CN116426424A - Chicken bacillus strain with salpingitis and application thereof - Google Patents
Chicken bacillus strain with salpingitis and application thereof Download PDFInfo
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Abstract
The invention discloses a chicken bacillus salpingitis (Gallibacterium salpingitidis) GS FC22 strain and application thereof. The chicken bacillus tubulosis GS FC22 strain is preserved in China Center for Type Culture Collection (CCTCC) at the year 2022, 07 and 11, and the preservation number is CCTCC NO: m20221076. The invention adopts the GS FC22 strain to successfully construct the model of the laying duck infected with chicken bacillosis, and proves the pathogenicity of the GS FC22 strain to the reproductive system of the laying duck; the GS FC22 strain is prepared into the inactivated vaccine, has the characteristics of low production cost, good safety, good immune effect and the like, can obviously improve the egg laying performance and clinical symptoms of the laying ducks after immunization, reduce the drop of the egg laying rate and the rise of the number of defective eggs, has better immune protection effect, and provides a new thought and method for preventing and controlling the reproductive tract diseases of the laying ducks.
Description
Technical Field
The invention belongs to the technical field of microorganisms. More particularly relates to a strain of chicken bacillus with salpingitis and application thereof.
Background
With the improvement of the living standard of substances, the public demand for eggs is increasingly vigorous, and the egg-poultry farming industry also presents a situation of vigorous development. Salpingitis caused by primary, secondary or co-infection of bacterial pathogenic microorganisms is an important hazard for the egg-laying poultry farming industry. Normally, a sterile environment is provided in the oviduct, and when the mucous membrane of the oviduct is mechanically damaged due to egg laying or other reasons, bacteria invade the oviduct in a retrograde manner through blood or through cloaca, thereby causing inflammation. Oviduct inflammation can cause oviduct dysfunction of egg fowls, abnormal egg white and eggshell formation, increased shell breaking proportion of thin shell eggs, abnormal eggs and sand shell eggs, even can prevent mature follicles from falling normally, and cause vitelline peritonitis, so that the death rate of egg fowls is increased. The bacterial pathogen causing the oviduct inflammation of the laying fowl mainly comprises escherichia coli, mycoplasma, chicken bacillus and the like.
The chicken bacillus is a gram-negative bacillus with blunt ends, no spores and no movement, mainly invades bird hosts such as chickens, turkeys, ducks, geese, pheasants, pigeons, peacocks, partridge and the like, is a common bacterium in the digestive tract and the respiratory tract of the birds, and has pathogenicity closely related to factors such as the age of the host, immune condition, environmental stress, heterologous pathogen infection pressure, mucous membrane integrity and the like. The biological classification of the genus chicken bacillus into the family pasteurelloidae mainly comprises: chicken bacillus of duck origin (Gallibacterium anatis), chicken bacillus of salpingitis (Gallibacterium salpingitidis), chicken bacillus of budgerigar (Gallibacterium melopsittaci) and chicken bacillus of trehalose fermentation (Gallibacterium trehalosifermen tans) 4 named species. The current research shows that the chicken bacillus is used as an opportunistic pathogen to mainly cause the appearance symptoms of the chicken such as hemorrhagic oophoritis, oophoroma rupture, salpingitis, peritonitis, perihepatitis, liver necrosis, pericarditis, air sac inflammation, and the like, and cause the appearance symptoms of lower laying rate and egg quality, higher mortality and the like, and the separation part of the chicken bacillus with salpingitis reported at home and abroad mainly comprises the external environment of the upper respiratory tract (nasal cavity, trachea), genital tract (cloaca, fallopian tube), digestive tract (rectum) and the like. At present, no report on the separation of chicken bacillus with salpingitis from egg-laying ducks is seen, and little research on chicken bacillus-induced egg-laying ducks diseases is also seen.
In terms of control of chicken bacillosis, on one hand, chicken bacillosis has general drug resistance to antibacterial drugs with good oral absorption, such as fluoroquinolones, tetracyclines, penicillins, sulfonamides and the like, and few drugs can be selected in the clinical treatment process. On the other hand, the current vaccine research and products related to the disease are few, and the requirements of the egg-laying poultry farming industry on the prevention and control of chicken bacillus can not be met. Therefore, the development of a safe and effective chicken bacillosis vaccine has positive significance for preventing and treating chicken bacillosis of laying fowls.
