Porcine pasteurella multocida high-temperature passage attenuated strain and application thereof

Abstract

The invention belongs to the field of vaccine microorganisms, and provides a swine pasteurella multocida attenuated strain PmA4-750, which is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 20251644. The invention further provides a preparation method of the swine pasteurella multocida attenuated strain PmA4-750. After alternate high-temperature continuous passage, three generations of whole-gene sequencing and pathogenicity contrast tests find that a vaccine candidate strain (PmA4-750 strain) has a section of specific insertion fragment with the length of about 43kb, and 14 genetic variation sites, including 10 InDel (insertion / deletion) and 4 SNP (single nucleotide polymorphism), exist in the vaccine candidate strain. The inserted genes may generate adaptive evolution of strains in the passage process of the strains, so that the pathogenic characteristics of the strains are changed, and the toxicity is promoted to be remarkably reduced. In addition, a piglet immune challenge protection test result shows that the PmA4-750 strain has an immune effect on swine pasteurellosis caused by type A, type D or type B pasteurella multocida. In addition, the invention also discloses a swine pasteurellosis live vaccine and a preparation method thereof.

Description

Technical Field

[0001] This invention belongs to the field of vaccine microbiology. Specifically, this invention relates to a porcine Pasteurella multocida strain (PmA4-750) that has been passaged at high temperatures, and the application of this strain in the preparation of a live vaccine against porcine Pasteurella multocida. Background Technology

[0002] Pasteurella multocida (Pm) is a key zoonotic pathogen, a Gram-negative bacterium capable of infecting various hosts including pigs, sheep, cattle, chickens, rabbits, horses, geese, and humans. It typically causes pasteurellosis when the host is malnourished or has weakened immunity, resulting in diseases such as fowl cholera, swine pneumonia, swine atrophic rhinitis, and respiratory diseases in cattle, posing a serious threat to animal and human health. This pathogen was officially named Pasteurella multocida in 1938. Based on differences in capsular antigen immunogenicity, Pasteurella multocida is classified into five capsular serotypes (A, B, D, E, and F).

[0003] Currently, outbreaks of Pasteurella multocida infection remain frequent in my country and other pig-producing countries worldwide, seriously threatening the healthy and stable development of the pig industry. In my country's pig herds, the predominant serotype has shifted from type B to types A and D (incidence >85%), while traditional vaccine strains (such as type B C44401 strain) show serotype mismatch, with a cross-protection rate of <40%.

[0004] my country lags behind in swine pasteurellosis control technology, currently relying mainly on traditional vaccines developed in the mid-20th century, such as inactivated swine pasteurellosis vaccines (type B C44401 strain) and live swine pasteurellosis vaccines (type B EO630 strain). The serotypes of these traditional vaccines do not match the currently prevalent serotypes. Recent research indicates that previous type B vaccines cannot provide effective cross-protection against type A and type D strains, posing a significant challenge to swine pasteurellosis control. Therefore, developing highly effective inactivated, subunit, or live vaccines against the currently prevalent type A and type D strains of Pasteurella multocida is an urgent need for the prevention and control of swine pasteurellosis. CN119530084A screened a highly virulent vaccine-producing strain from clinical isolates of type A Pasteurella multocida to prepare an inactivated vaccine. This vaccine was found to protect pigs against lethal doses of type A Pasteurella multocida and also provide partial protection against type F strains. However, traditional inactivated or subunit vaccines have limited immunogenicity, narrow protection (only against specific serotypes), require multiple vaccinations, and have serotype coverage of less than 40%, failing to induce mucosal immune responses. In contrast, live vaccines can mimic natural infection, stimulating multiple responses of mucosal, cellular, and humoral immunity, providing more comprehensive protection, covering multiple serotypes, expanding the scope of protection, effectively preventing disease, reducing morbidity and mortality in pig herds, and thus improving farming efficiency.

[0005] In the field of veterinary biological products and microbial vaccine research and development, high-temperature passage attenuation technology has become a widely used practice for developing attenuated strains of various pathogens. This technology involves continuously passaged pathogens under non-physiological high-temperature conditions, utilizing the selective pressure generated by the high temperature to gradually screen for strains with reduced virulence but stable immunogenicity. For example, Huazhong Agricultural University (CN10222026313B) successfully developed a bovine mycoplasma attenuated strain, MbovHB0801-150.2, using a single high temperature (41℃) for 150 consecutive passages. Animal experiments showed that the virulence of this strain was significantly reduced while maintaining good immunogenicity, making it suitable for use as a vaccine strain. This method represents the mainstream mode of current high-temperature passage attenuation technology, namely, constant high-temperature passage. However, a single temperature may limit sufficient variation in virulence genes. Summary of the Invention

[0006] For the reasons stated above, this invention provides a porcine Pasteurella multocida strain subjected to high-temperature passage and its application in the preparation of a live vaccine against porcine Pasteurella infection. This invention employs two different high-temperature alternating passages of the porcine wild-type Pasteurella multocida strain A. Compared to the traditional single-temperature mode, this alternating temperature fluctuation strategy can exert stronger environmental pressure on the pathogen, accelerate more comprehensive genomic adaptive mutations, and thus more thoroughly inactivate virulence genes.

