

[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 slide agglutination method was 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, 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 O9,12:-,- (Table 2). Further fermentation experiments of galactoside and ornithine were carried out, and the results were all negative. According to the antigenic formula combined with the biochemical fermentation results, the Salmonella was determined to be Salmonella Pullorum.

[0026] Table 2. Serological typing results of SP0936

[0027] Diagnostic serum polyA-I O2 O4 O7 O8 O9 Test results + - - - - + Diagnostic serum O10 O11 O12 O15 O19 polyH Test results - - + - - -

[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 SP0936 to 20 common drugs, and the results were determined according to the international bacterial resistance detection standards such as the Clinical and Laboratory Standards Institute (CLSI), the US National Antimicrobial Resistance Monitoring System (NARMS) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The results are shown in Table 3, which shows that SP0936 is sensitive to the 20 drugs tested (ampicillin, cefazolin, cefotaxime, ceftriaxone, ceftazidime, aztreonam, imipenem, streptomycin, gentamicin, kanamycin, amikacin, tetracycline, nalidixic acid, ciprofloxacin, sulfamethoxazole, trimethoprim, cotrimoxazole, chloramphenicol, polymyxin B, and rifampicin).

[0031] Table 3. SP0936 drug resistance test results

[0032] Types of medication Antimicrobial drugs Resistant (R) / Sensitive (S) Penicillins Ampicillin S Cephalosporins Cefazolin S Ceftriaxone S Ceftriaxone S Ceftazidime S Monobactams Aztreonam S Carbapenems Imipenem S Aminoglycosides Streptomycin R Gentamycin S Kanamycin S Amikacin S Tetracyclines tetracycline S Quinolones Nalidixic acid R Fluoroquinolones Ciprofloxacin R Folate metabolic pathway inhibitors Sulfadiazine S Trimethoprim S Co-trimoxazole S Chloramphenicol Chloramphenicol S Colistin Polymyxin B S Rifamycins Rifampin S

[0033] 5) Genetic stability test of SP0936

[0034] After the freeze-dried strain of SP0936 was resuscitated 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, there was no colony type variation when SP0936 was subcultured 30 times, the variation rate was 20% when it was subcultured 60 times, and the variation rate was about one-third after subcultured 90 times, so it has good genetic stability.

[0035] Table 4. SP0936 genetic stability test results

[0036] 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 4 8 50th Generation 50 6 12 60s 50 10 20 70s 50 11 22 80s 50 14 28 90s 50 17 34 100th Generation 50 17 34

[0037] Example 2 Construction of aroA gene deletion mutant of SP0936 using Red homologous recombination system

[0038] Primers were designed based on the upstream and downstream sequences of the aroA gene in the SP0936 genome sequence. Primers SP-ΔaroA-F / SP-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.

[0039] Table 5. aroA gene mutation primer sequences

[0040]

[0041] Note: The underlined part is a 50bp homologous sequence

[0042] 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. SP0936 competent cells were mixed with pKD46 plasmid and then electrotransformed. 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 SP0936-pKD46 strain. The SP0936-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 of SP0936ΔaroA:cat containing only chloramphenicol resistance. The pCP20 plasmid carrying the FLP recombinase was mixed with the SP0936ΔaroA:cat competent cells and then electrotransformed. The transformants were screened on LB plates containing ampicillin and chloramphenicol resistance, and 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 SP0936ΔaroA.

[0043] The genomic DNA of SP0936ΔaroA:CAT and SP0936ΔaroA was extracted, and the wild-type DNA was used as a control. The SP-ΔaroA-F / SP-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 1337 bp. After the chloramphenicol resistance plate identified the elimination of the resistance gene, the size of the target gene was about 323 bp, and the size of the wild-type amplification was about 1607 bp ( Figure 1 ).

[0044] Example 3 LD50 determination of SP0936 wild-type strain and SP0936 ΔaroA mutant strain

[0045] The wild-type SP0936 strain and the ΔaroA mutant SP0936 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 SP0936 strain was 10 5.91 CFU, the LD50 after the deletion of aroA gene is 10 7.29 CFU, the virulence decreased by dozens of times.

[0046] Table 6. LD50 test results of SP0936 wild-type strain and SP0936ΔaroA mutant strain

[0047]

[0048] Example 4 Determination of the immune efficacy of Salmonella pullorum SP0936ΔaroA attenuated live vaccine

[0049] To determine the immune efficacy of SP0936ΔaroA 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 4.21×10 6 CFU, 4.21×10 5 CFU, 4.21×10 4 CFU of SP0936ΔaroA and PBS (blank control group). Two weeks after immunization, the wild strain of SP0936 was used for challenge and the survival and death of each group were observed. As shown in Table 7, the SPF chickens were immunized with 4.21×10 5 After receiving SP0936ΔaroA live vaccine at doses of 100% or above, the immune protection rate reached 100%, indicating that SP0936ΔaroA attenuated live vaccine has good immunogenicity.

[0050] Table 7. Results of determination of immune efficacy of SP0936ΔaroA attenuated live vaccine

[0051] Immunization dose (CFU / mouse) Survival Survival rate <![CDATA[4.21×10 6 ]]> 10 100％ <![CDATA[4.21×10 5 ]]> 10 100％ <![CDATA[4.21×10 4 ]]> 5 50％ PBS control 0 0

[0052] 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 Salmonella Pullorum SP0936 of avian origin, 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 20241098; The Salmonella pullorum SP0936 is used in constructing a poultry attenuated vaccine strain, wherein the aroA gene of the poultry attenuated vaccine strain is deleted.

2. The use of Salmonella pullorum SP0936 in constructing an attenuated vaccine strain according to claim 1, characterized in that: Bacterial gene editing tools containing ampicillin, tetracycline, chloramphenicol, spectinomycin, neomycin, streptomycin, gentamicin and / or kanamycin resistance tags were used to construct mutant strains of Salmonella pullorum SP0936.

3. The use according to claim 2, 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.

4. A method for constructing a Salmonella pullorum attenuated vaccine strain, characterized in that: The Salmonella pullorum SP0936 of claim 1 is used as a parent strain, and the aroA gene of the attenuated vaccine strain of Salmonella pullorum is deleted.

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