Disclosure of Invention
The invention aims to provide a strong strain of chicken bacillus salpingitis (Gallibacterium salpingitidis) from an egg-laying duck, which is a representative chicken bacillus salpingitis obtained by the separation and identification of the inventor from the body cavity environment (liver) of the egg-laying duck, and provides good materials for the deep research of the pathogenic mechanism of chicken bacillus salpingitis in the egg-laying duck. The virulent strain can cause the original animal to generate salpingitis symptoms, can be further used for constructing an egg duck virus challenge model, is primarily applied to research on an inactivated vaccine of salpingitis chicken bacillus, and aims to solve the difficulty in prevention and control of salpingitis chicken bacillosis of egg ducks.
The first object of the present invention is to provide a chicken bacillus tubingensis (Gallibacterium salpingitidis) GS FC22 strain.
A second object of the present invention is to provide the use of the chicken Bacillus salpingitis GS FC22 strain.
The third object of the invention is to provide an inactivated vaccine for chicken bacillosis.
A fourth object of the present invention is to provide a method for constructing an animal model of chicken bacteriosis infection.
It is a fifth object of the present invention to provide the use of an animal model of chicken bacteriosis infection.
The above object of the present invention is achieved by the following technical scheme:
the invention separates and identifies a strain of chicken bacillus salpingitis (Gallibacterium salpingitidis) GS FC22 from the liver of a disease duck with typical systemic symptoms of salpingitis and peritonitis, and the strain is preserved in China center for type culture Collection (China, with the preservation number: cctccc NO: m20221076. The chicken bacillus tubal inflammatory GS FC22 strain provided by the invention has the following characteristics:
(1) Bacterial morphological characteristics: incubation on TSA plates containing 5% new born calf serum at 37℃for 24-48h under aerobic conditions, resulting in oval, smooth, bluish, opaque, shiny colonies of 1-2mm diameter; gram staining was negative and pink in color was seen under the microscope as Brevibacterium globosum; under electron microscopy with a resolution of 5. Mu.m, coryneform Brevibacterium in a cluster distribution appeared.
(2) Bacterial biochemical characteristics: the GS FC22 strain can ferment glucose, lactose, sucrose, phosphatase and catalase positively and generate a well-defined and completely transparent beta hemolytic ring on sheep blood plates.
According to the invention, the research shows that the strain GS FC22 of the chicken bacillus with salpingitis is adopted to attack the serology and etiology of the double-negative high-yield female duck, the oviduct mucous membrane of the female duck with the egg-laying duck is bloody and atrophic, the oviduct cheese-like substance is blocked, the pathological change of the egg-yolk peritonitis has pathogenicity to the reproductive system of the egg-laying duck, and the lesions such as perihepatitis, liver necrosis, pericarditis and air sac inflammation which appear when the egg-laying chicken is infected are not found, and the clinical symptoms of the disease differentiation with the chicken bacillosis of the egg-laying duck are shown, so that the chicken bacillosis egg-laying duck infection model with the chicken bacillus with salpingitis is successfully constructed. Further, chicken bacillus with salpingitis GS FC22 strain is prepared into an oil emulsion adjuvant inactivated vaccine and the egg-laying duck is immunized, and the result shows that the vaccine does not influence indexes such as feed intake, mental state, egg-laying performance and the like of the egg-laying duck, thus indicating no biological potential safety hazard. Further adopting GS FC22 strain inactivated vaccine to make primary immunity and secondary immunity of egg duck, then using homologous strain GS FC22 strain to make virus-killing. The result shows that after the GS FC22 strain inactivated vaccine is immunized twice, the laying duck has obvious protection effect on the attack of the GS FC22 strain of the homologous strain, can obviously improve the laying performance and clinical symptoms of the laying duck, and effectively avoid the clinical symptoms such as the drop of the laying rate, the rise of the number of defective eggs and the like, and shows that the chicken bacillus salpingitis inactivated vaccine prepared by using the GS FC22 strain of the chicken bacillus salpingitis has better immune protection effect.