[0007] One of the objectives of this invention is to provide a porcine-derived attenuated strain of Pasteurella multocida, classified and named Pasteurella multocida PmA4-750, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20251644.

[0008] Furthermore, whole-genome sequencing revealed 14 genetic variation sites in the PmA4-750 strain compared to its parent, the PmA4 strain, including 10 InDels (insertions / deletions) and 4 SNPs (single nucleotide polymorphisms). These variations occurring in the CDS region may alter the structure and function of the proteins encoded by the genes. Regarding structural variations, the F750 generation strain genome contained a specific insertion fragment of approximately 43 kb compared to the F0 generation. Functional prediction of these inserted genes revealed that some genes were associated with bacterial resistance, virulence factor synthesis, and environmental adaptation. It is speculated that these inserted genes may have led to adaptive evolution of the strain during passage, altering its pathogenic characteristics and significantly reducing virulence. This provides important reference value for screening key targets in subsequent live vaccine development.

[0009] A second objective of this invention is to provide a live porcine Pasteurella multocida vaccine comprising an effective dose of the PmA4-750 strain and a pharmaceutically acceptable lyophilized protectant.

[0010] Furthermore, the preparation method of the porcine Pasteurella multocida live vaccine includes the following steps:

[0011] (a) The wild-type strain PmA4 of Pasteurella multocida from pigs (preservation number: 202106118) was revived to obtain F1 generation cells;

[0012] (b) In TSA medium containing 3-8% newborn calf serum, culture at 41-43℃ for 10-14 hours to obtain F2 generation cells;

[0013] (c) Take the single colony obtained in step (b) and incubate it in the same culture medium as in step (b) at 44-46℃ for 10-14 hours;

[0014] (d) Repeat steps (b) and (c) to perform alternating generations, with a cumulative generation of ≥500 generations;

[0015] (e) The final generation strain was purified by ≥3 single-colony purifications to obtain a purified strain, which was named PmA4-750 strain.

[0016] (f) The PmA4-750 strain was freeze-dried and preserved under conditions containing 8-12% skim milk and 5-8% sucrose as a freeze-drying protectant.

[0017] Furthermore, in a live vaccine against porcine Pasteurella multocida, challenge with a homologous type A virulent strain (PmA4 strain) after immunization: 100% of the control group developed the disease, of which 80% died, while 100% of the immunized group were protected.

[0018] Furthermore, in a clinical challenge test of a porcine-derived Pasteurella multocida live vaccine against a highly virulent porcine type D strain (PmD6), 80% of the control group developed the disease, with 40% dying, while 80% of the immunized group remained protected. Similarly, in a challenge test against a highly virulent porcine type B strain (CVCC44401), 100% of the control group developed the disease, with 80% dying, while 80% of the immunized groups remained protected. Cross-protection tests demonstrated that this Pasteurella multocida live vaccine exhibits good cross-protection.

[0019] For a more detailed solution, please refer to the specific implementation examples.

[0020] The beneficial effects of this invention are:

[0021] The porcine Pasteurella multocida type A strain (PmA4 strain) used in this invention was isolated, purified, and identified as type A from lung tissue, synovial fluid, and spleen lesions collected from suspected infected pigs in a large-scale pig farm in Wuhan, Hubei Province in 2021. A live porcine Pasteurella multocida vaccine (PmA4-750 strain), obtained through alternating high-temperature passage attenuation, was prepared using this strain. This vaccine can be used to prevent swine pneumonia caused by type A Pasteurella multocida and also provides cross-protection against swine Pasteurella multocida caused by types D and B. It exhibits advantages such as good safety, high immunogenicity, long duration of immunity, and cross-protection. Attached Figure Description

[0022] Figure 1 The growth status of Pasteurella multocida on TSA plates;

[0023] Figure 2 Gram staining results;

[0024] Figure 3 A: Identification of the kmt I gene in Pasteurella multocida; B: Identification of the yellow membrane type A in Pasteurella multocida.

[0025] Figure 4 Preliminary assembly results of F0 and F750 generation Contig;

[0026] Figure 5 Statistical results of genome assembly quality;

[0027] Figure 6 Bandage loop topology diagram (red: free loop; blue: chromosome);

[0028] Figure 7 Comparison of positional changes of the annular structure between F0 and F750 generations;

[0029] Figure 8 Ten variant sites are located in the CDS region of the encoded protein;

[0030] Figure 9 A schematic diagram of the evolution mechanism of the 43kb insertion sequence. Detailed Implementation

[0031] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention.

[0032] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0033] Example 1: Passage of porcine Pasteurella multocida strain Pm-A4 and screening of attenuated strains

[0034] 1. Materials

[0035] 1.1 Source of the strain: In 2021, the strain was collected from the lung tissue, joint fluid and spleen of suspected infected pigs from large-scale pig farms in Wuhan, Hubei Province. It was isolated, purified and identified.

