Establishment method and application of pigeon salmonella acute infection model
An acute infection model of pigeon Salmonella was established by using pigeon-derived Salmonella typhimurium strain ST-JN-63 to infect young pigeons through multiple injections under the neck. This solved the problems of low model success rate and short disease course in existing technologies and enabled effective evaluation of drug efficacy.
Patent Information
- Application Number
- CN202511282474.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-09
- Publication Date
- 2025-12-16
AI Technical Summary
The lack of an effective acute infection model of Salmonella in pigeons in the current technology makes it difficult to evaluate the efficacy of drugs. Moreover, the existing methods have a short course of disease and a low success rate, which cannot meet the needs of the pigeon farming industry.
Pigeons were infected with the pigeon-derived Salmonella typhimurium strain ST-JN-63 via multiple injections under the neck. An acute infection model of pigeon Salmonella was established by combining specific disease diagnosis criteria.
It improves the success rate and uniformity of the model, prolongs the course of the disease, and can stably assist in the research and development of drugs to prevent and treat pigeon Salmonella infection, thus enabling effective evaluation of drug efficacy.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a method for establishing and applying an acute infection model of Salmonella pigeonis. Background Technology
[0002] In recent years, the pigeon farming industry has received increasing attention across various regions, with "small pigeons driving a large industry" becoming a significant force in rural revitalization. As the scale of meat pigeon farming expands, safe and efficient breeding has become a focus of industry attention. To meet the ever-increasing demand for meat pigeons, pigeon farms are expanding, but the environmental hygiene and feeding management levels of some farms need improvement. Among these issues, Salmonella infection and mortality rates in pigeons are rising. To effectively study Salmonella infection in pigeons, it is necessary to establish relevant animal models. Epidemiological evidence shows that Salmonella serotypes isolated from pigeons differ from those isolated from other hosts, indicating that the prevalent Salmonella serotypes in pigeon flocks exhibit high host adaptability.
[0003] Currently, there are few successful cases of acute Salmonella infection models in pigeons. Most literature uses mammalian endotoxin (LPS) challenge methods in mice or rats, but this method suffers from problems such as short disease course and difficulty in evaluating drug efficacy. Furthermore, the significant differences between rodents and poultry make it unsuitable for poultry drug research. Some researchers have also used methods such as eye drops, nasal drops, or oral administration to infect pigeons, but due to the selection of the infecting strain or the acidic environment of the gastric juice, no obvious symptoms appeared, making these methods unsuitable as effective Salmonella infection models in pigeons.
[0004] Given the above background, and the lack of reports on the successful construction of an acute Salmonella infection model in pigeons, it is urgent to construct a relevant model to assist in the research and development of new traditional Chinese veterinary medicine preparations for the prevention and treatment of this type of disease and to objectively evaluate their efficacy. Summary of the Invention
[0005] The purpose of this invention is to provide a method and application for establishing an acute infection model of Salmonella pigeonis, which solves the technical problems of low success rate and poor replication rate in existing modeling methods. The acute infection model of Salmonella pigeonis established by this method improves the success rate and uniformity of modeling, has a long disease course, and is stable. It can assist in the research and development of drugs to prevent and treat Salmonella pigeonis infection and can achieve effective and objective evaluation of drug efficacy.
[0006] This invention provides one of the following technical solutions:
[0007] A method for establishing an acute Salmonella infection model in pigeons includes the step of infecting young pigeons with pigeon-derived Salmonella Typhimurium obtained through isolation and screening by multi-point injection under the neck, thereby preparing an acute Salmonella infection model in pigeons; wherein the pigeon-derived Salmonella Typhimurium is the screened challenge strain ST-JN-63, which was deposited at the China General Microbiological Culture Collection Center on August 25, 2025, with the accession number: CGMCC No. 35198.
[0008] Furthermore, the 16S rRNA sequence of the pigeon-derived Salmonella typhimurium ST-JN-63 is shown in SEQ No. 1.
