CN106635849A - Method for building candida albicans infected chicken models - Google Patents
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- CN106635849A CN106635849A CN201611003951.XA CN201611003951A CN106635849A CN 106635849 A CN106635849 A CN 106635849A CN 201611003951 A CN201611003951 A CN 201611003951A CN 106635849 A CN106635849 A CN 106635849A
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- 239000000725 suspension Substances 0.000 claims abstract description 13
- 229920001817 Agar Polymers 0.000 claims abstract description 9
- 235000002595 Solanum tuberosum Nutrition 0.000 claims abstract description 9
- 244000061456 Solanum tuberosum Species 0.000 claims abstract description 9
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- 238000000227 grinding Methods 0.000 claims abstract description 4
- 239000006228 supernatant Substances 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 3
- 230000001580 bacterial effect Effects 0.000 claims description 15
- 239000007924 injection Substances 0.000 claims description 9
- 238000002347 injection Methods 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 8
- 238000011081 inoculation Methods 0.000 claims description 5
- 238000011010 flushing procedure Methods 0.000 claims description 3
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- 230000015572 biosynthetic process Effects 0.000 claims description 2
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- 238000005119 centrifugation Methods 0.000 claims 1
- 239000000243 solution Substances 0.000 abstract description 2
- 239000002244 precipitate Substances 0.000 abstract 3
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- 238000007865 diluting Methods 0.000 abstract 1
- 238000002255 vaccination Methods 0.000 abstract 1
- 241001465754 Metazoa Species 0.000 description 8
- 201000003984 candidiasis Diseases 0.000 description 8
- 210000002784 stomach Anatomy 0.000 description 7
- 238000010171 animal model Methods 0.000 description 6
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
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- 238000000926 separation method Methods 0.000 description 4
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
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- 241000700605 Viruses Species 0.000 description 2
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- 229940097572 chloromycetin Drugs 0.000 description 1
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- 229960003957 dexamethasone Drugs 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
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- 238000001764 infiltration Methods 0.000 description 1
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- 230000007774 longterm Effects 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/30—Bird
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0337—Animal models for infectious diseases
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- Animal Husbandry (AREA)
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Abstract
The invention provides a method for building candida albicans infected chicken models. The method includes steps of a, preparing candida albicans strain conidium suspension, to be more specific, inoculating clinically isolated candida albicans strains on potato agar culture media, culturing the candida albicans strains at the temperature of 25 DEG C for 2.5-4 days, washing candida albicans colonies by the aid of PBS (phosphate buffer solution), collecting washing solution with candida albicans conidia or hyphae, centrifugally discarding supernatant, collecting precipitates, grinding the precipitates and diluting the precipitates until the concentration of the candida albicans conidia reaches 1*10<6>/mL so as to obtain the conidium suspension for standby application; b, forming the models, to be more specific, acquiring just hatched chicks, injecting 1mL of conidium suspension into the crop of each chick when the chick is 7 days old, raising the vaccination chicks in coops and allowing the chicks to freely drink water and feed for 4-9 days so as to successfully build the models. The method has the advantages that the models are made from chicken infected by clinically isolated candida albicans (HE strains), and accordingly discussion on candida albicans infection of the chicken is close to reality and is accurate.
Description
Technical field
The invention belongs to technical field of pharmaceutical biotechnology, more particularly to the side that a kind of candida albicans infection chicken model is set up
Method.
Background technology
Candidiasiss are a kind of funguses caused by candidal candidiasis, mainly candida albicans infection
Disease, is Amphixenosises.People's mucosa, skin, tissue, organ etc. can be invaded and cause different degrees of candidiasises.With
Broad ectrum antibiotic, corticosteroidss medicine, anti-tumor chemotherapeutic and radiotherapy, the extensive application of Interventional diagnosis and treatment means, and because of disease
The reasons such as malicious infection, wasting diseasess reduce Abwehrkraft des Koepers, and the microbial infection of Candida albicans gradually increases, Resistant strain
Occur clinical treatment difficulty is increased.
Under field conditions (factors), various birdss are susceptible to suffer from, main harm poult.The cause of disease and the long-term symbiosis of body, are a kind of
Condition pathogen, has generation, sickness rate 51.5-5% and mortality rate 60-0% all over the world.Have previously been thought that birdss white is read
Pearl bacterium disease is often distributed, once outburst, you can bring about great losses.In recent years, as the swift and violent of broiler large-scale cultivation is sent out
Exhibition, candidiasises are widely present in broiler production, and new popular and infection characteristic is presented, and are brought to broiler production
Serious impact.
It is both at home and abroad to adopt laboratory animal mice and rabbit to set up candida albicans infection mould more, but mice and rabbit are not
Candida albicans natural reservoir (of bird flu viruses), although these experimental animal models have a reference value to studying candida albicans infection, but after all
Mice and rabbit with natural reservoir (of bird flu viruses) chicken be it is variant, therefore, using being clinically separated Candida albicans（HE strains）Infected chicken makees model, visits
Begging for chicken candida albicans infection can be closer actual, more accurate.More importantly also animal is provided for the research of people's candidiasises
Model.The drug sensitive test result of conventional Candida albicans shows, the curative effect after the result of In vitro chemo-drug sensitive test and vivo medicine-feeding
Sometimes it is not consistent.So, the candida albicans infection chicken model of foundation is for the pathogeny for inquiring into candida albicans infection；
The evaluation of premunition pathological process and candidiasiss medicine curative effect is respectively provided with significance.