[0036] 1.2 Culture Media and Reagents

[0037] 1.2.1 TSA solid culture medium

[0038] Weigh 40g of tryptic soy agar (TSA), dissolve it in 950mL of double-distilled water, autoclave at 121℃ for 15min, cool to about 45℃, add 50mL of filtered sterilized bovine serum, mix thoroughly and pour into a petri dish for later use.

[0039] 1.2.2 TSB liquid culture medium

[0040] Weigh 30g of Tryptic Soy Broth (TSB), dissolve it in 950mL of double-distilled water, autoclave at 121℃ for 15min, and set aside. Add serum before use.

[0041] 1.2.3 Reagents

[0042] Bacterial genomic DNA extraction kit (Beijing Zhuangmeng International), 2×Rapid Taq Master Mix (Nanjing Novozymes), Pasteurella multocida capsular typing primers (Nanjing Genscript Synthetic), biochemical identification kit (Hangzhou Microbiology Reagent): oxidase reagent, sugar fermentation tubes (glucose, sucrose, mannitol), urease reagent, etc., as well as PCR amplification reagent (TaKaRa) and Gram staining solution (Haibo Biotechnology).

[0043] 1.3 Instruments and equipment: PCR instrument (Applied Biosystems), low-temperature high-speed centrifuge (Eppendorf), gel imaging system (Bio-Rad), microplate reader (Thermo Fisher), sterile operating table (Suzhou Antai), etc.

[0044] 2 methods

[0045] 2.1 In vitro passage culture of Pm-A4 strain

[0046] The original strain of Pasteurella multocida A4, which was isolated and freeze-dried in clinical settings, was revived and activated. The revived and activated bacterial culture was used as the F1 generation. The F1 generation was then inoculated onto TSA plates (containing 5% healthy newborn calf serum) and incubated at 42°C for 12 hours to be harvested as the F2 generation. Then, typical single colonies were selected and inoculated onto TSA plates (containing 5% healthy newborn calf serum) and incubated at 45°C for 12 hours to be harvested as the F3 generation. The same method was used to continuously subculture the bacteria by changing the temperature. When the 750th generation was reached, three consecutive clonal purification and identification were performed. The finally obtained purified F750 generation strain was named the candidate attenuated strain PmA4-750 and freeze-dried for later use.

[0047] 2.2 PCR identification of strains from different generations

[0048] Primers for identifying the kmt I gene and the hyaD-hyaC gene capsular type were designed to target Pasteurella multocida. Specific primer sequences for identifying Pasteurella multocida and specific primer sequences for identifying the capsular type were designed. Primers P1 / P2 and P3 / P4 were used to identify and amplify the capsular type of different generations (F1, F250, F450, F750) of the strain.

[0049] 2.2.1 Primer sequences are shown in Table 1.

[0050] Table 1 Primer sequences for PCR amplification of Pasteurella multocida.

[0051]

[0052] 2.2.2 Reagent Kits: Both the viral nucleic acid extraction kit and the PCR kit are commercially available products.

[0053] 2.2.3 DNA extraction: DNA was extracted using a commercial nucleic acid extraction kit, following the instructions in the kit's manual.

[0054] 2.2.4 PCR reaction system

[0055] 2.2.4.1 PCR reaction system for seed determination: total volume 50 μl, genomic DNA 2 μl, 1.5 μl each of 10 μmol / L upstream and downstream primers (P1 / P2), 25 μl of 2×Accurate Taq Master Mix, and 20 μl of ultrapure water.

[0056] 2.2.4.2 Capsule-based PCR reaction system

[0057] The total volume is 50 μl, including 2 μl of genomic DNA, 4.0 μl each of 10 μmol / L upstream and downstream primers (P3 / P4), 20 μl of 2×Accurate Taq Master Mix, and 20 μl of ultrapure water.

[0058] 2.2.5 PCR reaction procedure

[0059] 2.2.5.1 The fixed-type PCR reaction program is as follows: pre-denaturation at 95℃ for 5 minutes; denaturation at 95℃ for 30 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 1 minute, for a total of 30 cycles; and finally extension at 72℃ for 10 minutes to end the reaction.

[0060] 2.2.5.2 Capsule-type PCR reaction program: 95℃ pre-denaturation for 10 minutes; 95℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 1 minute, for a total of 30 cycles; final extension at 72℃ for 10 minutes to end the reaction.

[0061] 2.2.6 Electrophoresis Detection

[0062] After PCR amplification, 5.0 μl of the PCR product was subjected to electrophoresis on a 1.0% agarose gel containing nucleic acid dye, with DNA Marker as a reference. Electrophoresis was performed at 120V for 25–30 minutes. After electrophoresis, the sample was observed under a UV lamp.

[0063] 2.3 Purity testing of different generations of seedlings

[0064] Different generations (F1, F250, F450, F650, F750) of strains were tested for purity according to the appendix of the current Chinese Veterinary Pharmacopoeia, Part III.

[0065] 2.4 Examination of biological characteristics of strains from different generations

[0066] 2.4.1 Morphological observation

[0067] After the culture was completed, the strains of different generations (F1, F250, F450, F650, F750) were Gram stained and the morphology of the cells was observed under a microscope.

