CN109929780B - Humanized cryptobacter pus and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of microorganisms, and discloses a swine-derived cryptococcus pyogenes and application thereof, wherein the swine-derived cryptococcus pyogenes is TPY JX18, and the preservation number is CCTCC NO: m2019042; the pig-sourced cryptococcus pyogenes is preserved in the China center for type culture collection in 2019, 1 month and 14 days; the address is China center for type culture Collection in eight Wuhan universities in flood and mountain areas of Wuhan city, Hubei province. The strain TPY JX18 contains multiple virulence factors, causes nursery pigs and mice to die acutely, has stronger pathogenicity and immunogenicity, and can be used for researching the molecular mechanism of virulence factors on host pathogenicity or for researching a subunit vaccine of cryptobacter pyogenes; the invention provides research materials for discovering new pathogenicity, pathogenic mechanism and epidemiology of the cryptobacter pyogenes.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a swine cryptococcus pyogenes and application thereof.
Background
Currently, the current state of the art commonly used in the industry is such that:
cryptococcus pyogenes (Trueperella pyogenes) is a gram-positive brevibacterium, with the strongest virulence among the species Cryptococcus. As a mucous membrane indigenous bacterium in the bodies of economic livestock (cattle, sheep, pigs and the like), cryptobacter pyogenes can invade a host through skin or mucous membrane wounds to cause adjacent tissue and organ infection, also can cause cryptobacter pneumophila and chest diseases through respiratory tract infection, is manifested by subcutaneous abscess, pneumonia, endometritis, mastitis, arthritis and the like, and can cause death of the livestock due to sepsis septicemia in severe cases. The resistance of animals is reduced and traumatic infection is a direct cause of the disease, flies are main transmission media, and the bacteria invade to cause diseases when the living environment of the animals is poor or the autoimmunity of the animals is low. The discovered virulence factors of the cryptococcus pyogenes comprise hemolysin PLO, collagen binding protein CbpA, neuraminidase NanP and NanH, pilin FimA/FimC/FimE/FimG and the like. The pathogenicity of the strain carrying different virulence factors to animals is different, wherein the pathogenicity of the strain carrying PLO and NanP is obviously stronger than that of the strain carrying other virulence factors.
The cryptobacter pyogenes as a conditional pathogenic bacterium can be widely spread among livestock, the health development of the breeding industry faces major challenges due to the lack of prevention means or misdiagnosis at present, and huge economic losses are caused.
In recent years, with the vigorous development of the pig breeding industry, modern pig farms gradually tend to be large-scale intensive, the problems of swinery diseases are more complicated, especially the occurrence of bacterial diseases is also gradually frequent, and the pig farms pose a potential threat to the pig breeding industry. According to the report of the literature, the cryptobacter pyogenes is widely distributed in China and can cause various diseases of pigs, such as subcutaneous abscess, endocarditis, endometritis, pneumonia, arthritis, meningitis and the like. The cryptobacter pyogenes causes a large range of pathological changes, so that a large number of clinical symptoms are shown. Often, abscesses only cause physical weakness in animals, and endocarditis, bronchopneumonia and adhesive peritonitis can be fatal, with fever. Clinically, the cases of swine systemic septicemia and acute death caused by cryptobacter pyogenes are rarely reported at home and abroad. In addition, the pathogenicity, pathogenic mechanism and epidemiology of cryptobacter pyogenes are still under the initial stage, and the role of virulence factors in infecting human and animal organisms and the immunogenicity thereof are also in need of intensive research.
In summary, the problems of the prior art are as follows:
(1) viral diseases are often regarded more seriously by current veterinary practitioners in disease diagnosis, however, in production practice, single or mixed bacterial infection also poses a great threat to disease prevention and control in large-scale pig farms, and particularly the problem of antibiotic abuse caused thereby is worthy of attention. The cryptococcus pyogenes belongs to conditional pathogenic bacteria and can be widely spread among livestock, but currently, the cryptococcus pyogenes is lack of systematic research, so that a prevention means (for example, an effective cryptococcus pyogenes vaccine is lacked) is lacked or misdiagnosis is caused, and the healthy development of the breeding industry faces a great challenge.