[0009] Furthermore, the serotype of the pigeon-derived Salmonella typhimurium ST-JN-63 is O:4, H:i.
[0010] Furthermore, the above-mentioned establishment method includes the following operational steps:
[0011] (1) Selected pigeons that showed no Salmonella infection after antigen and antibody testing were raised in a routine environment. The skin on the neck of the pigeons was disinfected, and multiple subcutaneous inoculations were performed with the pigeon-derived Salmonella Typhimurium solution. The inoculation dose was 1 mL × 10⁻⁶. 8 cfu / mL - 1.5mL × 10 8 cfu / mL;
[0012] (2) Observe the mental state, detoxification and mortality of the young pigeons daily, collect cotton swabs to detect Salmonella shedding, and perform necropsy and organ PCR detection on the dead young pigeons; according to the disease judgment criteria, the pigeon Salmonella acute infection model of the tested young pigeons after challenge was successfully established.
[0013] Furthermore, in step (1), the young pigeons are 30-33 days old.
[0014] Further, the preparation of the pigeon-derived Salmonella typhimurium bacterial suspension in step (1) is as follows: the pigeon-derived Salmonella typhimurium stored at -80℃ is streaked onto SS solid medium and cultured overnight at 37℃; after picking a single colony and checking that it is a pure culture by smear, a single colony is picked and inoculated into fresh LB liquid medium and cultured for 22 hours at 37℃ and 180r / min on a shaker to obtain the bacterial suspension, which is then placed at 4℃ for later use.
[0015] Furthermore, in step (1), the young pigeons are deprived of water for 0.5 hours before the challenge and resumed eating and drinking 0.5 hours after the challenge.
[0016] Furthermore, the criteria for determining the onset of illness in step (2) are as follows:
[0017] Criterion 1: Onset and death within 4-10 days;
[0018] Standard 2: Necropsy of dead young pigeons showed copper-black liver, swollen spleen and kidneys, pasty vent, and intestinal bleeding;
[0019] Standard 3: Two days after the challenge, the cloacal swab smear was positive, and the serum antibody test was positive;
[0020] When the challenged pigeons exhibit symptoms of Standard 3 and Standard 1 or Standard 2, the challenged pigeons are considered to be diseased, thus completing the establishment of the pigeon typhoid Salmonella challenge model.
[0021] The present invention provides the following second technical solution:
[0022] The acute infection model of pigeon Salmonella established by the aforementioned method can be used as a treatment model to screen drugs for pigeon and rodent Salmonella typhimurium infection.
[0023] Furthermore, the acute infection model of Salmonella pigeon established by the aforementioned method can be used as a treatment model to evaluate or assess the effectiveness of vaccines against Salmonella typhimurium infection.
[0024] The beneficial effects of this invention are:
[0025] This invention discloses a method for establishing an acute Salmonella infection model in pigeons. The method involves challenging the pigeons with the selected pigeon-derived Salmonella typhimurium strain ST-JN-63, preferably using a multi-site subcutaneous inoculation route in the neck, which improves the success rate and uniformity of model establishment, achieving a 100% bacterial shedding rate. The disease incidence in pigeons after challenge is evaluated according to the disease assessment criteria mentioned in the method, thus completing the establishment of the pigeon Salmonella challenge model.
[0026] The pigeon-derived Salmonella typhimurium strain ST-JN-63 of this invention is a highly pathogenic strain obtained through screening from multiple isolated strains. Using this ST-JN-63 strain for challenge via existing intraperitoneal-muscular injection, an acute infection model of Salmonella typhimurium in pigeons was successfully established, unlike the standard strain which could not establish an infection model. After further optimizing the infection route to multiple injections under the neck, the course of the disease was prolonged by 7 days compared to the acute infection model of Salmonella typhimurium in pigeons established by intraperitoneal-muscular injection challenge with the ST-JN-63 strain. This provides a basis for subsequent screening of therapeutic drugs. Attached Figure Description
[0027] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:
[0028] Figure 1 This is a transmission electron microscope image of the pigeon-derived Salmonella typhimurium of the present invention;
[0029] Figure 2 This is a schematic diagram of the whole genome analysis of pigeon-derived Salmonella typhimurium ST-JN-63 in this invention;
[0030] Figure 3 A graph showing the changes in mortality rates of pigeons infected with different challenge methods of pigeon-derived Salmonella typhimurium ST-JN-63.