Therefore, the invention provides application of the strain of the chicken bacillus tubulosis GS FC22 in preparing a chicken bacillosis vaccine of the laying duck or constructing an animal model of chicken bacillosis infected by the laying duck.
Further, the vaccine is an inactivated vaccine.
The invention provides an inactivated vaccine for chicken bacillosis, which contains chicken bacillus salpingitis (Gallibacterium salpingitidis) GS FC22 strain.
Preferably, the vaccine further comprises a vaccine adjuvant.
More preferably, the vaccine adjuvant is a water-in-oil-in-water adjuvant; the water-in-oil-in-water adjuvant adopted by the invention is a conventional adjuvant in the field, and can be prepared by a method disclosed by the prior art, and the main components of the w/o/w adjuvant used by the invention are as follows: white oil, lecithin, tween-80 and Span-80.
The invention provides a method for constructing an egg-laying duck model infected with chicken bacillosis, which adopts a chicken bacillus with salpingitis GS FC22 strain to infect an egg-laying duck to obtain the egg-laying duck model infected with chicken bacillosis.
Preferably, the method of infection is chest intramuscular injection.
Preferably, the half-size infection of the GS FC22 strain is 2X 10 6 CFU/mL。
The invention also provides application of the egg-laying duck model obtained by the method for constructing the egg-laying duck model infected with chicken bacillosis in drug screening, vaccine effect evaluation or pathogenesis research.
The invention has the following beneficial effects:
the invention firstly separates and identifies the strain GS FC22 of the chicken bacillus salpingitis (Gallibacterium salpingitidis) from the liver of the disease duck with typical systemic symptoms of salpingitis and peritonitis, and can successfully construct an egg duck model infected with the chicken bacillus salpingitis by adopting the strain GS FC22, thereby proving the pathogenicity of the chicken bacillus salpingitis to the reproductive system of the egg duck, further enriching the clinical data of the chicken bacillus pingitis and providing a new direction for diagnosing the reproductive tract diseases of the egg duck.
The invention prepares the GS FC22 strain into the oil emulsion adjuvant inactivated vaccine, has the characteristics of low production cost, good safety, good immune effect and the like, does not influence indexes such as feed intake, mental state, egg laying performance and the like of the laying duck, does not find salpingitis after the section inspection, and has no biological potential safety hazard; after the GS FC22 strain inactivated vaccine is adopted for immunization of the laying ducks, the laying performance and clinical symptoms of the laying ducks can be obviously improved, the drop of the laying rate and the rise of the number of defective eggs can be obviously reduced, and the vaccine has a better immune protection effect and a good market prospect.
The invention provides excellent scientific research materials for deeply researching infection and pathogenic mechanism of chicken bacillus with salpingitis, provides a new thought for preventing and controlling reproductive tract diseases of the laying ducks, provides a new method for application research of chicken bacillosis, and has important value for effectively preventing and controlling reproductive system diseases of the laying ducks.
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FIG. 1 is a gram of a strain in an etiology identification experiment.
FIG. 2 shows the morphology of strains under electron microscopy in an etiology identification experiment.
Fig. 3 is a morphology of oviduct section of different treatment groups in an animal regression experiment.
FIG. 4 is a graph showing pathological changes 7 days after challenge in the experimental group in animal regression experiments.
Figure 5 shows daily egg production for each group within 3 weeks after challenge in the immunoprotection experiment.
FIG. 6 is a statistical chart of the quality of eggs after toxin challenge in each group of immunoprotection experiments (wherein "defective eggs" are shell eggs, malformed eggs, striped eggs, broken eggs excluding artificial injury; 7d "indicates the cumulative number of eggs laid in 1-7d, 14d" indicates the cumulative number of eggs laid in 8-14d, and 21d "indicates the cumulative number of eggs laid in 15-21 d).