[0068] 2.4.2 Biochemical Characteristic Testing

[0069] After cultivation, different generations of bacterial strains (F1, F250, F450, F650, F750) were inoculated into bacterial identification biochemical tubes for oxidase, catalase, sugar fermentation (glucose, sucrose, mannitol), and urease tests. The biochemical reaction results were observed and recorded and compared with the standard biochemical characteristics of Pasteurella multocida.

[0070] 2.4.3 Testing of Culture Characteristics

[0071] After cultivation, strains of different generations (F1, F250, F450, F650, and F750) were inoculated onto TSA plates containing 5% healthy newborn calf serum and incubated at 36–37°C for 16–22 hours, and colony morphology was observed. Simultaneously, TSB medium (containing 5% healthy newborn calf serum) was inoculated, and after fermentation at 37°C for 7–8 hours, the bacterial culture was harvested. Viable bacterial counts were performed on TSA plates containing 5% healthy newborn calf serum according to the appendix of the current Chinese Veterinary Pharmacopoeia, Part III.

[0072] 2.4.4 Serological Characteristic Tests

[0073] According to the NY / T 564-2016 standard, strains identified as PmA group positive by PCR at different generations (F1, F250, F450, F650, F750) were retested using the Caeter capsular grouping method to further confirm the consistency of Pasteurella multocida serotypes.

[0074] 3 Results

[0075] 3.1 Results of Purity and Biological Characteristics Tests of Strains from Different Generations Strains from different generations (F1, F250, F450, F650, F750) were tested for purity and biological characteristics according to the appendices of the current Chinese Veterinary Pharmacopoeia, Part III. The specific results are shown in Table 2.

[0076] Table 2. Results of purity and biological characteristics tests of strains from different generations.

[0077]

[0078] The results of the purity and biological characteristics tests of strains from different generations are shown in Table 2. Figure 1 Growth status of Pasteurella multocida on TSA plates and Figure 2Gram staining results showed that all passaged strains were pure, with all being Gram-negative, and their morphology was characterized by short rods or coccobacilli with intense staining at both poles and a slightly convex center. The results indicate that the morphological characteristics of the parent strain of *Pasteurella multocida* (strain PmA4) remained stable during passage, without significant changes. In biochemical tests, the oxidase, xylose, sorbitol, nitrate reduction, arabinose, lactose, and urease tests all showed consistent results, completely consistent with the biochemical characteristics of the *Pasteurella multocida* type A standard strain. This further confirms the stability of the passaged strain in terms of biochemical characteristics. Figure 3 A is the identification of the kmt I gene of Pasteurella multocida in pigs. Figure 3 B represents the identification of type A of Pasteurella multocida in the yellow membrane of pigs. As can be seen from the figure, both PCR identification (species identification and capsular serotyping) and Caeter typing serotyping identification identified it as type A.

[0079] The feline calicivirus strain isolated in this invention is named Pasteurella multocida strain PmA4-750, and was deposited at the China Center for Type Culture Collection on July 21, 2025, with accession number CCTCC NO:M20251644.

[0080] Example 2. Full-length genome sequencing and comparative analysis of genetic variations of PmA4-750 strain

[0081] 1. Materials and Methods

[0082] 1.1 Source of strains

[0083] The porcine Pasteurella multocida strains F0 (original clinical isolates) and F750 (strains after 750 consecutive passages in the laboratory) used in this embodiment were both provided by Wuhan Keqian Biotechnology Co., Ltd. The original strains (F0) were isolated from clinically ill pigs. The F750 strain was obtained by continuously passaged the F0 strains in vitro 750 times under laboratory conditions.

[0084] 1.2 Genome Sequencing

[0085] High-coverage whole-genome sequencing of two generations of strains was performed using the PacBio RS II sequencing platform. Before sequencing, the strains were rigorously purified; subsequently, high-integrity, high-molecular-weight genomic DNA was extracted using specialized methods, and single-molecule real-time sequencing libraries were constructed. After sequencing, data error correction and assembly were performed using Flye software, while QUAST software was used to check the assembly quality to ensure the accuracy and reliability of the genome sequence.

[0086] 1.3 Genomic loop assay

[0087] By consulting the assembly_info.txt document in the fly assembly output folder, we obtained the circularization prompt information; at the same time, we used the bandage software v1.0.0 to construct the assembly diagram and visually observe the circularization of contigs, thus doubly verifying the authenticity and integrity of the genomic circular structure.

[0088] 1.4 Extraction of circular sequences

[0089] The samtools tool was used to accurately extract the circular structure sequence of the assembled genome, preparing for in-depth analysis of the characteristics of the circular structure.

[0090] 1.5 Genetic Variation Detection

[0091] The Mummer v 4.0.0beta2 software was used to compare genomes (snp, indel, structural variation) and perform detailed comparisons of the F0 generation and F750 generation strain genomes. Single nucleotide polymorphisms (SNPs), insertions and deletions (InDel), and structural variations were systematically detected.

