A strain of Gallibacillus salpingitis and its application

Abstract

The present invention discloses a Gallibacterium salpingitidis GS FC22 strain and its application. The Gallibacterium salpingitidis GS FC22 strain was deposited in the China Center for Type Culture Collection on July 11, 2022, with a deposit number of CCTCC NO: M 20221076. The present invention successfully constructs a laying duck model infected with Gallibacterium salpingitidis using the GS FC22 strain, confirming the pathogenicity of the GS FC22 strain to the reproductive system of laying ducks; the GS FC22 strain is prepared into an inactivated vaccine, which has the characteristics of low production cost, good safety, and good immune effect. After immunization, it can significantly improve the egg-laying performance and clinical symptoms of laying ducks, reduce the decline in egg production rate and the increase in the number of defective eggs, and has a good immune protection effect, providing new ideas and methods for the prevention and control of reproductive tract diseases of laying ducks.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and more specifically relates to a Gallibacillus salpingitis strain and application thereof. Background Art

[0002] With the improvement of material living standards, the public's demand for poultry eggs is growing, and the egg-laying poultry farming industry is also booming. Salpingitis caused by primary, secondary or co-infection of bacterial pathogens is an important hazard to the egg-laying poultry farming industry. Under normal circumstances, the oviduct is a sterile environment. When the oviduct mucosa is mechanically damaged due to egg laying or other reasons, bacteria invade the oviduct through the blood or through the cloaca, causing inflammation. Salpingitis can cause dysfunction of the oviduct of laying poultry, abnormal formation of egg white and eggshell, and an increase in the proportion of thin-shelled eggs, deformed eggs, sandy-shelled eggs and broken eggs. It can even block the normal fall of mature follicles, causing yolk peritonitis and increasing the mortality rate of laying poultry. The bacterial pathogens that cause inflammation of the oviduct of laying poultry chickens are mainly Escherichia coli, Mycoplasma, Gallinaceus, etc.

[0003] Gallibacillus is a Gram-negative coccobacillus with blunt ends, no spores, and no motility. It mainly attacks avian hosts such as chickens, turkeys, ducks, geese, pheasants, pigeons, peacocks, and partridges. It is a common bacterium in the digestive tract and respiratory tract of poultry. Its pathogenicity is closely related to factors such as the host's age, immune status, environmental stress, heterologous pathogen infection pressure, and mucosal integrity. Gallibacillus is biologically classified as Pasteurellaceae, mainly including four named species: Gallibacterium anatis, Gallibacterium salpingitidis, Gallibacterium melopsittaci, and Gallibacterium trehalosifermen tans. Current research shows that Gallibacillus, as an opportunistic pathogen, mainly causes hemorrhagic oophoritis, ovarian follicle rupture, salpingitis, peritonitis, perihepatitis, liver necrosis, pericarditis, and air sac inflammation in laying hens, resulting in apparent symptoms such as decreased egg production and egg quality, and increased mortality. The isolation sites of Gallibacillus salpingitis reported at home and abroad are mainly in the upper respiratory tract (nasal cavity, trachea), reproductive tract (cloaca, fallopian tube), digestive tract (rectum) and other extracorporeal environments. At present, there are no reports on the isolation of Gallibacillus salpingitis from laying ducks, and there are very few studies on diseases caused by Gallibacillus salpingitis in laying ducks.

[0004] As for the prevention and control of Gallibacillus, on the one hand, Gallibacillus is generally resistant to oral well-absorbed antimicrobial drugs such as fluoroquinolones, tetracyclines, penicillins and sulfonamides, and there are few drugs available for clinical treatment. On the other hand, there are very few vaccine studies and products for this disease, which are far from meeting the needs of the egg-laying poultry breeding industry for the prevention and control of Gallibacillus. Therefore, the development of a safe and effective vaccine for Gallibacillus is of positive significance for the prevention and control of Gallibacillus in egg-laying poultry. Summary of the invention

[0005] The invention aims to provide a highly virulent strain of Gallibacterium salpingitidis from laying ducks, wherein the inventors have isolated and identified a representative Gallibacterium salpingitidis strain from the intracavitary environment (liver) of laying ducks, providing good material for in-depth research on the pathogenic mechanism of Gallibacterium salpingitidis in laying ducks. The highly virulent strain can cause salpingitis symptoms in the original animals, and can be used to construct a laying duck challenge model, and is preliminarily applied to the research of Gallibacterium salpingitidis inactivated vaccine, aiming to solve the problem of prevention and control of Gallibacterium salpingitidis disease in laying ducks.