Fig. 7 is an external view of eggshells after challenge in each group of immunoprotection experiments.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The preparation method of the solid culture medium adopted by the invention comprises the following steps: 40g tryptone soy agar TSA was dissolved in 950mL deionized water, sterilized at 121℃for 15 minutes, added with 50mL fetal bovine serum, and plated in sterile dishes.
The preparation method of the liquid culture medium adopted by the invention comprises the following steps: 30g of peptone soybean broth TSB was dissolved in 950mL of deionized water, sterilized at 121℃for 15 minutes, added with 50mL of sterile fetal bovine serum, and stored at 4 ℃.
The preparation method of the fresh blood agar culture medium adopted by the invention comprises the following steps: 40g tryptone soy agar TSA was dissolved in 950mL deionized water, sterilized at 121℃for 15 minutes, added with 50mL of sterilized defibrinated sheep blood, and dispensed into sterile plates to prepare plates.
The vaccine adjuvant adopted by the invention is a water-in-oil-in-water adjuvant, is a conventional adjuvant in the field, can be prepared by a method disclosed by the prior art, and comprises the following main components: white oil, lecithin, tween-80 and Span-80.
Example 1 isolation and identification of strains
1. Isolated culture
The sample is derived from the explosion peritonitis and oviduct diseases of a duck in Jiangxi province, abnormal egg and sand shell egg are produced, and the laying rate is suddenly reduced. Shearing liver tissue blocks of the diseased female ducks, soaking the liver tissue blocks in 75% ethanol solution for 1min, and removing pathogenic microorganisms on the surfaces of the livers; the liver tissue block is placed in a sterile EP tube and washed twice with 1.5mL of sterilized normal saline; grinding the liver tissue blocks, coating the liver tissue blocks on a TSA plate containing 5% fetal bovine serum, and standing and culturing at a constant temperature of 37 ℃ for 24 hours; the purified single colony is streaked on a TSA plate containing 5% sheep blood, and the single colony is subjected to stationary culture at a constant temperature of 37 ℃ for 24 hours.
Incubating for 24-48h at 37deg.C under aerobic conditions on TSA plate containing 5% fetal bovine serum to give 1-2mm diameter oval, smooth, light blue, opaque, glossy single colony; a well-defined, fully transparent beta-hemolytic loop of 2-3 colony diameter size was formed on TSA plates containing 5% sheep blood.
2. Etiology identification
A plurality of purified colonies were scraped with a bacteria-receiving ring and subjected to gram staining microscopy. The single colony after purification is selected and placed in an EP tube, after being resuspended by 0.5mL physiological saline, a TSA plate of 5% fetal bovine serum is coated, and the TSA plate is lightly covered on plate agar by a cover glass, after standing and culturing for 24 hours at the constant temperature of 37 ℃, the thalli transferred on the cover glass are sent for electron microscope analysis, and the single colony after purification is selected and inoculated with glucose, mannitol, sucrose, lactose, fructose, catalase, phosphatase and oxidase respectively in a trace biochemical identification tube, and is cultured for 24 hours to 48 hours at the temperature of 37 ℃ and judged according to the instruction book of the kit.
As a result, as shown in FIG. 1, brevibacterium pink was seen under a 1000-fold field of view with an oil mirror, and gram staining was negative; under the electron microscope field of view with a resolution of 5 μm, as shown in FIG. 2, coryneform Brevibacterium in a cluster distribution can be seen; the isolated bacteria can ferment glucose, lactose, sucrose, fructose and mannitol; catalase, phosphatase, oxidase, and indigo substrate were tested positive.
3. Biological identification
Selecting a single colony after purification, inoculating the single colony into a TSB liquid culture medium containing 5% fetal bovine serum, culturing for 24 hours at a constant temperature of 37 ℃ under 180r/min shaking, taking 2mL of bacterial liquid, centrifuging for 5 minutes at 5000r/min, removing supernatant, freezing in liquid nitrogen for 2-4 hours, transporting and storing dry ice, carrying out whole genome sequencing analysis by Guangzhou Diao biotechnology, and carrying out BLAST homology comparison analysis on a 16S rRNA sequence released by injection of a sequencing file.