[0031] Figure 4 Clinical symptoms of pigeons infected with ST-JN-63 Salmonella typhimurium in the control group and pigeon-derived Salmonella typhimurium;
[0032] Figure 5 Anatomical images of test pigeons infected with ST-JN-63 Salmonella typhimurium from pigeons in the control group and pigeon source;
[0033] Figure 6 Figure showing the weight changes of pigeons infected with pigeon-derived Salmonella typhimurium ST-JN-63.
[0034] Figure 7 The bacterial shedding status of pigeons infected with ST-JN-63 Salmonella typhimurium in the control group and pigeons was recorded.
[0035] in, Figure 4 The left and right images in the middle represent the blank control group and the Salmonella model group (pigeon-derived Salmonella typhimurium ST-JN-63 model group), respectively. Figure 7 The left and right sides of the middle image represent the blank control group and the Salmonella model group (pigeon-derived Salmonella typhimurium ST-JN-63 model group), respectively. The upper culture dish in the right image shows the culture results of cloacal swabs on SS medium, while the lower culture dish in the right image shows the culture results of bacteria isolated from the liver and intestines on SS differential medium. Detailed Implementation
[0036] To clearly illustrate the technical features of this solution, the invention will be described in detail below through specific embodiments and in conjunction with the accompanying drawings. The scope of this invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to this invention without departing from its spirit and scope.
[0037] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.
[0038] As one implementation method, a method for establishing an acute infection model of Salmonella pigeonis is provided, comprising the following steps:
[0039] S1. Screening of challenge strains
[0040] 1. Strain Isolation and Identification: Suspected Salmonella infection samples were collected from a pigeon farm in Shandong Province. Autopsy revealed a characteristic bronze-colored liver; blackening of the abdomen and internal organs; enlarged heart with peripheral necrosis; thickened intestinal wall filled with green, yellowish-green, or grayish-green foamy, pasty contents; samples were collected aseptically. The collected samples were inoculated into buffered peptone water (BPW) for pre-enrichment and cultured at 37°C with shaking at 130 rpm for 12 h; then transferred to RVS broth and cultured overnight at 34°C with shaking at 130 rpm; RVS broth was then streaked onto SS agar and HE agar, and incubated at 37°C for 18 h, with colony morphology observed. Subsequently, Gram staining was performed on the selected strains, and the bacterial culture was transported on dry ice to Beijing Novogene Technology Co., Ltd. for whole-genome sequencing analysis. BLAST homology analysis was performed on the 16S rRNA sequences annotated in the sequencing files.
[0041] Three strains, ST-JN-63, ST-JN-09, and ST-JN-29, were isolated from the above.
[0042] 2. Biological identification of the strain:
[0043] After extracting the bacterial DNA, the DNA fragments were amplified by PCR, and the amplified products were purified. Sequencing was performed using universal primers (such as the sequences shown in SEQ No. 2 and SEQ No. 3).
[0044] The 16S rRNA sequence of the isolated ST-JN-63 strain is shown in SEQ No. 1. BLAST alignment and housekeeping gene assays revealed that the housekeeping gene of ST-JN-63 strain showed 100% homology with *Salmonella Typhimurium* strain SJTUF10452 (Sequence ID: CP047535.1) via BLAST comparison analysis using the NCBI database. Based on its colony morphology, molecular biological characteristics, and identification using a Salmonella serotyping kit, the strain was confirmed to be *Salmonella* genus, serotype O:4, H:i.