[0006] The first object of the present invention is to provide a Gallibacterium salpingitidis GS FC22 strain.

[0007] The second object of the present invention is to provide the application of Gallibacillus GS FC22 strain.

[0008] The third object of the present invention is to provide an inactivated vaccine for Gallibacillus disease.

[0009] The fourth object of the present invention is to provide a method for constructing an animal model of Gallibacillus infection.

[0010] The fifth object of the present invention is to provide an application of an animal model infected with Gallibacillus infection.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention isolates and identifies a Gallibacterium salpingitidis GS FC22 strain from the liver of a sick duck with typical systemic symptoms of salpingitis and peritonitis. The strain has been deposited in the China Center for Type Culture Collection on July 11, 2022, with a deposit number of CCTCC NO: M 20221076. The Gallibacterium salpingitidis GS FC22 strain provided by the present invention has the following characteristics:

[0013] (1) Bacterial morphological characteristics: On TSA plates containing 5% newborn calf serum, incubated at 37°C under aerobic conditions for 24-48 h, oval, smooth, light blue, opaque, shiny colonies with a diameter of 1-2 mm were produced; Gram staining was negative, and pink short rods were visible in clusters under a microscope; under an electron microscope with a resolution of 5 μm, rod-shaped short rods appeared in clusters.

[0014] (2) Bacterial biochemical characteristics: GS FC22 strain can ferment glucose, lactose, and sucrose, is positive for phosphatase and catalase, and produces a clearly defined, completely transparent β-hemolytic ring on a sheep blood agar plate.

[0015] The present invention shows that the GS FC22 strain of Gallibacillus salpingitis is used to attack the high-yielding ducks with double negative serological and etiological tests. The oviduct mucosa of the laying ducks is bleeding and atrophy, the oviduct is blocked by caseous substances, and the pathological changes of yolk peritonitis are pathogenic to the reproductive system of laying ducks. No lesions such as perihepatitis, liver necrosis, pericarditis, and air sac inflammation are found when the laying hens are infected. Clinical symptoms that are different from the Gallibacillus disease of laying hens are shown, thereby successfully constructing a Gallibacillus disease laying duck infection model infected with salpingitis Gallibacillus. The GS FC22 strain of Gallibacillus salpingitis is further prepared into an oil-emulsion adjuvant inactivated vaccine and immunized laying ducks. The results show that the vaccine does not affect the feed intake, mental state, egg-laying performance and other indicators of laying ducks, indicating that there is no biosafety hazard. The GS FC22 strain inactivated vaccine is further used to perform the first and second immunizations on laying ducks, and then the homologous strain GS FC22 strain is used to attack the virus. The results showed that after two immunizations with the inactivated GS FC22 strain vaccine, the laying ducks had a significant protective effect against the attack of the homologous strain GS FC22 strain, and the egg-laying performance and clinical symptoms of the laying ducks were significantly improved, and clinical symptoms such as a decrease in egg production rate and an increase in the number of defective eggs were effectively avoided. This indicates that the inactivated Gallibacillus sp. vaccine prepared using the Gallibacillus sp. GS FC22 strain provided by the present invention has a good immune protection effect.

[0016] Therefore, the present invention provides the use of Gallibacillus GS FC22 strain in preparing a laying duck Gallibacillus disease vaccine or in constructing an animal model of laying ducks infected with Gallibacillus disease.

[0017] Furthermore, the vaccine is an inactivated vaccine.