(2) The cryptobacter pyogenes infects pigs to cause a large range of pathological changes, and present clinical symptoms are very many, usually, local abscesses only cause physical weakness of animals, and clinically, cases of liver and lung suppurative nodules, systemic septicemia and meningitis caused by the cryptobacter pyogenes are rarely reported at home and abroad. However, as the situation of swine diseases becomes more complex, cases lethal to crypt pyogenes gradually increase, and the pathogenesis of crypt pyogenes is deeply studied.
The difficulty of solving the technical problems is as follows:
since the cryptococcus pyogenes can infect not only economic animals (cattle, sheep, pigs, and the like), but also domestic pets (cats, dogs, and the like) and wild animals (more than ten species of antelope, camel, elephant, deer, and the like), and the clinical symptoms caused by the cryptococcus pyogenes are numerous, the research on the cryptococcus pyogenes is relatively scattered and superficial at present, and particularly, the research on the molecular mechanism of the cryptococcus pyogenes infecting pigs and killing the pigs is only in the initial stage, and theoretical data are lacked. With the increasing number of lethal cases of cryptobacter pyogenes, intensive research and discussion on pathogenicity, pathogenesis and epidemiology of the cryptobacter pyogenes by scientific research workers are urgently needed, so that the research and development of effective vaccines are promoted.
The significance of solving the technical problems is as follows:
the isolated cryptococcus pyogenes TPY JX18 has multiple virulence factors, causes typical clinical symptoms and acute death of nursery pigs and mice, has strong pathogenicity, can be used as a standard strain for scientific research, and provides research materials for researching pathogenicity, pathogenesis and epidemiology of the cryptococcus pyogenes, such as researching molecular mechanism of virulence factors on host pathogenicity, or for researching subunit vaccine of the cryptococcus pyogenes, or establishing an animal infection model and the like. The bacterial strain can lay a foundation for deeply researching the infection mechanism of the cryptococcus pyogenes, and can also provide research materials for vaccine development of the cryptococcus pyogenes, pathogenic mechanism research and development of a diagnosis method.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a swine cryptococcus pyogenes and application thereof.
The invention is realized in such a way that the swine cryptococcus pyogenes is a swine cryptococcus pyogenes TPY JX18(Trueperella pyogenenes TPY JX18) with the preservation number of CCTCC NO: m2019042; the swine-origin cryptobacter pyogenes (Trueperella pyogenes TPY JX18) was deposited in the chinese collection of type cultures at 1 month and 14 days 2019; the address is China center for type culture Collection in eight Wuhan universities in flood and mountain areas of Wuhan city, Hubei province.
Further, the swine-origin cryptobacter pyogenes bacteria belong to the genus cryptobacter, gram-positive brevibacterium corynebacterium, do not exercise, do not resist acid, do not have spores, are aerobic or facultative anaerobic, and have a growth temperature of 37 ℃.
Another object of the present invention is to provide a method for obtaining swine-origin cryptococcus pyogenes, comprising:
step one, preparation of a culture medium:
TSB liquid medium: dissolving 30g tryptone soybean broth (TSB, purchased from BD Difco, USA) in 1000mL ultrapure water, sterilizing at 121 deg.C for 15min, adding 50mL fetal calf serum, and storing at 4 deg.C;
TSA solid medium: dissolving tryptone soy agar (TSA, purchased from BD Difco)40g in 1000mL of ultrapure water, sterilizing at 121 ℃ for 15min, adding fetal calf serum 50mL, and subpackaging in sterile plates to prepare plates;
fresh blood agar medium: dissolving 40g tryptone soy agar TSA in 1000mL of ultrapure water, sterilizing at 121 ℃ for 15min, adding 50mL of sterile defibered sheep blood, and preparing into a sterile flat plate;
and step two, isolated culture of the strain:
aseptically collecting lung tissues of sick pigs, grinding, inoculating on a TSA solid culture medium, culturing at constant temperature of 37 ℃ for 24h, and growing single colonies with needle-tip-shaped size, transparency, roundness, smoothness and regular edges; after purification inoculation, gram staining is carried out, and the bacteria are gram positive brevibacterium, single in or in clusters; culturing the cells on a TSA culture medium added with 5% fresh blood for 48 hours to obtain beta-hemolytic rings with the size of 2-3 times of the diameter of a colony.