[0045] The housekeeping gene sequences of ST-JN-63 strain are as follows: thrA (SEQ No. 4), purE (SEQ No. 5), sucA (SEQ No. 6), hisD (SEQ No. 7), aroC (SEQ No. 8), hemD (SEQ No. 9), and dnaN (SEQ No. 10). Primer sequences for each housekeeping gene are shown in Table 1 below.
[0046] Table 1. Primers for Steward Gene Sequencing
[0047]
[0048]
[0049] 3. Electron microscopy identification of the strain: Select the isolated ST-JN-63 strain, and aspirate 1×10⁻⁶ micrograms of bacterial suspension. 6 The bacteria were cultured at 37°C with shaking at 280 rpm (cfu / mL) until the end of the logarithmic growth phase. The bacterial pellets were collected by centrifugation at 3000 rpm for 5 min, the supernatant was removed, and the bacteria were washed three times with PBS. The pellets were then collected by centrifugation at 3000 rpm for 15 min, and fixed overnight at 4°C with 1 mL of 2.5% glutaraldehyde. Afterward, the pellets were gently washed with PBS, stained with 2% phosphotungstic acid solution for 10 min, and then dehydrated using a gradient of ethanol (60–100%). The pellets were then sprayed onto a copper mesh, and the submicroscopic morphology of the bacteria was observed and photographed using a transmission electron microscope (TEM). Results are as follows: Figure 1 As shown, two levels of enlarged rod-shaped bacteria are visible.
[0050] Whole-genome sequencing revealed that this strain contained 57 genes with high virulence, 26 lethal genes, and 554 genes with low virulence. (See [link to source]). Figure 2 .
[0051] 4. Antibiotic susceptibility testing of isolated bacteria:
[0052] Antimicrobial susceptibility was assessed using the Kirby-Bauer disk diffusion method and judgment criteria recommended by the Clinical Laboratory Standards Institute (CLSI). The identified isolate ST-JN-63 was inoculated onto LB nutrient agar plates and incubated at 37°C for 24 hours. Three to five single colonies were collected using a sterile swab and diluted in 1 mL of physiological saline to adjust the bacterial concentration to 0.5 McFarland turbidity. Another sterile swab was used to soak the bacterial suspension and evenly spread onto the surface of fresh LB medium. After the plate dried at room temperature for 5 minutes, antimicrobial susceptibility discs were applied to the agar surface using forceps. Within 15 minutes of applying the discs, the plate was inverted and placed in an incubator, incubated at 37°C for 24 hours. The plate was then removed, and the diameter of the inhibition zone was measured. The results were interpreted as sensitive (S), intermediate (I), or resistant (R) when the edge of the inhibition zone was no longer visible to the naked eye.
[0053] The results of antibiotic susceptibility analysis are shown in Table 2 below. The isolated strain ST-JN-63 was resistant to polymyxin B, ampicillin, ciprofloxacin, ofloxacin, enrofloxacin, chloramphenicol, cefotaxime, doxycycline, tetracycline, polymyxin B, trimethoprim-sulfamethoxazole, and erythromycin; moderately sensitive to streptomycin; and sensitive to amoxicillin, ceftazidime, florfenicol, and gentamicin.
[0054] Table 2 Antimicrobial susceptibility testing of pigeon-derived Salmonella strain ST-JN-63
[0055]
[0056] S2. Selection of Infection Targets and Infection Initiation
[0057] 5. Animal reintroduction experiment
[0058] 5.1 The purified ST-JN-63 strain and two other clinically isolated Salmonella typhimurium strains were used to...
[0059] ST-JN-09, ST-JN-29, and the Salmonella Typhimurium standard strain ATCC14028 were activated and prepared for use.