[0092] 2 Results

[0093] 2.1 Genome assembly quality

[0094] After assembling the original data using Flye software, the F0 generation strain genome yielded 5 contigs, with the longest contig reaching 2409518 bp, completely covering the full length of the standard genome of *Pasteurella multocida* (approximately 2.4 Mb). The N50 value reached 2409518 bp, demonstrating good assembly continuity. The F750 generation strain genome yielded 3 contigs, with the longest contig reaching 2452524 bp, also covering the full length of *Pasteurella multocida*, with an N50 value of 2452524 bp. Specific assembly indices comparing the contig assembly length distribution and N50 values ​​between the F0 and F750 generations are as follows: Figure 4 As shown, the specific genome assembly quality statistics are as follows: Figure 5 As shown.

[0095] 2.2 Identification of genome circularity

[0096] By examining the assembly_info.txt file, circumstance indications were found in the genomes of both the F0 and F750 generations of strains; combined with the bandage software, the circumstance status of contigs was examined as follows: Figure 6 As shown, the F0 generation exhibits a circular contig (length ≈ 15 kb) independent of the main chromosome, and the bandage map indicates that it is a free element. Figure 6(Red arrow in the image); F750 generation: circular structure integrated into the main chromosome ( Figure 6 (The blue lines in the diagram indicate this). It was confirmed that circular structures independent of the main chromosome exist in the genomes of both generations of strains. This discovery is significant for understanding genome organization and genetic stability, and provides crucial clues for further analysis of the dynamic changes in these circular structures during strain propagation.

[0097] 2.3 Detection and Analysis of Ring Structures

[0098] Sequencing results as follows Figure 7 As shown, the alignment position of the circular structure on the genome in the F750 generation strain differs significantly from that in the F0 generation. In the F0 generation, the circular structure was located at genomic coordinates [1,500,000–1,515,000, as shown on the left side of the red box], while in the F750 generation it migrated to [2,200,000–2,215,000, as shown on the right side of the red box]. This shift suggests that the circular element underwent genomic integration during passage. This change indicates that the impact of this structural positional shift must be fully considered in subsequent genome alignment analyses.

[0099] 2.4 Results of Genomic Comparison

[0100] A comprehensive genome alignment of the F750 and F0 generations of strains was performed using Mummer v4.0.0beta2 software, detecting a total of 14 genetic variation sites, including 10 InDels (insertions / deletions) and 4 SNPs (single nucleotide polymorphisms). Figure 8 Table 3 shows 10 variant sites located in the CDS region encoding proteins (including 7 missense mutations, 2 nonsense mutations, and 1 frameshift mutation), while Table 3 shows 4 additional variant sites located in the intergenic region. These variants in the CDS region may significantly affect the biological characteristics of the strain by altering the structure and function of the proteins encoded by the genes (such as changes in amino acid sequence, premature introduction of stop codons, etc.), for example, affecting metabolic pathways and the activity of pathogenic factors. As for the 4 variant sites located in the intergenic region, although they do not directly encode proteins, they may still indirectly affect the physiological functions of the strain by affecting the transcriptional regulation of genes (such as promoter region binding efficiency, changes in transcription factor binding sites, etc.).

[0101] Table 3. Four variant sites located in intergenic regions

[0102]

[0103] 2.5 Analysis of Structural Variation (SV)

[0104] 2.5.1 Origin of Inserted Sequences

[0105] Regarding structural variation, the F750 generation genome contained a specific insertion fragment of approximately 43 kb compared to the F0 generation. Further analysis revealed a 15 kb extrachromosomal circular DNA element in the F0 generation. BLAST alignment showed that only 75% of the sequence (approximately 11.25 kb) of this element was homologous to the F0 generation's main chromosome, while the remaining 25% (approximately 3.75 kb) was a unique sequence. This indicates that the element existed in the F0 generation in a mixed form of "partial integration + partial free space"; while... Figure 9 The schematic diagram of the evolution mechanism of the 43kd insertion sequence shows that the full-length sequence (15kb) of the circular element was 100% integrated into the main chromosome in the F750 generation, and there were direct repeat sequences on the flanking side of the integration site, suggesting that after homologous recombination-mediated integration, the region was amplified, eventually forming a 43kb insertion fragment.

[0106] 2.5.2 Amplification and Structure of Inserted Sequences

[0107] Table 4 shows that after removing the homologous sequence (contig_4) already present in the F0 generation, the 43kb insert fragment was identified as containing two functional gene clusters. The first insert region is 1020bp long, located in the genome [coordinate interval 2,100,500–2,101,520], containing 8 genes: geneA, geneB, ..., geneH. The second insert region is 1311bp long, located in the genome [coordinate interval 2,102,800–2,104,111], covering 10 genes: geneI, geneJ, ..., geneR. Functional prediction of these inserted genes revealed that some genes are related to bacterial drug resistance, virulence factor synthesis, and environmental adaptation. It is speculated that these inserted genes may promote adaptive evolution of the strain during strain passaging, altering its pathogenic characteristics. This has important reference value for screening key targets in subsequent live vaccine development.

[0108] Table 4F750 contains two major inputs, covering specific results for 18 genes.

[0109]

[0110] Example 3. Evaluation of pathogenicity and virulence reversion of PmA4-750 strain in piglets.

[0111] 1. Materials

[0112] 1.1 The strains Pasteurella multocida PmA4 and Pasteurella multocida PmA4-750 were both provided by Wuhan Keqian Biotechnology Co., Ltd.

