Bacillus mucilaginosus for inhibiting systemic infection, pneumonia and neurologic type of chicken salmonella pullorum and application thereof

Abstract

The application discloses a bacillus mucilaginosus for inhibiting systemic infection type, pneumonia type and neurotype chicken pullorum salmonella and application thereof. The bacillus mucilaginosus for inhibiting systemic infection type, pneumonia type and neurotype chicken pullorum salmonella is named Anti-S.gallinarum, is preserved in the China Center for Type Culture Collection, and has a preservation number of CCTCC NO: M20251300.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products, specifically to a strain of mucilaginous spores that inhibits systemic, pneumonia, and neurogenic Salmonella pullorum and its applications. Background Technology

[0002] In recent years, with the rapid development of my country's chicken farming industry, the incidence of pullorum disease has been on the rise, making prevention and control a serious challenge. Salmonella pullorum is highly adaptable to a specific host (chicken), exhibiting high pathogenicity and infectivity. Infection can lead to acute systemic disease outbreaks, with mortality rates reaching 40%-80% in chicks under 10 days old. Adult chickens typically become carriers of the pathogen. This disease not only causes huge economic losses but also poses a serious threat to the healthy development of the poultry industry and public health safety.

[0003] When chicks are infected with Salmonella pullorum, they mainly exhibit three types of symptoms: systemic infection, pneumonia, and neurological. Among them, the systemic infection type often presents as acute septicemia, and the chicks often die rapidly before external symptoms can appear. The pneumonia type is mainly characterized by open-mouth breathing, difficulty breathing, and eventually death from heart failure. The neurological type is mainly characterized by motor dysfunction, such as head tilting, head tilting back, beak touching the ground, and circling, which seriously affects the economic benefits of farmers.

[0004] Current methods for preventing and controlling Salmonella pullorum in chickens have the following main shortcomings: 1. Limitations of chemical drug prevention and treatment Increased risk of chemical drug residues: Long-term ingestion of antibiotic residues from animal products through the food chain leads to their accumulation in the human body, posing potential or direct harm to human health. Increasingly strong antibiotic resistance in pathogens: Salmonella pullorum exhibits strong adaptability and mutation capabilities, developing resistance to commonly used antibiotics. Over time, the therapeutic effect of antibiotics against Salmonella pullorum declines, resulting in the use of higher doses of antibiotics and the accumulation of higher doses of antibiotic residues, creating a vicious cycle. With the increasing prominence of antibiotic resistance, chemical drug control measures face new challenges. Inability to eliminate respiratory colonizing bacteria: While chemical drugs can inhibit the proliferation of Salmonella, they cannot eliminate its adhesion and colonization on respiratory epithelial cells.

[0005] 2. Immunological defects of traditional vaccines Live attenuated vaccines: Live attenuated vaccines may mutate and regain virulence; inactivated vaccines may cause infection if inactivated incompletely. Live vaccines have stringent storage and transportation requirements (requiring storage at -20℃), limiting their application in grassroots farms. Inactivated vaccines: Rely on humoral immunity to induce IgG antibodies, but cannot activate respiratory mucosal immunity (insufficient sIgA secretion), resulting in limited clearance of colonized Salmonella. Subunit vaccines: Currently, the epidemiological characteristics of pullorum disease in chickens are complex and variable. Salmonella pathogens have strong adaptability and mutation capabilities. Salmonella has O antigen, H antigen, and Vi antigen, exhibiting significant antigenic variation. There are currently more than 280 serotypes in my country, and new serotypes are constantly emerging, resulting in generally poor cross-reaction protection. Gene-deleted live vaccines: Although they can reduce pathogen colonization through competitive exclusion, there is a risk of reversion to virulence.

[0006] 3. Current Status and Challenges of Biological Control Technology In response to the shortcomings of chemical drugs and vaccines, probiotics have become a research hotspot due to their advantages such as no drug resistance and no residues. However, current research faces significant technical bottlenecks: Single antibacterial target function: Most reported probiotics (such as lactic acid bacteria and Bacillus) focus on the regulation of intestinal health ecology and only conduct research on Salmonella pullorum in chickens with a certain symptom type. In addition, there are many types of Salmonella serotypes, resulting in an overall unsatisfactory prevention and control effect.