[0018] The invention provides an inactivated vaccine for Gallibacillus disease, which contains Gallibacterium salpingitidis GS FC22 strain.

[0019] Preferably, the vaccine further contains a vaccine adjuvant.

[0020] More preferably, the vaccine adjuvant is a water-in-oil-in-water adjuvant; the water-in-oil-in-water adjuvant used in the present invention is a conventional adjuvant in the art and can be prepared by the method disclosed in the prior art. The main components of the w / o / w adjuvant used in the present invention are: white oil, lecithin, Tween-80 and Span-80.

[0021] The invention provides a method for constructing a laying duck model infected with Gallibacillus sp., and the laying duck model infected with Gallibacillus sp. is obtained by infecting the laying duck with a Gallibacillus sp. GSFC22 strain.

[0022] Preferably, the infection method is intramuscular injection into the chest.

[0023] Preferably, the 50% infection dose of the GS FC22 strain is 2×10 6 CFU / mL.

[0024] The present invention also provides an application of an egg-laying duck model obtained by the method of constructing an egg-laying duck model infected with Gallibacillus disease in drug screening, vaccine effect evaluation or pathogenic mechanism research.

[0025] The present invention has the following beneficial effects:

[0026] The present invention isolates and identifies a Gallibacterium salpingitidis GS FC22 strain for the first time from the liver of a sick duck with typical systemic symptoms of salpingitis and peritonitis. The GS FC22 strain can be used to successfully construct a laying duck model infected with Gallibacterium salpingitidis, confirming the pathogenicity of Gallibacterium salpingitidis to the reproductive system of laying ducks, further enriching the clinical data of Gallibacterium salpingitidis, and providing a new direction for the diagnosis of reproductive tract diseases of laying ducks.

[0027] The present invention prepares the GS FC22 strain into an oil-emulsion adjuvant inactivated vaccine, which has the characteristics of low production cost, good safety, good immune effect, etc., will not affect the feed intake, mental state, egg-laying performance and other indicators of laying ducks, and no oviduct inflammation is found after autopsy, and there is no biosafety hazard; after immunizing laying ducks with the GS FC22 strain inactivated vaccine, the egg-laying performance and clinical symptoms of laying ducks can be significantly improved, the decline in egg production rate and the increase in the number of defective eggs can be significantly reduced, the vaccine has a good immune protection effect, and has a good market prospect.

[0028] The present invention provides excellent scientific research materials for in-depth research on the infection and pathogenic mechanism of Gallibacillus salpingitis, provides new ideas for the prevention and control of reproductive tract diseases in laying ducks, and provides a new method for the applied research of Gallibacillus salpingitis, which is of great value for the effective prevention and control of reproductive system diseases in laying ducks. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1This is a Gram staining microscopic examination of the strain in the pathogen identification experiment.

[0030] Figure 2 This is the morphology of the strain under an electron microscope in the pathogen identification experiment.

[0031] Figure 3 These are the morphological diagrams of the oviduct autopsies of different treatment groups in the animal regression experiment.

[0032] Figure 4 This is a graph of pathological changes in the experimental group in the animal regression experiment 7 days after infection.

[0033] Figure 5 This is the daily egg production of each group within 3 weeks after the virus attack in the immune protection experiment.

[0034] Figure 6 This is a statistical chart of egg quality after virus attack in each group in the immune protection experiment (where "defective eggs" are sandy-shelled eggs, deformed eggs, and striped eggs, excluding broken eggs caused by human damage; "7d" means the cumulative number of eggs laid within 1-7 days, "14d" means the cumulative number of eggs laid within 8-14 days, and "21d" means the cumulative number of eggs laid within 15-21 days).

[0035] Figure 7 This is the appearance of the eggshell after each group was challenged with the virus in the immune protection experiment. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0038] The solid culture medium preparation method adopted by the present invention is as follows: 40g of tryptone soy agar TSA is dissolved in 950mL of deionized water, sterilized at 121°C for 15 minutes, 50mL of fetal bovine serum is added, and the mixture is divided into sterile plates to prepare flat plates.