[0113] 1.2 Culture Media and Reagents

[0114] 1.2.1 TSA solid culture medium: Weigh 40g of tryptic soy agar (TSA), dissolve it in 950mL of double-distilled water, autoclave at 121℃ for 15min, cool to about 45℃, add 50mL of filtered sterilized bovine serum, mix thoroughly and pour into a petri dish for later use.

[0115] 1.2.2 TSB liquid culture medium: Weigh 30g of tryptic soy broth powder (TSB), dissolve it in 950mL of double-distilled water, autoclave at 121℃ for 15min and set aside. Add serum before use.

[0116] 1.3 Healthy, susceptible piglets aged 35–42 days were purchased from a pig farm in Wuhan.

[0117] 2 methods

[0118] 2.1 Test Plan

[0119] Fifteen piglets were divided into three groups of five. The cultured PmA4 strain F1 generation was serially diluted seven times (2-fold). Three appropriate dilutions (expected to induce 0-100% mortality) were used to further dilute the bacterial suspension. Each dilution was administered intraperitoneally to one group of piglets (2 mL / pig). Viable bacteria were counted to determine the actual bacterial load. Clinical symptoms and mortality were observed and recorded after challenge, and observation continued for 14 days. The F250, F450, F650, and F750 generations of the strain were cultured and inoculated into piglets using the same method.

[0120] 2.2 Preparation of bacterial suspension for challenge

[0121] Pasteurella multocida PmA4 strain F1, F450, and F750 generations were inoculated onto TSA plates containing 5% healthy newborn calf serum and incubated at 37°C for 24 hours. Single colonies were then selected and passaged on TSA plates and incubated at 37°C for 16–18 hours. Then, suitable typical colonies were selected and inoculated into TSB (containing 5% healthy newborn calf serum) liquid medium and incubated at 37°C for 10–12 hours. Finally, 1% of the inoculum was transferred to TSB (containing 5% healthy newborn calf serum) liquid medium and incubated at 37°C for 8 hours, followed by incubation at 2–8°C. Simultaneously, samples were taken for viable counts according to the appendix of the current Chinese Veterinary Pharmacopoeia, Part III.

[0122] 2.3 Rebound in toxicity

[0123] Using F750 generation basal seed culture purified and identified three times, 2-5 healthy, susceptible pigs aged 35-42 days were selected. Each pig was intraperitoneally injected with an appropriate dose of live bacteria. Tissue samples with the highest bacterial count were collected at appropriate times. The mixed tissue samples were appropriately processed and used as inoculum. Microbiological identification and bacterial count determination were performed using suitable methods. The processed inoculum was then inoculated into the same number of subcultures using the same route and dose as the first subculture. After each subculture, the inoculated animals were observed for appropriate time to check for clinical symptoms and pathological changes due to virulence reversion of the vaccine strain (especially comparing changes between the last subculture and the first subculture). Subcultures should be continuous for at least 5 generations, with at least 5 animals in the last subculture, and the observation period should be at least 21 days.

[0124] 3 Results

[0125] 3.1 Pathogenicity test evaluation

[0126] According to the experimental protocol, the F1, F450 and F750 generations were each diluted with three appropriate serial dilutions of bacterial solution. Each serial dilution was used to inoculate a group of piglets intraperitoneally (2 mL / head). Clinical symptoms and mortality were observed and recorded daily after challenge for 14 days. The specific experimental results are shown in Table 5.

[0127] Table 5. Toxicity (LD50) of different generations of the strain to piglets.

[0128]

[0129] As shown in Table 5, the challenge dose for the F1 generation strain was 2.0 × 10⁻⁶. 9 At CFU, 5 / 5 of the strains developed symptoms, and 3 / 5 of them died; the challenge dose for the F250 generation strain was 5.0 × 10⁻⁶. 9 During CFU administration, 4 / 5 of the strains developed symptoms, with 2 / 5 of them dying; the challenge dose for the F450 generation strain was 1.0 × 10⁻⁶. 10 During CFU administration, 3 / 5 of the strains developed symptoms, with 1 / 5 dying; the challenge dose for the F650 generation strain was 2.0 × 10⁻⁶. 10 At CFU, 3 / 5 of the strains developed symptoms, with 0 / 5 dying; the challenge dose for the F750 generation strain was 4.0 × 10⁻⁶. 10 During CFU administration, 3 / 5 of the vaccine candidates developed the disease, with 0 / 5 of them dying. The experimental results showed that the virulence of the vaccine candidate strain (PmA4-750 strain) after attenuation through high-temperature passage was significantly reduced, approximately 20 times less virulence than the parent wild-type strain (PmA4 strain).