[0060] The bacterial culture was prepared as follows: each of the above-mentioned Salmonella Typhimurium strains stored at -80℃ was streaked onto SS differential medium and incubated overnight at 37℃; after a single colony was picked and a smear was checked to confirm that it was a pure culture, a single colony was picked and inoculated into fresh SS liquid medium and incubated at 37℃ and 180r / min for 22h on a shaker; the resulting bacterial culture was then placed at 4℃ for later use.
[0061] 5.2 Pigeon chicks aged 30-33 days without Salmonella infection, tested for antigen and antibody, were selected and divided into a blank control group and a Salmonella treatment group. They were raised in a conventional environment. Water was withheld 0.5 hours before challenge and resumed 0.5 hours after challenge. The pigeons' necks were disinfected with 75% alcohol using cotton swabs. The pigeon Salmonella treatment group received intraperitoneal intramuscular injections of 1.5 mL × 10g of pigeon-derived Salmonella typhimurium bacterial solution. 8 cfu / mL, blank control group subcutaneously inoculated with PBS;
[0062] 5.3 After challenge, the mental state, water intake, and mortality of the pigeons were observed daily, and autopsies and organ PCR tests were performed on the dead pigeons. The criteria for determining the disease incidence in pigeons after challenge were as follows:
[0063] Criterion 1: Onset and death within 4–8 days;
[0064] Standard 2: Necropsy of dead pigeons shows typical symptoms such as bronze-colored liver or white spots, intestinal bleeding, swollen spleen, atrophied thymus, pasty vent, and green loose stools.
[0065] Standard 3: Cloacal swab and PCR test showed positive results for Salmonella.
[0066] When the challenged pigeons meet the criteria 3 and 1 or 2, the challenged pigeons are considered to be diseased, thus completing the establishment of the pigeon Salmonella challenge model.
[0067] The PCR detection conditions and primer sequences used for identification are as follows:
[0068] PCR reaction: 20 μL reaction mixture: 10 μL 2×TaqMix, 1 μL template, 0.4 μL invA F, 0.4 μL invA R, 1 μL STM4497F, 1 μL STM4497R, 6.2 μL sterile water. Reaction conditions: 95℃ for 5 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 5 min. PCR products were photographed using a gel imaging system after agarose gel electrophoresis.
[0069] Table 3 Primers for multiplex PCR identification of Salmonella:
[0070]
[0071] The results showed that, compared with Salmonella Typhimurium ST-JN-09 and ST-JN-29, strain ST-JN-63 showed the best clinical symptoms in pigeons after intraperitoneal injection modeling, but mortality began to occur on the second day after model establishment. The ATCC14028 standard strain failed to successfully establish an acute Salmonella infection model. Ultimately, pigeon-derived Salmonella strain ST-JN-63 was selected for modeling.
[0072] The pigeon-derived Salmonella typhimurium strain ST-JN-63 isolated and identified above was deposited at the China General Microbiological Culture Collection Center on August 25, 2025, with the accession number: CGMCC No. 35198.
[0073] S3, Optimized attack method
[0074] The challenge method for strain ST-JN-63 was further optimized by changing the intraperitoneal-intramuscular injection to multiple subcutaneous injections of 1 mL × 10 μL of ST-JN-63 Salmonella bacterial solution into the neck. 8 cfu / mL - 1.5mL × 10 8 The control group received subcutaneous PBS at cfu / mL, and the results were compared with a model established using intramuscular challenge. The results showed that, compared with intraperitoneal intramuscular injection, multiple subcutaneous injections at the neck prolonged the disease course in pigeons by 7 days. This provides a basis for subsequent screening of therapeutic drugs. See [link to statistics on survival rates for different challenge methods]. Figure 3 .
[0075] 6. Clinical symptoms and necropsy findings
[0076] Compared with the blank control group, pigeons in the challenge group (multiple subcutaneous injections of ST-JN-63 Salmonella in the neck) exhibited lethargy, decreased appetite, emaciation, and excreted green watery feces, dying on the 4th day after infection. On the 6th day after infection, necropsy revealed typical symptoms in the challenge group, including bronze-colored liver, intestinal hemorrhage, splenomegaly, thymus atrophy, pasty vent, and green watery feces; see [link to relevant documentation]. Figure 4 , Figure 5 , Figure 6 .