[0039] The liquid culture medium preparation method adopted by the present invention is as follows: 30g of peptone soy broth TSB is dissolved in 950mL of deionized water, sterilized at 121°C for 15 minutes, 50mL of sterile fetal bovine serum is added, and stored at 4°C.

[0040] The preparation method of the fresh blood agar culture medium adopted by the present invention is as follows: 40g of tryptone soy agar TSA is dissolved in 950mL of deionized water, sterilized at 121°C for 15 minutes, 50mL of sterile defibrinated sheep blood is added, and the mixture is divided into sterile plates to prepare flat plates.

[0041] The vaccine adjuvant used in the present invention is a water-in-oil-in-water adjuvant, which is a conventional adjuvant in the art and can be prepared by the method disclosed in the prior art. The main components of the water-in-oil-in-water adjuvant (w / o / w adjuvant) used in the present invention are: white oil, lecithin, Tween-80 and Span-80.

[0042] Example 1 Isolation and identification of strains

[0043] 1. Isolation and culture

[0044] The samples came from a duck farm in Jiangxi Province where peritonitis and oviduct disease broke out in ducks, resulting in deformed eggs, sandy shell eggs, and a sudden drop in egg production. Cut the liver tissue blocks of the diseased ducks and soak them in 75% ethanol solution for 1 minute to remove pathogenic microorganisms on the liver surface; place the liver tissue blocks in a sterile EP tube and wash them twice with 1.5mL sterile saline; grind the liver tissue blocks and spread them on TSA plates containing 5% fetal bovine serum, and incubate them at a constant temperature of 37℃ for 24h; pick the purified single colony and streak it on a TSA plate containing 5% sheep blood, and incubate them at a constant temperature of 37℃ for 24h.

[0045] On TSA plates containing 5% fetal bovine serum, incubate at 37°C under aerobic conditions for 24-48 hours to produce single oval, smooth, light blue, opaque, shiny colonies with a diameter of 1-2 mm; on TSA plates containing 5% sheep blood, a clearly defined, completely transparent β-hemolytic ring 2-3 times the size of the colony diameter is formed.

[0046] 2. Pathogen identification

[0047] Use a bacterial loop to scrape multiple purified colonies for Gram staining microscopy. Pick a purified single colony and place it in an EP tube. After resuspending with 0.5 mL of saline, apply it to a TSA plate with 5% fetal bovine serum, and gently cover it on the plate agar with a cover glass. After culturing at 37°C for 24 hours, send the bacteria transferred on the cover glass for electron microscopy analysis. Pick a purified single colony and inoculate it into glucose, mannitol, sucrose, lactose, fructose, catalase, phosphatase and oxidase microbiochemical identification tubes, culture at 37°C for 24h-48h, and make judgments according to the instructions of the kit.

[0048] The results are as follows Figure 1 As shown in the figure, pink short rods can be seen under the oil microscope at 1000 times magnification, and Gram staining is negative; under the electron microscope with a resolution of 5 μm, Figure 2 As shown, rod-shaped short bacteria can be seen distributed in clusters; the isolated bacteria can ferment glucose, lactose, sucrose, fructose, and mannitol; the catalase, phosphatase, oxidase, and indigo matrix tests are positive.

[0049] 3. Biological identification

[0050] A single colony after purification was picked and inoculated into TSB liquid culture medium containing 5% fetal bovine serum. After culturing at 37°C and 180 r / min for 24 h, 2 mL of bacterial solution was centrifuged at 5000 r / min for 5 min, the supernatant was removed, and the culture was frozen in liquid nitrogen for 2-4 h. It was then transported and stored on dry ice and sent to Guangzhou Kidio Biotechnology Co., Ltd. for whole genome sequencing analysis. The 16S rRNA sequence annotated in the sequencing file was subjected to BLAST homology comparison analysis.