[0130] 3.2 Evaluation of toxicity reversion test

[0131] The basal strain of porcine Pasteurella multocida PmA4-750, F1 generation, was used at 3.0 × 10⁻⁶. 10Five healthy piglets aged 28-35 days were inoculated intramuscularly with CFU / head dose (60 times the immunization dose). Four days after inoculation, lung tissue from each piglet was processed, mixed in equal volumes, and then inoculated into five more healthy piglets aged 28-35 days. This process was repeated for five generations in the pigs. The fifth generation was observed daily for 21 days to study the reversion of virulence of porcine Pasteurella multocida strain PmA4-750 in vivo. Results showed that during the observation period, the body temperature of the pigs in each generation did not exceed 40.5℃, and no clinical symptoms such as lethargy, coughing, dyspnea, emaciation, lameness, or rough coat were observed. Necropsy revealed no typical pathological changes characteristic of Pasteurella multocida. Viable bacterial counts in the lung suspensions from each generation were consistently not less than 5.0 × 10⁻⁶. 8 CFU / ml, PCR identification results showed that the isolated colonies were all *Pasteurella multocida* strain PmA4-750. Sequencing results showed that after the porcine *Pasteurella multocida* strain PmA4-750 was passaged to the 5th generation in vivo, the *Pasteurella multocida* isolated from lung tissue was identical to the F1 generation strain in genome sequencing, showing no variation. HE staining results showed no histological lesions in the lungs of pigs at any generation. These results indicate that *Pasteurella multocida* strain PmA4-750 can continuously reproduce in pigs for 5 generations, is genetically stable, does not exhibit virulence reversion, and has high safety.

[0132] Example 4: Immunogenicity test of PmA4-750 strain

[0133] 1. Materials

[0134] 1.1 Vaccine: Live vaccine against porcine Pasteurella multocida (PmA4-750 strain), provided by Wuhan Keqian Biotechnology Co., Ltd.

[0135] 1.2 Culture Media and Reagents

[0136] 1.2.1 TSA solid culture medium

[0137] Weigh 40g of tryptic soy agar (TSA), dissolve it in 950mL of double-distilled water, autoclave at 121℃ for 15min, cool to about 45℃, add 50mL of filtered sterilized bovine serum, mix thoroughly and pour into a petri dish for later use.

[0138] 1.2.2 TSB liquid culture medium

[0139] Weigh 30g of Tryptic Soy Broth (TSB), dissolve it in 950mL of double-distilled water, autoclave at 121℃ for 15min, and set aside. Add serum before use.

[0140] 1.3 Healthy, susceptible piglets aged 35–42 days were purchased from a pig farm in Wuhan.

[0141] 2 methods

[0142] 2.1 Test Plan

[0143] Ten healthy, susceptible piglets aged 35–42 days were used. Five of these piglets were administered 2.0 ml (5.0 × 10 ml) of a solution diluted with 20% aluminum hydroxide saline and injected intramuscularly into the neck muscles, according to the dosage indicated on the bottle label. 8 A live vaccine against Pasteurella multocida infection in pigs (CFU) was administered. Five pigs were not vaccinated and served as a challenge control group. Twenty-one days after vaccination, each of the five control pigs was intraperitoneally injected with 2.0 ml of 1 MLD (20 CFU-30 CFU) of highly virulent Pasteurella multocida A4 strain. The pigs were continuously observed for 14 days after challenge, and clinical symptoms and mortality were recorded.

[0144] 2.2 Preparation of bacterial suspension for challenge

[0145] Pasteurella multocida strain PmA4 was inoculated onto TSA plates containing 5% healthy newborn calf serum and incubated at 37°C for 24 hours. Single colonies were then selected and passaged on TSA plates and incubated at 37°C for 16–18 hours. Then, suitable typical colonies were selected and inoculated into TSB (containing 5% healthy newborn calf serum) liquid medium and incubated at 37°C for 10–12 hours. Finally, 1% of the inoculum was transferred to TSB (containing 5% healthy newborn calf serum) liquid medium and incubated at 37°C for 8 hours. After incubation at 2–8°C, samples were taken for viable counts according to the appendix of the current Chinese Veterinary Pharmacopoeia, Part III.

[0146] 3 Results

[0147] According to the experimental protocol, 21 days after vaccination, each of the pigs, along with 5 control pigs, was intraperitoneally injected with 1 MLD (2.5 × 10⁻⁶). 9 2.0 ml of highly virulent Pasteurella multocida strain A4 strain (CFU) was used to challenge the bacteria. Clinical symptoms and mortality were observed and recorded after the challenge. The specific results are shown in Table 6.

[0148] Table 6. Immunogenicity test results of PmA4-750 strain

[0149]

[0150] As shown in Table 6, 21 days after inoculation, when challenged with a virulent strain of Pasteurella multocida A4, 5 / 5 of the immunized group were protected, while 5 / 5 of the control group developed the disease, with 4 / 5 dying. The experimental results indicate that strain PmA4-750 exhibits good immunogenicity.

[0151] Example 5: Cross-protection test of porcine Pasteurella multocida live vaccine (PmA4-750 strain)

[0152] 1. Materials

[0153] 1.1 Vaccine: Porcine Pasteurella multocida live vaccine (PmA4-750 strain), provided by Wuhan Keqian Biotechnology Co., Ltd.

[0154] 1.2 Culture Media and Reagents

[0155] 1.2.1 TSA solid culture medium

[0156] Weigh 40g of tryptic soy agar (TSA), dissolve it in 950mL of double-distilled water, autoclave at 121℃ for 15min, cool to about 45℃, add 50mL of filtered sterilized bovine serum, mix thoroughly and pour into a petri dish for later use.