The isolated bacteria were found to have the highest homology to the Gallibacterium salpingitidis strain CCUG 15564 strain (Sequence ID: NR_ 044612.1) by BLAST comparison with NCBI database: 98%. Combining its colony morphology, physiological and biochemical characteristics, and molecular biology characteristics, and referring to Bergey's Manual of Systematic Bacteriology (second edition), it was determined that this strain is a chicken bacillus (Gallibacterium) -Gallibacterium salpingitis (Gallibacterium salpingitidis), NCBI accession no: CP103875, identified and designated as a chicken bacillus salpingitis (Gallibacterium salpingitidis) GS FC22 strain, and deposited at the chinese collection for typical cultures at 2022, month 07 and 11, with a deposit number of cctccc NO: m20221076, the preservation address is the university of Wuhan in the mountain area of Wuhan City of Hubei province.
Example 2 animal regression experiments
The separated and purified GS FC22 strain is inoculated into TSB culture medium added with 5% fetal bovine serum, and shake culture is carried out for 24 hours at 37 ℃ and 180 r/min. Centrifuging the culture solution at 5000r/min for 5min, removing supernatant, and re-suspending the bacteria with sterile phosphate buffer salt solution. According to 2X 10 respectively 4 CFU/mL、2×10 5 CFU/mL、2×10 6 CFU/mL、2×10 7 CFU/mL、2×10 8 CFU/mL concentration, chest intramuscular injection, toxicity-counteracting 160-day-old serology and etiology detection double-negative high-yield female ducks, 1mL for each feather injection, physiological saline control group, and continuous observation record of 7d.
The result shows that compared with a control group, after the treatment group of the GS FC22 strain with different concentrations attacks the virus for 3 days, the egg yield of different degrees is reduced, and the inferior eggs are increased; the section of the experimental group can be seen in the hemorrhage of mucous membrane of fallopian tube and atrophy of mucous membrane, as shown in figure 3. After 7d, the pathological changes such as blocking of the cheesy material of the oviduct, vitelline peritonitis and the like appear, and the pathological changes such as oviduct mucous membrane bleeding, salpingo uterine folds disappearance, cloacal swelling bleeding and the like appear, as shown in figure 4. At the same time, the half-number infection (EID 50) of the strain was 2×10 6 CFU/mL. Prove the pathogenicity of GS FC22 strain to the reproductive system of the laying duck, and successfully construct the chicken bacillus disease egg infected with the chicken bacillus with salpingitisDuck infection model.
EXAMPLE 3 preparation of inactivated antigen
The separated and purified GS FC22 strain is inoculated into TSB culture medium added with 5% fetal bovine serum, and shake culture is carried out for 24 hours at 37 ℃ and 180 r/min. Centrifuging culture broth at 5000r/min for 5min, removing supernatant, and suspending the microorganism to 2×10 with sterile phosphate buffer salt solution 8 CFU/mL, 0.4% formaldehyde was added, and the mixture was allowed to stand at 37℃for inactivation for 48 hours, with stirring 1 time every 4 hours.
And (3) coating the inactivated bacterial liquid on a TSA plate containing 5% fetal bovine serum, culturing at 37 ℃ for 24 hours, and carrying out live bacteria detection to ensure that the inactivation is thorough and no bacterial colony grows on the plate. Then fully and uniformly mixed with water-in-oil-in-water adjuvant to make the final concentration of chicken bacillus salpingitis GS FC22 strain be 2X 10 8 CFU/mL to obtain the inactivated vaccine of the GS FC22 strain.
30 feathers of egg ducks with the age of 160 days and the average weight of 2.5kg are selected and randomly divided into 3 groups, wherein each group of 10 feathers is respectively injected with 2mL of GS FC22 strain inactivated vaccine, 2mL of adjuvant and 2mL of sterile physiological saline, feed intake, mental state and egg laying performance of the egg ducks are recorded, and the health condition of oviducts is checked after 7d.
The results show that compared with the control group, the indexes of feed intake, mental state, egg laying performance and the like of the egg ducks in the vaccine immune group and the adjuvant immune group are not different, and oviduct inflammation is not found after the split inspection. Thus, vaccine immunization has no biological safety hidden trouble.