[0051] The BLAST comparison analysis of the NCBI database found that the isolated bacteria had the highest homology with the Gallibacterium salpingitidis strain CCUG 15564 strain (Sequence ID: NR_044612.1): 98%. Combined with its colony morphological characteristics, physiological and biochemical characteristics, and molecular biological characteristics, and referring to Bergey's Manual of Systematic Bacteriology (Second Edition), it was determined that the strain was Gallibacterium-Gallibacterium salpingitidis, with an NCBI accession number of CP103875. The strain was identified and named Gallibacterium salpingitidis GS FC22 strain, and was deposited in the China Center for Type Culture Collection on July 11, 2022, with a deposit number of CCTCC NO: M 20221076, and the deposit address is Wuhan University, Bayi Road, Hongshan District, Wuhan City, Hubei Province.

[0052] Example 2 Animal regression experiment

[0053] The isolated and purified GS FC22 strain was inoculated into TSB medium with 5% fetal bovine serum and cultured at 37°C and 180r / min for 24h. The culture solution was centrifuged at 5000r / min for 5min, the supernatant was removed, and the cells were resuspended in sterile phosphate buffered saline. 4 CFU / mL, 2×10 5 CFU / mL, 2×10 6 CFU / mL, 2×10 7 CFU / mL, 2×10 8 The concentration of CFU / mL was measured by chest intramuscular injection. High-producing ducks with double negative serological and pathogen tests at 160 days of age were infected with the virus, and each duck was injected with 1 mL. A normal saline control group was set up, and the results were observed and recorded for 7 consecutive days.

[0054] The results showed that compared with the control group, the egg production of the groups treated with different concentrations of GS FC22 strains decreased and the number of low-quality eggs increased to varying degrees 3 days after infection. The autopsy of the experimental group showed bleeding and atrophy of the oviduct mucosa. Figure 3 7 days later, the autopsy showed that the fallopian tube was blocked by caseous substances, yolk peritonitis and other pathological changes, such as bleeding of the fallopian tube mucosa, disappearance of the folds of the uterine part of the fallopian tube, redness, swelling and bleeding of the cloaca, etc. Figure 4 As shown. At the same time, the median infection dose (EID 50) of this strain was 2×10 6 The pathogenicity of GS FC22 strain to the reproductive system of laying ducks was confirmed, and the infection model of laying ducks infected with Gallibacillus gallinarum was successfully established.

[0055] Example 3 Preparation of inactivated antigens

[0056] The isolated and purified GS FC22 strain was inoculated into TSB medium supplemented with 5% fetal bovine serum and cultured at 37°C and 180 r / min for 24 h. The culture solution was centrifuged at 5000 r / min for 5 min, the supernatant was removed, and the cells were resuspended to 2×10 8 CFU / mL, add formaldehyde to a final concentration of 0.4%, and inactivate at 37°C for 48 hours, stirring once every 4 hours.

[0057] The inactivated bacterial solution was spread on a TSA plate containing 5% fetal bovine serum and cultured at 37°C for 24 hours. The live bacteria test was performed to ensure that the inactivation was complete and no colonies grew on the plate. Then the solution was fully mixed with the water-in-oil-in-water adjuvant to make the final concentration of Gallinaria gallinarum GS FC22 strain 2×10 8 CFU / mL, and the GS FC22 strain inactivated vaccine was obtained.

[0058] Thirty laying ducks of 160 days old and weighing 2.5 kg were selected and randomly divided into 3 groups, with 10 ducks in each group. They were injected with 2 mL of GSFC22 inactivated vaccine, 2 mL of adjuvant, and 2 mL of sterile saline respectively. The feed intake, mental state, and egg-laying performance of the laying ducks were recorded. After 7 days, they were dissected to check the health of the oviduct.

[0059] The results showed that compared with the control group, there was no difference in feed intake, mental state, egg-laying performance and other indicators between the vaccine-immunized group and the adjuvant-immunized group, and no oviduct inflammation was found after autopsy. This shows that vaccine immunization does not pose a biosafety risk.