[0157] 1.2.2 TSB liquid culture medium

[0158] Weigh 30g of Tryptic Soy Broth (TSB), dissolve it in 950mL of double-distilled water, autoclave at 121℃ for 15min, and set aside. Add serum before use.

[0159] 1.3 Healthy, susceptible piglets aged 35–42 days were purchased from a pig farm in Wuhan.

[0160] 2 methods

[0161] 2.1 Test Plan

[0162] Twenty healthy, susceptible piglets aged 35–42 days were divided into four groups of five piglets each. Two groups were labeled with the number of piglets per vial. The vials were diluted with 20% aluminum hydroxide saline solution, and each piglet was inoculated intramuscularly with 2.0 ml (5.0 × 10 ml) of the solution in the neck muscles. 8 CFU (Porcine Pasteurella multocida) live vaccine (PmA4-750 strain). Two other groups were not vaccinated and served as challenge control groups. Twenty-one days post-vaccination, five pigs from group 1, along with five control pigs, were each injected intraperitoneally with 1 MLD (1.0 × 10⁻⁶). 10 2.0 ml of highly virulent strain of Pasteurella multocida type D (CFU) from pigs was administered; 5 pigs from group 2, along with the control group, were each injected intraperitoneally with 1 ml of (1.0 × 10⁻⁶) of the strain. 9 2.0 ml of highly virulent Pasteurella multocida type B strain (CFU) from pigs was used to observe and record clinical symptoms and mortality after challenge, and the observation continued for 14 days.

[0163] 2.2 Preparation of bacterial suspension for challenge

[0164] Porcine Pasteurella multocida strains D and B were inoculated onto TSA plates containing 5% healthy newborn calf serum and incubated at 37°C for 24 hours. Single colonies were then selected and passaged on TSA plates and incubated at 37°C for 16–18 hours. Then, suitable typical colonies were selected and inoculated into TSB (containing 5% healthy newborn calf serum) liquid medium and incubated at 37°C for 10–12 hours. Finally, 1% of the inoculum was transferred to TSB (containing 5% healthy newborn calf serum) liquid medium and incubated at 37°C for 8 hours. After incubation at 2–8°C, samples were taken for viable counts according to the appendix of the current Chinese Veterinary Pharmacopoeia, Part III.

[0165] 3 Results

[0166] According to the experimental protocol, 21 days after vaccination, five pigs from group 1, along with five control pigs, were selected and each pig was intraperitoneally injected with 1 MLD (1.0 × 10⁻⁶). 10 2.0 ml of highly virulent strain of Pasteurella multocida type D (CFU) from pigs was administered; 5 pigs from group 2, along with the control group, were each injected intraperitoneally with 1 ml of (1.0 × 10⁻⁶) of the strain. 9 2.0 ml of highly virulent strain of Pasteurella multocida type B (CFU) from pigs was used to observe and record clinical symptoms and mortality after challenge. The specific results are shown in Tables 7 and 8.

[0167] Table 7. Results of Cross-Protection Test and Type D Challenge Test of Porcine Pasteurella M. m. Live Vaccine (PmA4-750 strain)

[0168]

[0169] Table 8. Results of cross-protection test and type B challenge test of porcine Pasteurella multocida live vaccine (PmA4-750 strain).

[0170]

[0171] As shown in Tables 7 and 8, 21 days post-vaccination, when challenged with virulent porcine Pasteurella multocida strain D (PmD6) and virulent porcine Pasteurella multocida strain B (CVCC44401), respectively, 4 / 5 of the D-type control group developed the disease, with 2 / 5 dying; in the B-type control group, 5 / 5 developed the disease, with 4 / 5 dying; and 4 / 5 of the immunized groups showed protection. The cross-protection test results indicate that this live Pasteurella multocida vaccine provides good cross-protection.

[0172] Although the above embodiments have described the present invention and its implementation in detail, it should be noted that for those skilled in the art, any changes, modifications, substitutions, combinations, simplifications, etc., made to the corresponding conditions without departing from the technical principles of the present invention should be considered as equivalent substitutions, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A porcine Pasteurella multocida strain subjected to high-temperature passage and attenuated, characterized in that, The strain was classified and named Pasteurella multocida PmA4-750, and is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20251644.

2. A live vaccine against porcine Pasteurella multocida infection, characterized in that, The product includes an effective dose of the PmA4-750 strain of claim 1 and a pharmaceutically acceptable lyophilization protectant.

3. A method for preparing a live vaccine against porcine Pasteurella multocida, characterized in that, The PmA4-750 strain described in claim 1 was freeze-dried and preserved under conditions containing 8-12% skim milk and 5-8% sucrose as a freeze-drying protectant.

4. The application of the strain described in claim 1 in the preparation of porcine Pasteurella multocida vaccine, characterized in that, The antibodies produced by this strain in animals have cross-protective activity against serotypes A, D, or B of Pasteurella multocida, and can provide immune protection for all three porcine serotypes A, B, and D simultaneously.

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