[0007] The mechanism of action is unclear: Existing studies have focused on the inhibition zone phenotype of the strain, but there is insufficient research on deeper mechanisms such as host immune regulation (how it interacts with the host immune system at the molecular level) and biofilm disruption (such as inhibiting the expression of QS-related genes).

[0008] Barriers to industrial application: Most strains have poor heat resistance (requiring coating treatment) and weak bile salt resistance (unable to pass through the acidic environment of the stomach), resulting in a survival rate of less than 30% in actual production.

[0009] In summary, the epidemiological characteristics of pullorum disease in chickens are complex and variable, with new serotypes constantly emerging. In addition, traditional prevention and control methods have certain shortcomings. Therefore, there is an urgent need to strengthen the research and development of safe, effective, multi-target synergistic antibacterial agents with strong stress resistance. Summary of the Invention

[0010] This invention aims to overcome the shortcomings of existing technologies and provide a strain of *Salmonella mucilaginosa* that inhibits systemic, pneumonia, and neurogenic *Salmonella pullorum*, as well as its applications. Mucilaginous Bacillus Anti-S. gallinarum has significant inhibitory activity against systemic, pneumonia, and neurogenic Salmonella pullorum, and its ability to improve production performance, enhance immunity, and reduce intestinal pathogen load has been confirmed through broiler challenge experiments.

[0011] To achieve the above objectives, the technical solution provided by this invention is as follows: The mucilaginous spore-forming bacterium that inhibits systemic, pneumonia, and neurogenic Salmonella pullorum was named *Anti-S. gallinarum* and deposited at the China Center for Type Culture Collection on June 9, 2025, with accession number CCTCC NO: M20251300. This mucilaginous spore-forming bacterium inhibiting systemic, pneumonia, and neurogenic Salmonella pullorum was isolated from the intestinal digestive tract of healthy chickens.

[0012] The systemic, pneumonia, and neurogenic Salmonella strains were purchased strains, namely SAL02657, SAL03395, and SAL03405 Salmonella strains, respectively. They are all deposited at the National Center for Biotechnology Information in the United States, with accession numbers SAMN29205455, SAMN29205583, and SAMN29205592, respectively.

[0013] The *Bacillus mucilaginosus* strain that inhibits systemic, pneumonia, and neurogenic *Salmonella pullorum* can be used to prepare feed additives. The amount of *Bacillus mucilaginosus* strain that inhibits systemic, pneumonia, and neurogenic *Salmonella pullorum* added to feed as a feed additive is ≥8.0 × 10⁻⁶. 9 CFU / kg.

[0014] The described *Salmonella mucilaginosa* strain, which inhibits systemic, pneumococcal, and neuropathic forms of Salmonella pullorum, can be used to prevent and treat systemic (SAL02657), pneumococcal (SAL03395), and neuropathic (SAL03405) forms of Salmonella pullorum disease in chickens. It can also be used to prepare vaccines for the prevention and treatment of these three pathogenic symptom types (systemic, pneumococcal, and neuropathic) of Salmonella pullorum disease in chickens.

[0015] The present invention will be further described below: The *Anti-S. gallinarum* strain of this invention, which inhibits systemic, pneumonia, and neurogenic *Salmonella pullorum*, exhibits a multi-target synergistic antibacterial effect: ① Competitive inhibition: The formed biofilm can seize the attachment sites of the respiratory mucosa, blocking the adhesion and colonization of *Salmonella pullorum* and preventing intestinal diseases; ② Chemical defense: It produces organic acids, antimicrobial peptides, and other metabolites that disrupt the integrity of pathogen cell membranes, leading to ion leakage and leakage of contents; ③ Metabolic interference: Through specific immunity, it enhances the secretion of IFN-γ and other substances in the body, interfering with the growth and metabolism of *Salmonella*. It can activate the immune regulatory network, acting as a non-specific immunomodulatory factor to enhance the phagocytic capacity of phagocytes and the antibody production capacity of humoral cells: ① Enhanced innate immunity: Activation of the TLR2 / 4-NF-κB pathway, inducing macrophages to secrete pro-inflammatory factors such as IL-2; ② Adaptive immune regulation: Promoting Th1 immune responses (IFN-γ↑), inhibiting Th2 responses, and balancing the body's immune microenvironment; ③ Enhanced mucosal immunity: Stimulating the secretion of sIgA by Pear-like lymph nodes in the intestine, blocking the transmucosal transmission of pathogens along the respiratory-MALT axis. It can also optimize environmental adaptability: The spore structure can withstand the high temperatures of feed pelleting (survival rate >90% at 80℃ for 30 minutes), and improves tolerance to gastric acid and bile salts.