[0077] Compared to the blank control group, the challenge group (multiple subcutaneous injections of ST-JN-63 Salmonella in the neck) began shedding bacteria after 24 hours. Cloacal swabs cultured on SS medium showed typical Salmonella colonies with a black, circular center and a transparent periphery. Furthermore, bacteria isolated from the liver and intestines of the challenge group also showed typical Salmonella colonies with a black, circular center and a transparent periphery in SS differential medium. No bacteria grew in the liver and cloacal swab samples from the blank control group pigeons. See also Figure 7 .
[0078] Table 3 Organ index of pigeons infected with pigeon-derived Salmonella ST-JN-63 strain
[0079]
[0080]
[0081] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.
Claims
1. A method for establishing an acute infection model of Salmonella pigeonis, characterized in that, The procedure includes the steps of infecting pigeons with a pigeon-derived Salmonella typhimurium obtained through isolation and screening by injecting it into the neck of young pigeons at multiple points to prepare an acute infection model of Salmonella typhimurium; the pigeon-derived Salmonella typhimurium was deposited at the China General Microbiological Culture Collection Center on August 25, 2025, with the accession number: CGMCC No. 35198.
2. The method for establishing according to claim 1, characterized in that, Includes the following steps: (1) Select pigeons free of Salmonella infection and raise them in a normal environment. Disinfect the skin on the neck of the pigeons and inoculate multiple points subcutaneously in the neck with the aforementioned pigeon-derived Salmonella Typhimurium bacterial suspension. The inoculation dose is 1 mL × 10 8 cfu / mL-1.5mL×10 8 cfu / mL; (2) Observe the mental state, detoxification and mortality of the young pigeons daily, collect cotton swabs to detect Salmonella shedding, and perform necropsy and organ PCR detection on the dead young pigeons; according to the disease judgment criteria, the pigeon Salmonella acute infection model of the tested young pigeons after challenge was successfully established.
3. The method for establishing according to claim 2, characterized in that, Step (1) The young pigeons are 30-33 days old.
4. The method for establishing according to claim 2, characterized in that, Step (1) Preparation of pigeon-derived Salmonella Typhimurium bacterial suspension is as follows: The pigeon-derived Salmonella Typhimurium stored at -80℃ is streaked onto SS solid medium and cultured overnight at 37℃; after picking a single colony and checking that it is a pure culture by smear, a single colony is picked and inoculated into fresh LB liquid medium and cultured for 22 hours at 37℃ and 180r / min on a shaker to obtain bacterial suspension, which is then placed at 4℃ for later use.
5. The method for establishing according to claim 2, characterized in that, In step (1), the young pigeons were deprived of water 0.5 hours before the challenge and resumed eating and drinking 0.5 hours after the challenge.
6. The method for establishing according to claim 2, characterized in that, The criteria for determining the onset of the disease in step (2) are as follows: Criterion 1: Onset and death within 4-10 days; Standard 2: Necropsy of dead young pigeons showed copper-black liver, swollen spleen and kidneys, pasty vent, and intestinal bleeding; Standard 3: Two days after the challenge, the cloacal swab smear was positive, and the serum antibody test was positive; When the challenged pigeons exhibit symptoms of Standard 3 and Standard 1 or Standard 2, the challenged pigeons are considered to be diseased, thus completing the establishment of the pigeon typhoid Salmonella challenge model.
7. The application of the acute infection model of Salmonella typhimurium in pigeons established by the method described in claim 1 as a treatment model for screening drugs for Salmonella typhimurium infection in pigeons and rodents.
8. The acute infection model of pigeon Salmonella established by the method described in claim 1 can be used as a treatment model for evaluating or assessing the effectiveness of pigeon and rodent Salmonella typhi vaccines.