Further, after the acquisition of the cryptobacter pyogenes from swine origin, the following steps are required:
identification of the strains:
extracting genome DNA from the subcultured strain, and extracting according to the instruction of a bacterial genome DNA extraction kit; synthesizing a general primer for detecting the cryptobacter pyogenes, carrying out PCR reaction, and then identifying.
Further, after the acquisition of the pig-sourced cryptobacter pyogenes, the PCR identification of virulence factors is required:
the method comprises the steps of detecting a virulence factor hemolysin gene plo, a collagen binding protein cbpA, neuraminidase genes nanP and nanH and a pilus gene fimA/fimC/fimE/fimG of the cryptococcus pyogenes by using a virulence factor detection primer for synthesizing the cryptococcus pyogenes, and carrying out PCR reaction and then identifying.
The invention also aims to provide an animal model construction method for verifying the virulence effect of the pig-sourced cryptobacter pyogenes, which comprises the following steps:
determining the pathogenicity of the cryptococcus pyogenes TPY JX18 by using a 4-5-week-old female Kunming mouse, and taking physiological saline as a negative reference; the inoculation dose is 8X 108CFU/only, 3X 108CFU/only, 1 × 108CFU/only, 5X 107CFU/only, 3X 107Each dose group was inoculated with 8 mice in the abdominal cavity, each inoculated with 0.5mL of bacterial suspension; the pathogenicity of the strain to the mice was analyzed for 7 days.
The invention also aims to provide a vaccine prepared by using the swine-origin cryptococcus pyogenes.
In the invention, the bacteria are separated from the lungs of nursery pigs in a certain pig farm in Pingxi province, the body temperature of sick pigs is raised to about 41 ℃, the sick pigs are shortness of breath, anorexia, emaciation and limb weakness, the course of disease is short, and the sick pigs die urgently within a few days. Dissecting suppurative necrotic foci formed after lung marginal consolidation of dead pigs, separating to obtain a strain of slow-growing bacteria, and performing smear staphyloscopy and 16S rRNA detection to confirm that the separated bacteria is cryptococcus pyogenes, which is named as TPY JX 18.
The characteristics of bacteria are as follows: the bacillus belongs to the genus cryptococcus, gram-positive corynebacterium parvum, does not move, is not acid-resistant, does not have spores, is aerobic or facultative anaerobic, and has the optimal growth temperature of 37 ℃. The serum or blood can accelerate the proliferation of bacteria. After 24h growth on TSA medium supplemented with 5% fetal bovine serum, needle-like wet microcolonies were visible. After 48h of growth on TSA medium added with 5% fresh blood, beta-hemolytic ring 2-3 times the diameter of the colony can be seen.
Through PCR amplification and agarose gel electrophoresis identification, the cryptobacter pyogenes has a plurality of virulence factors: hemolysin PLO, neuraminidase NanP/NanH, pilin fimA/fimC/fimE. By using 8X 1088 female Kunming mice with the age of 4-5 weeks are inoculated by CFU, the abdomens of all inoculated mice are suppurative, all the inoculated mice are attacked and died within 3 days, the liver enlargement and the meningeal hemorrhage of the mice are found by a autopsy examination, and the result of a histopathological section proves that the bacterium can cause intracerebral hemorrhage of the mice. The strain can be used as a standard virulent strain for tests of researchers, and is particularly suitable for replicating pathological models by using nursery pigs and mice as research objects.
In summary, the advantages and positive effects of the invention are:
the strain TPY JX18 provided by the invention has strong virulence, carries various virulence factors, has typical and lethal pathogenic capability on pigs and mice, can be used as a standard test strain of researchers, and provides a new application prospect for research, diagnosis, treatment and vaccine development of live pig diseases caused by cryptobacter pyogenes.
The strain TPY JX18 is separated from the lungs of nursery pigs which die in typical respiratory symptoms, no other bacteria grow when the strain is separated on a TSA culture medium added with 5% fetal calf serum, and the strain can be used for establishing a pig pneumonia pathological model caused by infection of cryptobacter pyogenes.