[0060] Example 4 Immune protection test

[0061] 60 120-day-old laying ducks that were negative in serological and etiological tests were evenly divided into an inactivated vaccine immunization group, an adjuvant immunization group and a blank control group. The preparation of the inactivated vaccine immunization group, the adjuvant immunization group and the blank control group was the same as in Example 3.

[0062] For the first vaccination, 0.5 mL (1×10 8 CFU / mL; after an interval of 14 days, the second immunization was carried out, and each feather was immunized with 1 mL (2×10 8 CFU / mL). 21 days after the second immunization, the inactivated vaccine immunization group, adjuvant group and blank control group were injected with 1 mL (2×10 9 CFU / mL) live bacteria of Gallinarum Gallinarum GS FC22. Swabs of duck oviducts were collected for pathogen detection before the first immunization, before the challenge and 7 days after the challenge. The health status and egg-laying performance of laying ducks were continuously monitored after the challenge. Three weeks after the challenge, oviducts of ducks in each group were taken for clinical pathological analysis.

[0063] The results showed that before the first vaccination and before the virus challenge, the fallopian tube swab test for Gallinarum gallinulitis in the immunized group, adjuvant group and control group was negative; 7 days after the virus challenge, the inactivated vaccine immunized group was negative, while the adjuvant immunized group and the blank control group were positive. The daily egg production of each group within 3 weeks after the virus challenge was as follows Figure 5 As shown in the figure, compared with the immunized group, the laying ducks in the adjuvant group and the control group showed different degrees of egg production decline; further statistics on the egg quality showed that the number of defective eggs increased significantly, such as Figure 6 As shown in Figure 1, at 14 days, the number of defective eggs such as thin-shelled eggs, sandy-shelled eggs, and deformed eggs in the adjuvant group and the control group increased significantly; and the eggshell quality of qualified eggs also deteriorated, with apparent symptoms such as Figure 7 As shown. At the same time, 21 days after the challenge, autopsy found that 8 ducks in the adjuvant immunization group and 5 ducks in the blank control group had a series of pathological changes of salpingitis. The morphological structure of the oviducts in the immunized ducks was normal, and no oviduct inflammation was found after autopsy. It can be seen that the use of GS FC22 strain to prepare inactivated vaccines and the first and second immunizations of laying ducks can effectively prevent GS FC22 strain infection and avoid the influence of GS FC22 strain on the feed intake, mental state, egg-laying performance and other indicators of laying ducks. It can be seen that the inactivated vaccine prepared by the GS FC22 strain of Gallinaria gallinarum provided by the present invention has a good immune protection effect on laying ducks.

[0064] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Gallinarum salpingitis Gallibacterium salpingitidis ) GS FC22 strain, characterized in that The strain was deposited in the China Center for Type Culture Collection on July 11, 2022, with the deposit number being CCTCC NO: M 20221076.

2. The Gallibacillus sp. according to claim 1 ( Gallibacterium salpingitidis )Application of GS FC22 strain in the preparation of Gallibacillus infection vaccine for laying ducks.

3. The Gallibacillus sp. according to claim 1 ( Gallibacterium salpingitidis ) Application of GS FC22 strain in constructing an animal model of laying ducks infected with Gallibacillus infection.

4. The application according to claim 2, characterized in that: The vaccine is an inactivated vaccine.

5. An inactivated vaccine for Gallibacillus infection in laying ducks, characterized in that: Contains the Gallibacillus GSFC22 strain of claim 1.

6. The inactivated vaccine according to claim 5, characterized in that It also contains vaccine adjuvants.

7. The inactivated vaccine according to claim 6, characterized in that The adjuvant is a water-in-oil-in-water adjuvant.

8. A method for constructing a laying duck model infected with Gallibacillus infection, characterized in that: The Gallibacillus GS FC22 strain described in claim 1 is used to infect laying ducks to obtain a laying duck model infected with Gallibacillus disease.

9. The method according to claim 8, characterized in that: The infection method is chest muscle injection.

10. The method according to claim 8, characterized in that The median infection volume of the GS FC22 strain was 2×10 6 CFU / mL.

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