[0016] Experiments have shown that the *Anti-S. gallinarum* strain described in this invention, which inhibits systemic, pneumonia, and neurogenic Salmonella pullorum, exhibits superior resistance to viral infection both in vitro and in vivo. In vitro, the inhibition zone diameters of the fermentation broth supernatant against systemic, pneumonia, and neurogenic Salmonella pullorum strains were 1.30 cm, 1.22 cm, and 1.15 cm, respectively. In vivo, on days 7 and 21 post-challenge, compared with the control groups challenged by SAL02657, SAL03395, and SAL03405, the Salmonella content in the cecal digesta of broilers challenged by *Bacillus pilaris* Anti-S. gallinarum + SAL02657, *Bacillus pilaris* Anti-S. gallinarum + SAL02657, and *Bacillus pilaris* Anti-S. gallinarum + SAL02657 were all significantly reduced. The pathogenic symptoms of systemic, pneumonia, and neurogenic Salmonella pullorum were all significantly improved, and the mortality rate was significantly reduced. The *Anti-S. gallinarum* strain described in this invention, which inhibits systemic, pneumonia-type, and neurogenic *Salmonella pullorum*, significantly improves broiler growth performance, increasing daily weight gain, improving feed conversion ratio, reducing diarrhea rate, and lowering mortality. In terms of immune protection, serum IgG and IgA levels are increased, IFN-γ concentration is elevated, and IL-2 levels are upregulated. Regarding safety and cost-effectiveness, it poses no risk of drug residues and offers outstanding cost-effectiveness.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Its antibacterial efficacy is significantly superior to chemical drugs and traditional probiotics, and it has a multi-target synergistic antibacterial function against multiple targets: The inhibition zone diameters of *Anti-S. gallinarum* against *Salmonella pullorum* strains SAL02657 (systemic infection type), SAL03395 (pneumonia type), and SAL03405 (neural type) are 1.30 cm, 1.22 cm, and 1.15 cm, respectively, far exceeding existing bacillus preparations (inhibition zone <1 cm); it has an inhibitory effect on the biofilm formation stages (early adhesion, mid-term matrix synthesis, and late maturation) of *Salmonella pullorum* strains SAL02657, SAL03395, and SAL03405, while traditional antibiotics only act on the proliferation stage. Anti-S. gallinarum exhibits strong inhibitory effects against Salmonella pullorum SAL02657, SAL03395, and SAL03405, making it suitable for controlling mixed infections of these three strains. Furthermore, Anti-S. gallinarum demonstrates multi-target synergistic effects (competitive inhibition + biofilm disruption + metabolic interference), avoiding the development of drug resistance from single targets.

[0018] 2. Overcoming the limitations of traditional vaccines in immunomodulation: Significantly increases respiratory mucosal IgA levels, while traditional vaccines (such as whole-cell inactivated vaccines) only induce serum IgG antibodies; inhibits Th2 inflammatory factors and promotes Th1 immune responses, which is superior to the unidirectional immune activation of subunit vaccines.