The strain TPY JX18 contains multiple virulence factors, causes nursery pigs and mice to die acutely, has stronger pathogenicity and immunogenicity, and can be used for researching the molecular mechanism of virulence factors on host pathogenicity or researching a subunit vaccine of cryptobacter pyogenes.
After the strain TPY JX18 infects a mouse, pathological damage is caused to the brain of the mouse, and an unusual intracranial hemorrhage symptom appears, thereby providing research materials for discovering new pathogenicity, pathogenic mechanism and epidemiology of the cryptobacter pyogenes.
Drawings
FIG. 1 is a graph of suppurative necrotic foci formed after lung margin consolidation of dead nursery pigs according to an embodiment of the present invention.
FIG. 2 is a graph of the gram stain microscopic results (1000X) provided by the examples of the present invention.
FIG. 3 is a PCR identification chart of 16S rRNA gene of Cryptococcus pyogenes TPY JX18 provided in the embodiment of the present invention.
FIG. 4 is a diagram of the PCR identification of virulence factors of Cryptococcus pyogenes TPY JX18 (representing a negative control) provided by the embodiments of the present invention.
FIG. 5 is a graph of meningeal congestion symptoms (visible as a distinct bleeding point) in infected mice provided by examples of the invention.
FIG. 6 is a photograph of a pathological section of brain tissue from an infected mouse (200X, in which significant red blood cell aggregation is visible at the bleeding point) provided in an example of the present invention.
FIG. 7 is a flowchart of a method for obtaining Proseclotus pyogenes from swine according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Clinically, the cases of swine systemic septicemia and acute death caused by cryptobacter pyogenes are rarely reported at home and abroad. In addition, the pathogenicity, pathogenic mechanism and epidemiology of cryptobacter pyogenes are still under the initial stage, and the role of virulence factors in infecting human and animal organisms and the immunogenicity thereof are also in need of intensive research.
In order to solve the above technical problems, the present invention will be described in detail with reference to specific embodiments.
The pig source cryptococcus pyogenes TPY JX18 provided by the embodiment of the invention has the preservation number of CCTCC NO: m2019042. The swine-origin cryptococcus pyogenes (Trueperella pyogenenes TPY JX18) is preserved in the China center for type culture preservation in 2019, 1 month and 14 days, and is addressed to the China center for type culture preservation in the eight Wuhan universities in the flood mountain area of Wuhan city, Hubei province.
The strain is separated from the lung of a nursery pig in a certain pig farm in Pingxi province, the body temperature of a sick pig is raised to about 41 ℃, the sick pig has the disadvantages of rapid respiration, anorexia, emaciation, limb weakness, short course of disease and acute death within a few days. A suppurative necrotic focus formed after lung marginal consolidation of a pig killed by a caesarean section is dissected, a strain of bacteria which grows slowly is obtained by separation, and the separated bacteria is proved to be cryptococcus pyogenes by smear staphyloscopy and 16S rRNA detection and is named as TPY JX18(Trueperella pyogenes TPY JX 18).
The characteristics of bacteria are as follows: the bacillus belongs to the genus cryptococcus, gram-positive corynebacterium parvum, does not move, is not acid-resistant, does not have spores, is aerobic or facultative anaerobic, and has the optimal growth temperature of 37 ℃. The serum or blood can accelerate the proliferation of bacteria. After 24h growth on TSA medium supplemented with 5% fetal bovine serum, needle-like wet microcolonies were visible. After 48h of growth on TSA medium added with 5% fresh blood, beta-hemolytic ring 2-3 times the diameter of the colony can be seen.
Through PCR amplification and agarose gel electrophoresis identification, the cryptobacter pyogenes has a plurality of virulence factors: hemolysin plo, neuraminidase nanP/nanH, pilin fimA/fimC/fimE.