EXAMPLE 4 immunoprotection test
The preparation of the inactivated vaccine immune group, the adjuvant immune group and the blank control group is the same as that of example 3.
First, 0.5mL (1×10) of muscle immunity per feather chest 8 CFU/mL); after 14 days of interval, a second immunization was performed with 1mL (2X 10) per chest muscle immunization 8 CFU/mL). After 21 days of secondary immunization, the inactivated vaccine immune group, adjuvant group and blank group were injected simultaneously with 1mL (2X 10) 9 CFU/mL) live chicken bacillus salpingitis GS FC 22. Before the first timeAnd respectively collecting duck oviduct swabs before and 7d after the virus attack to perform pathogen detection, continuously monitoring the health condition and egg laying performance of the egg ducks after the virus attack, and taking the duck oviducts of each group after 3 weeks of the virus attack to perform clinical pathological analysis.
The results show that before first-time and before virus attack, the detection of the oviduct swab and the oviduct chicken bacillus with salpingitis in the immune group, the adjuvant group and the control group is negative; 7d after the challenge, the inactivated vaccine immune group is negative, and the adjuvant immune group and the blank control group are positive. The daily egg production of each group within 3 weeks after the challenge is shown in fig. 5, and egg production of the adjuvant group and the control group egg ducks is reduced to different degrees compared with the immune group; further counting the quality of the eggs, and finding that the number of the defective eggs is obviously increased, as shown in fig. 6, the number of defective eggs such as thin-shelled eggs, sand-shelled eggs, malformed eggs and the like of the adjuvant group and the control group is obviously increased at 14 d; and the eggshell quality of the qualified eggs is also deteriorated, and the apparent symptoms thereof are shown in fig. 7. Meanwhile, 21d after toxin attack, 8 feathers appear in an adjuvant immune group and 5 feathers appear in a blank control group in a splitting examination, the morphological structure of oviduct of an immune group duck group is normal, and oviduct inflammation is not found after splitting examination. Therefore, the inactivated vaccine is prepared by adopting the GS FC22 strain, and the first immunization and the second immunization are carried out on the laying ducks, so that the infection of the GS FC22 strain can be effectively prevented, and the influence of the GS FC22 strain on indexes such as feed intake, mental state and egg laying performance of the laying ducks is avoided. Therefore, the inactivated vaccine prepared by the chicken bacillus salpingitis GS FC22 strain has better immune protection effect on the laying ducks.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A chicken bacillus salpingitis (Gallibacterium salpingitidis) GS FC22 strain, characterized in that said strain has been deposited in the China center for type culture collection (CCTCC NO) at 2022, 07, 11: m20221076.
2. Use of a strain of chicken bacillus salpingitis (Gallibacterium salpingitidis) GS FC22 as claimed in claim 1 for the preparation of a chicken bacillosis vaccine for laying ducks.
3. Use of a strain of chicken bacillus salpingitis (Gallibacterium salpingitidis) GS FC22 as claimed in claim 1 in the construction of an animal model of chicken bacillosis infected with laying ducks.
4. The use according to claim 2, wherein the vaccine is an inactivated vaccine.
5. An inactivated vaccine against egg duck chicken bacillosis, which is characterized by comprising the chicken bacillus tubal inflammatory GS FC22 strain of claim 1.
6. An inactivated vaccine according to claim 5, further comprising a vaccine adjuvant, preferably the adjuvant is a water-in-oil-in-water adjuvant.
7. A method for constructing a model of an egg-laying duck infected with chicken bacillosis, which is characterized in that the egg-laying duck is infected with chicken bacillosis by adopting the chicken bacillosis with salpingitis GS FC22 strain of claim 1.
8. The method of claim 7, wherein the method of infection is chest intramuscular injection.
9. The method according to claim 7, wherein the half-infectious amount of the GS FC22 strain is 2X 10 6 CFU/mL。
10. Use of the egg-laying duck model obtained by the method of any one of claims 7-9 in drug screening, vaccine effect evaluation or pathogenesis research.
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