[0019] 3. It has good tolerability, safety, economy and sustainability: The spore structure makes the survival rate of the preparation >90% after granulation at 80℃, and the bile salt tolerance is improved to 0.3% concentration, which solves the bottleneck of traditional probiotic industrial application; Anti-S. gallinarum has no residue risk and outstanding cost-effectiveness (the cost of prevention and control per chicken throughout the process is <0.2 yuan, which is 33%-40% lower than the antibiotic program (¥0.5-0.6 / chicken)). At the same time, the metabolites of Bacillus (such as organic acids) can improve the ammonia nitrogen emission of manure, which is in line with the trend of green farming. Detailed Implementation

[0020] 1. Isolation and identification of antibacterial strains highly resistant to Salmonella pullorum SAL02657 (systemic infection type), SAL03395 (pneumonia type), and SAL03405 (neurological type):

[0021] Bacillus spores were isolated from the intestinal chyme of healthy chickens using LB medium. Antibacterial experiments were conducted on *Salmonella pullorum* strains SAL02657, SAL03395, and SAL03405. The specific screening method was as follows: 300 μL of activated *Salmonella pullorum* strains (purchased from Nanjing Kangyou Biotechnology Co., Ltd., all deposited at the National Center for Biotechnology Information, USA, accession numbers: SAMN29205455, SAMN29205583, and SAMN29205592, respectively) were evenly spread on LB agar plates. Four Oxford cups were then placed on the plates, arranged in a square pattern for later use. *Bacillus spores* (…) Mucilaginous Bacillus After centrifuging the fermentation broth of *Anti-S. gallinarum* at 12000 r / m for 1-3 min, 250 mL of the supernatant was pipetted into Oxford cups on LB agar plates. Sterile water was used as a control. The plates were incubated at 37℃ for 24 h. The size and clarity of the inhibition zones were observed, and the size of the inhibition zones was measured with calipers to determine the antibacterial activity of the tested bacteria against *Salmonella pullorum* SAL02657, SAL03395, and SAL03405. Combined with bacterial molecular biological identification (16S rDNA sequencing), a *Geloidobacterium* strain exhibiting strong inhibitory activity against *Salmonella pullorum* SAL02657, SAL03395, and SAL03405 was selected and named *Anti-S. gallinarum*. It was deposited at the China Center for Type Culture Collection (CCTCC) on June 9, 2025, with accession number CCTCC NO: M20251300.

[0022] 2. Evaluation of the best results obtained from the target bacteria:

[0023] The antibacterial replication of a strain of *Anti-S. gallinarum* that inhibited *Salmonella pullorum* strains SAL02657, SAL03395, and SAL03405 (selected from screening) was verified, and a challenge experiment was conducted in broilers. The results are as follows: 2.1. Bacillus mucilaginosus ( Mucilaginous Bacillus Antibacterial activity verification of S. gallinarum: Take 8 LB solid nutrient agar plates, and spread 300 μL of activated Salmonella pullorum suspensions (SAL02657, SAL03395, and SAL03405) evenly on each plate. Spread two plates for each pathogenic bacterium. Then place four Oxford cups on each plate, arranged in a square pattern for later use. (Bacillus mucilaginosus) Mucilaginous BacillusAfter centrifuging the fermentation broth of *Anti-S. gallinarum* at 12000 r / m for 1-3 min, 250 mL of the supernatant was pipetted into Oxford cups on LB agar plates containing *Salmonella pullorum* bacterial suspensions of SAL02657, SAL03395, and SAL03405. Sterile water was used as a control. The plates were incubated at 37℃ for 24 h. The results showed that *Bacillus gallinarum* (…) Mucilaginous Bacillus The inhibition zone diameters of Anti-S. gallinarum against Salmonella pullorum strains SAL02657, SAL03395, and SAL03405 were 1.30 cm, 1.22 cm, and 1.15 cm, respectively, while no inhibition zone was observed in the sterile water control.