As shown in fig. 7, the method for obtaining humanized cryptococcus pyogenes provided by the embodiments of the present invention includes:
s101, preparation of a culture medium: TSB liquid medium: dissolving 30g tryptone soybean broth (TSB, purchased from BD Difco, USA) in 1000mL ultrapure water, sterilizing at 121 deg.C for 15min, adding 50mL fetal calf serum, and storing at 4 deg.C; TSA solid medium: dissolving tryptone soy agar (TSA, purchased from BD Difco)40g in 1000mL of ultrapure water, sterilizing at 121 ℃ for 15min, adding fetal calf serum 50mL, and subpackaging in sterile plates to prepare plates; fresh blood agar medium: dissolving 40g tryptone soy agar TSA in 1000mL of ultrapure water, sterilizing at 121 ℃ for 15min, adding 50mL of sterile defibered sheep blood, and making into sterile plates.
S102, isolated culture of the strain: aseptically collecting lung tissues of sick pigs, grinding, inoculating on a TSA solid culture medium, culturing at constant temperature of 37 ℃ for 24h, and growing single colonies with needle-tip-shaped size, transparency, roundness, smoothness and regular edges; after purification inoculation, gram staining is carried out, and the bacteria are gram positive brevibacterium, single in or in clusters; culturing the cells on a TSA culture medium added with 5% fresh blood for 48 hours to obtain beta-hemolytic rings with the size of 2-3 times of the diameter of a colony.
In the embodiment of the invention, after the acquisition of the swine-origin cryptobacter pyogenes, the following steps are required:
identification of the strains:
extracting genome DNA from the subcultured strain, and extracting according to the instruction of a bacterial genome DNA extraction kit; synthesizing a general primer for detecting the cryptobacter pyogenes, carrying out PCR reaction, and then identifying.
In the embodiment of the invention, after the acquisition of the swine-origin cryptobacter pyogenes, the PCR identification of virulence factors is also needed:
the method comprises the steps of detecting a virulence factor hemolysin gene plo, a collagen binding protein cbpA, neuraminidase genes nanP and nanH and a pilus gene fimA/fimC/fimE/fimG of the cryptococcus pyogenes by using a virulence factor detection primer for synthesizing the cryptococcus pyogenes, and carrying out PCR reaction and then identifying.
In an embodiment of the present invention, an animal model construction method for verifying the virulence effect of the pig-sourced cryptobacter pyogenes is provided, and the animal model construction method includes:
determining the pathogenicity of the cryptococcus pyogenes TPY JX18 by using a 4-5-week-old female Kunming mouse, and taking physiological saline as a negative reference; the inoculation dose is 8X 108CFU/only, 3X 108CFU/only, 1 × 108CFU/only, 5X 107CFU/only, 3X 107Each dose group was inoculated with 8 mice in the abdominal cavity, each inoculated with 0.5mL of bacterial suspension; the pathogenicity of the strain to the mice was analyzed for 7 days.
The strain can be used as a standard virulent strain for tests of researchers, and is particularly suitable for replicating pathological models by using nursery pigs and mice as research objects.
The invention is further described with reference to specific examples.
Example 1: isolation and identification of Cryptobacterium pyogenes
1.1 Collection of pathological materials
The diseased material comes from the pig farm of Pingxi Pingxiang city of Jiangxi province, the nursery pig dies of illness, the diseased pig has high fever, asthma, anorexia, emaciation and limb weakness, the lung of the diseased pig is collected after death (see figure 1), and the diseased pig is transported at low temperature and stored at 20 ℃ below zero.
1.2 preparation of the culture Medium
TSB liquid medium: tryptone soy broth (TSB, available from BD Difco, USA) 30g was dissolved in 1000mL of ultrapure water, sterilized at 121 deg.C for 15min, and then added with fetal bovine serum (available from Hangzhou Sijiqing Co., Ltd.) 50mL and stored at 4 deg.C for further use.
TSA solid medium: tryptone soy agar (TSA, available from BD Difco, USA) 40g is dissolved in 1000mL of ultrapure water, sterilized at 121 deg.C for 15min, added with fetal bovine serum 50mL, and distributed in sterile plates to make plates.
Fresh blood agar medium: dissolving Tryptone Soy Agar (TSA)40g in 1000mL of ultrapure water, sterilizing at 121 deg.C for 15min, adding sterilized defibrinated sheep blood 50mL, and making into sterile plate.
1.3 isolation and culture of the Strain
Aseptically collecting lung tissues of sick pigs, grinding, inoculating on a TSA solid culture medium, culturing at constant temperature of 37 ℃ for 24h, and growing single colonies with pinpoint shape, transparent shape, round shape, smooth shape and regular edges. After the purification inoculation, gram staining was performed (see FIG. 2), and the bacteria were gram-positive Brevibacterium, solitary or clumping. After culturing for 48h on a TSA culture medium added with 5% fresh blood, beta-hemolytic ring with the size 2-3 times of the colony diameter can be seen.
1.4 identification of the strains
Genomic DNA was extracted from the subcultured strain and extracted according to the instructions of a bacterial genomic DNA extraction kit (purchased from Takara, Inc., Dalian). A detection universal primer for Cryptococcus pyogenes was synthesized according to the reference (Duchenne, 2016), and PCR was carried out according to the conventional method, and the results of the identification are shown in FIG. 3. The primers were synthesized by Biotechnology engineering (Shanghai) Inc., and the sequences of the primers are shown in Table 1.
TABLE 1 primers for PCR amplification of Cryptobacterium pyogenes
1.5 PCR identification of virulence factors
Primers for detecting the virulence factor of cryptobacter pyogenes are synthesized according to a reference (e.silva, 2008), the sequences of the primers are shown in table 1, the main virulence factor hemolysin gene plo, collagen binding protein cbpA, neuraminidase genes nanP and nanH, and pilus genes fimA/fimC/fimE/fimG of the cryptobacter pyogenes are detected, and PCR reaction is carried out according to a conventional method. The electrophoresis chart is shown in fig. 4, and the identification result shows that hemolysin gene plo, neuraminidase gene nanP and nanH, pilus gene fimA/fimC/fimE, collagen-binding protein-free gene cbpA and pilus gene fimG exist in the cryptococcus pyogenes isolate TPY JX18, which indicates that the cryptococcus pyogenes isolate has strong pathogenicity.
Example 2: toxicity test of Cryptobacterium pyogenes TPY JX18
2.1 infection test of Cryptobacterium pyogenes TPY JX18 on mice
The pathogenicity of cryptococcus pyogenes TPY JX18 was determined using 4-5 week old female Kunming mice, and physiological saline was used as a negative reference. The inoculation dose is 8X 108CFU/only, 3X 108CFU/only, 1 × 108CFU/only, 5X 107CFU/only, 3X 107CFU/mouse, 8 mice per dose group were inoculated intraperitoneally, each with 0.5mL of bacterial suspension. The pathogenicity of the strain to mice observed for 7 days is shown in Table 2.
TABLE 2 lethality of Cryptobacterium pyogenes TPY JX18 in mice
The onset mice initially appear as depressed spirit, crouched into clusters and reduced appetite. Highest inoculation dose (8X 10)8CFU) caused abdominal suppuration in all vaccinated mice, all morbidity and mortality within 3 days. At higher inoculum doses (3X 10)8CFU、1×108CFU、5×107CFU), the mice have symptoms of weight loss, rough fur and abdominal suppuration, and die continuously within 7 days, and the suppuration parts of the mice which are not dead are healed, so that the symptoms are relieved and improved. When dead mice are subjected to autopsy, the liver is swollen and brown, and other organ tissues have no obvious lesion. However, brain tissue from mice at the highest infectious dose was dissected and meningeal congestion was found, with bleeding sites (see FIG. 5).
2.2 pathological section Observation of tissue injury
The highest inoculation dose (8X 10)8CFU) brain, lung lobes, liver tissues of mice under infection. Fully fixing in 4% paraformaldehyde solution, dehydrating, embedding in paraffin, making into slices (4 μm thick), and HE staining. Under an optical microscope (microscope: NIKON Eclipse ci, imaging System: NIKON digital sight DS-FI 2). Compared with the lung of the control group of mice, the bronchial epithelium structure of the attacking group of mice is complete, the morphological structure of epithelial cells is normal and closely arranged, the alveolar wall is not obviously thickened, and a small amount of inflammatory cell infiltration can be seen in the tissues. Compared with the liver of the control group of mice, a great amount of hepatocyte balloon degeneration, cell swelling, nucleus centering, vacuolation in cytoplasm, local necrosis of hepatocyte, nucleolysis and inflammatory cell infiltration can be seen in the liver tissue of the challenge group of mice. Compared with the brain of the control group of mice, more neurons were found to shrink, the staining was deepened, the cytoplasm of the nucleus was not demarcated clearly, and more erythrocytes were found in the brain tissue of the challenge group of mice (see fig. 6).