[0024] 2.2, Bacillus mucilaginosus ( Mucilaginous Bacillus Anti-S. gallinarum challenge experiment in broilers: The experiment was a single-factor experiment. 336 22-day-old Huainan yellow broiler chickens were randomly divided into 7 treatment groups, with 6 replicates in each treatment group and 8 chickens (half male and half female) in each replicate. There was no significant difference in initial body weight among the replicate groups. P >0.05). Treatment group 1 was the blank control group: fed a basal diet without any treatment; Treatment group 2 was the SAL02657 positive challenge control group: fed a basal diet and challenged with systemic Salmonella pullorum strain SAL02657. Treatment group 3 was the experimental group: the basal diet was supplemented with Bacillus mucilaginosus Anti-S. gallinarum fermentation broth at a dosage of 10. 10 Treatment group 4, a SAL03395-positive control group, was fed a basal diet and challenged with Salmonella pullorum strain SAL02657. Treatment group 5 was the experimental group, fed a basal diet supplemented with Bacillus mucilaginosus Anti-S. gallinarum fermentation broth at a dosage of 10 CFU / kg feed. Treatment group 5 was the experimental group, fed a basal diet supplemented with Bacillus mucilaginosus Anti-S. gallinarum fermentation broth at a dosage of 10 CFU / kg feed. 10 CFU / kg feed was used to challenge *Salmonella pullorum* strain SAL03395 (pneumoniae type). Treatment group 6 served as the SAL03405 positive challenge control group: fed a basal diet and challenged with *Salmonella pullorum* strain SAL03405 (neurogenic type). Treatment group 7 was the experimental group: supplemented with *Bacillus mucilaginosus* Anti-... S.gallinarum Fermentation broth, dosage added: 10 10 Chickens were challenged with Salmonella pullorum strain SAL03405 (CFU / kg feed) for 21 days. They had free access to feed and water.

[0025] A. Bacillus mucilaginosus Anti- S.gallinarum Effects on broiler growth performance The results of this experiment (see Table 1) show that, compared with the SAL02657 challenge control group, the average daily weight gain of the Bacillus mucilaginosus + SAL02657 challenge group was significantly increased. P <0.05%, feed conversion ratio, diarrhea rate, and mortality rate were all significantly reduced ( P <0.05), there were no significant differences in mean final weight and feed intake; compared with the blank control group, there were no significant differences in mean final weight, mean daily weight gain, feed intake, feed conversion ratio, diarrhea rate, and mortality in the *Bacillus lentigines* Anti-S. gallinarum + SAL02657 challenge group. Compared with the SAL03395 challenge control group, the mean final weight, daily weight gain, and feed intake in the *Bacillus lentigines* Anti-S. gallinarum + SAL03395 challenge group were significantly increased (…). P< 0.05), feed conversion ratio, diarrhea rate and mortality were significantly reduced ( P< 0.05); Compared with the blank control group, there were no significant differences in mean final weight, mean daily weight gain, feed intake, feed conversion ratio, diarrhea rate, and mortality in the Bacillus mucilaginosus + SAL03395 challenge group. Compared with the SAL03405 challenge control group, the mean daily weight gain and feed intake in the Bacillus mucilaginosus + SAL03405 challenge group were significantly increased ( P< 0.05), feed conversion ratio, diarrhea rate and mortality were significantly reduced ( P< (0.05); Compared with the blank control group, there were no significant differences in mean final weight, mean daily weight gain, feed intake, feed conversion ratio, diarrhea rate, and mortality rate in the Bacillus mucilaginosus + SAL03405 challenge groups. Therefore, each experimental group showed significant disease prevention effects compared with the corresponding challenge control group, and improved broiler production performance and economic benefits.

[0026] Table 1. Bacillus mucilaginosus Anti- S.gallinarum Effects on broiler growth performance

[0027] Note: Data in the same row whose shoulder labels do not contain the same letter indicates significant differences. P <0.05).

[0028] B. Effects of *Anti-S. gallinarum* on serum antibodies and cytokines in broilers. During the experiment, on day 7 of challenge, blood was collected from the anterior vena cava of broilers, serum was separated, and the levels of serum antibodies and cytokines were detected. The results (see Table 2) showed that, compared with the challenge control group, the *Bacillus mucilaginosus* + SAL02657 challenge group significantly increased the levels of serum antibodies IgG and IgA in broilers. P <0.05), and the levels of IFN-γ and IL-2 in serum; compared with the challenge control group, the Bacillus mucilaginosus + SAL03395 challenge test group significantly increased the levels of serum antibodies IgG and IgA in broilers ( P <0.05), and the levels of IFN-γ and IL-2 in serum; compared with the challenge control group, the Bacillus mucilaginosus + SAL03405 challenge test group significantly increased the levels of serum antibodies IgG and IgA in broilers ( P <0.05), and the levels of IFN-γ and IL-2 in serum. Therefore, *Anti-S. gallinarum* enhances animal immune performance by increasing humoral and cellular immunity, thereby reducing the negative effects of *Salmonella pullorum* and maintaining the health of broilers.