Although cryptococcus pyogenes TPY JX18 can cause lung lesions and respiratory symptoms of nursery pigs, the strain has no substantial influence on the lung of mice. From the results of clinical autopsy and histopathological section, the cryptobacter pyogenes TPY JX18 mainly causes serious pathological damage to the liver and brain of the mice and causes acute death of the mice. Therefore, the strain has different pathogenic modes for different hosts (pigs and mice), and can be used as a typical research material for researching the pathogenicity, pathogenic mechanism and epidemiology of the cryptobacter pyogenes.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (4)
1. The humanized cryptococcus pyogenes is characterized in that the humanized cryptococcus pyogenes is TPY JX18 with the preservation number of CCTCC NO: m2019042; the pig-sourced cryptococcus pyogenes is preserved in the China center for type culture collection in 2019, 1 month and 14 days; the address is China center for type culture Collection in eight Wuhan universities in flood and mountain areas of Wuhan city, Hubei province; the pig-derived cryptococcus pyogenes contains hemolysin gene plo, neuraminidase genes nanP and nanH, pilus gene fimA/fimC/fimE, collagen-free binding protein gene cbpA and pilus gene fimG.
2. The swine-origin cryptobacter pyogenes according to claim 1, wherein the swine-origin cryptobacter pyogenes bacterium belongs to the genus cryptobacter, gram-positive brevibacterium corynebacterium, nonmotile, nonacid-fast, spore-free, aerobic or facultative anaerobic, and has a growth temperature of 37 ℃.
3. An animal model construction method for verifying the virulence effect of the pig-derived cryptobacter pyogenes as set forth in claim 1, wherein the animal model construction method comprises:
determining the pathogenicity of the cryptococcus pyogenes TPY JX18 by using a 4-5-week-old female Kunming mouse, and taking physiological saline as a negative reference; the inoculation dose is 8X 108CFU/only, 3X 108CFU/only, 1 × 108CFU/only, 5X 107CFU/only, 3X 107Each dose group was inoculated with 8 mice in the abdominal cavity, each inoculated with 0.5mL of bacterial suspension; the pathogenicity of the strain to mice was analyzed 7 days later.
4. A vaccine prepared from the swine-origin Cryptococcus pyogenes of claim 1.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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-
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Non-Patent Citations (8)
| Title |
|---|
| Aminoglycoside resistance of Trueperella pyogenes isolated from pigs in China;Wen-Long DONG 等;《The Journal of Veterinary Medical Science》;20171002;第1-17页 * |
| Trueperella pyogenes infections in swine: clinical course and pathology;L.S. Jarosz等;《Polish Journal of Veterinary Sciences》;20141231;第17卷(第2期);第395-404页 * |
| 一株猪源化脓隐秘杆菌的分离与鉴定;陈蕾等;《养猪》;20111231(第05期);第111-112页 * |
| 吉林省8株猪源化脓隐秘杆菌主要毒力基因的鉴定及耐药性分析;董文龙等;《中国兽医科学》;20160115(第01期);第69-73页 * |
| 猪化脓隐秘杆菌的分离与鉴定;鲁杏华等;《中国畜牧兽医》;20090520(第05期);第149-152页 * |
| 猪化脓隐秘杆菌的分离鉴定及病原特性研究;张乐宜 等;《动物医学进展》;20141231;第35卷(第5期);摘要，第45页右栏倒数第二段，第47页左栏第2段至右栏第2段 * |
| 猪源化脓隐秘杆菌的分离鉴定及病理组织学观察;董文龙等;《上海交通大学学报(农业科学版)》;20160625(第03期);第22-27页 * |
| 生猪脓包化脓隐秘杆菌的分离鉴定与防控分析;刘晓波等;《湖南畜牧兽医》;20150415(第02期);第1-4页 * |
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