[0029] Table 2. Effects of *Anti-S. gallinarum* on serum antibodies and cytokines in broilers.

[0030] Note: Data in the same row whose shoulder labels do not contain the same letter indicates significant differences. P <0.05).

[0031] C. Bacillus mucilaginosus Anti- S.gallinaru Effect of m on total Salmonella content in the cecum of broilers During the experiment, on days 7 and 21 of challenge, cecal digesta samples from broilers were collected in 10 mL centrifuge tubes, flash-frozen in liquid nitrogen, and transferred to the laboratory for testing. Total Salmonella content was determined using the dilution plate count method. 1 g of fecal sample was accurately weighed and placed in an Erlenmeyer flask containing 99 mL of sterile water. The sample was shaken to mix, then serially diluted 10-fold using a vortex mixer. 0.1 mL of the diluted solution was spread onto XLD medium, with an equal volume of sterile water used as a blank control. This process was repeated three times. The number of colonies was observed and recorded, and the colony count was calculated based on the dilution. The number of microorganisms in the feces was expressed as 1 g CFU / g. Salmonella colonies were cultured aerobically at 37°C for 36 h on XLD medium. Colonies were pink or yellow, with or without a black center.

[0032] The experimental results (see Table 3) showed that, compared with the challenge control group, the *Bacillus mucilaginosus* + SAL02657 challenge group significantly reduced the total *Salmonella* content in the cecum of broilers on days 7 and 21. P <0.05; Compared with the blank control group, the Bacillus mucilaginosus + SAL02657 challenge group significantly reduced the total Salmonella content in the cecum of broilers on day 21 ( P <0.05%. Compared with the challenge control group, the Bacillus mucilaginosus + SAL03395 challenge group significantly reduced the total Salmonella content in the cecum of broilers on days 7 and 21. P <0.05%. Compared with the challenge control group, the *Bacillus mucilaginosus* + SAL03405 challenge group significantly reduced the total *Salmonella* content in the cecum of broilers on days 7 and 21. P <0.05).

[0033] Therefore, Bacillus mucilaginosus can significantly reduce the number of Salmonella cecum in the SAL02657, SAL03395 and SAL03405 challenge control groups, and the number of Salmonella cecum in the control group is reduced by 11.37%, 8.48% and 6.50% respectively compared with the blank control group, which has a promoting effect on maintaining the intestinal health of livestock and poultry and improving the ecological environment.

[0034] Table 3. Effects of *Anti-S. gallinarum* on Salmonella content in the cecal bursa of broilers.

[0035] Note: Data in the same row whose shoulder labels do not contain the same letter indicates significant differences. P <0.05).

Claims

1. A mucilaginous spore strain that inhibits systemic, pneumonia, and neurogenic Salmonella pullorum, named Anti-S. gallinarum, is deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: M20251300.

2. The *Bacillus subtilis* strain that inhibits systemic, pneumonia-type, and neurogenic *Salmonella pullorum* as described in claim 1, characterized in that... The mucoid spores that inhibit systemic, pneumonia, and neurogenic Salmonella pullorum were isolated from the intestinal chyme of healthy chickens.

3. The application of the Bacillus mucilaginosus, as described in claim 1, which inhibits systemic, pneumonia, and neurogenic Salmonella pullorum in chickens, in the preparation of feed additives.

4. The application as described in claim 3, characterized in that, The *Bacillus mucilaginosus* strain that inhibits systemic, pneumonia, and neurogenic *Salmonella pullorum* is added to the feed at a dosage of 8.0 × 10⁻⁶. 9 CFU / kg.

5. The application of the mucoid Bacillus that inhibits systemic, pneumonia and neurogenic Salmonella pullorum as described in claim 1 in the prevention and treatment of mixed infections of systemic, pneumonia and neurogenic Salmonella pullorum.

6. The use of the mucoid Bacillus that inhibits systemic, pneumococcal, and neurogenic Salmonella pullorum as described in claim 1 in the preparation of vaccines against systemic, pneumococcal, and neurogenic Salmonella pullorum disease.

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