The content of the invention
The present invention is not enough to solve prior art, there is provided a kind of method that candida albicans infection chicken model is set up, is chicken
Candida albicans infection prevention and control provide basis, provide animal model for the research of people's candidiasiss.
The technical solution used in the present invention is：A kind of method that candida albicans infection chicken model is set up, including following step
Suddenly：The preparation of a, Candida albicans bacterial strain conidiospore suspension：The Candida albicans bacterial strain being clinically separated is seeded in into potato agar
In culture medium, at 24 ~ 31 DEG C of temperature, cultivate 2.5 ~ 4 days；Candida albicans bacterium colony is rinsed with PBS again, white is collected
Candidiasises conidium or the flushing liquor of mycelia, 5000 turns/min are centrifuged 5 minutes；Abandon supernatant, collect precipitation, grinding；Use PBS
Buffer is diluted to the conidial concentration of Candida albicans bacterial strain for 0.5 × 106~2×106/ mL, makes conidiospore suspension standby
With；
B, formation model：Shell age chickling is just gone out, has observed 7, during 7 age in days, the above-mentioned conidiospore suspension 1mL of crop intracapsular injection will
Inoculation chicken in cage raising, free water and feeding 4 ~ 9 days, modeling success.
Further, the concentration of the PBS is 0.01mol/L.
Further, the conidial concentration of the Candida albicans bacterial strain is 1 × 106 /mL。
Further, in a steps, Candida albicans bacterial strain is seeded in the temperature in potato agar culture medium for 28 DEG C
Under, incubation time is 3 days.
What the present invention was obtained has the beneficial effect that：The present invention is adopted and is clinically separated Candida albicans（HE strains）Infected chicken does mould
Type, inquiring into chicken candida albicans infection can be closer actual, more accurate.Infectious condition of the present invention is easily-controllable, and infection rate is high, and infection is steady
It is fixed.
Description of the drawings
Fig. 1 is experimental group chicken stomach photo of the present invention；
Fig. 2 is experimental group chicken stomach culture Candida albicans glow of the setting sun piece of the present invention；
Fig. 3 is that experimental group chicken stomach of the present invention infects Candida albicans Gram’s staining photo；
Fig. 4 is experimental group chicken stomach histopathology photo of the present invention；
Fig. 5 is blank control group chicken stomach photo of the present invention.
Specific embodiment
Confirmatory experiment：1st, experimental strain：Candida albicans HE bacterial strains are clinical separation strain, on potato agar flat board,
Bacterium colony is circle, and milky, the back side do not have pigmentation.Under the microscope, thalline is not of uniform size, is the ferment of circular or ellipse
Female sample bacterium.The identification of bacterial strain Jing biochemical tests confirms and 16sRNA gene sequencing confirms.
2nd, laboratory animal：Healthy 7 Japanese instar chickling 16, is provided by Hebei University Of Engineering's laboratory animal breeding farm, average body
Weigh 30 ± 4g, male and female half and half.
3rd, the packet of laboratory animal and bacterial strain are processed：2 groups are randomly divided into by body weight, sex, 8 per group, A groups are blank right
According to, give glucocorticoid process but be not inoculated with Candida albicans；B groups give glucocorticoid process for experimental group, while connecing
Plant Candida albicans HE strains；
4th, preparation of the inoculation with Candida albicans bacterial strain conidiospore suspension：The detached bacterial strain from clinical pathological material of disease, Jing biochemical tests
Identification confirms and 16sRNA gene sequencing confirms as Candida albicans, is seeded in potato agar culture medium, in 28 DEG C of temperature
Under, cultivate 3 days；It is 0.01mol/LPBS wash buffer Candida albicans bacterium colonies with concentration again, collects Candida albicans mitogenetic
Spore or the flushing liquor of mycelia, 5000 turns/min are centrifuged 5 minutes；Abandon supernatant, collect precipitation, grinding；Diluted with PBS
It is 1 × 10 to the conidial concentration of Candida albicans bacterial strain6/ mL, makes conidiospore suspension standby；
5th, animal model：From healthy chick, crop intracapsular injection 1mL Candida albicans conidiospore suspensions.Blank control group is noted
Penetrate physiological saline solution.Dexamethasone injection 7.5mg/kg lumbar injections, the next day duplicate injection 1 time, co-injection 2 times.Daily
Search for food after observation experiment animal inoculation, the clinical symptoms such as growth.Animal is put to death in the 5th day, 7 days, 9 days, 11 days, drawing materials, it is big to carry out
Body pathological changes, culture and histopathological examination confirm model construction success or not.
6th, separation and Culture：After animal being put to death within 5th day, 7 days, 9 days, 11 days after inoculation, take crop, apply mucosa with cotton swab and connect
Plant in the potato agar culture medium containing chloromycetin and cycloheximide.
7th, tissue pathology checking：At animal after death, the tissue such as clip crop, neutral formalin are fixed, specimens paraffin embedding slices, OK
HE is dyeed.
8th, B groups experimental group experimental result
1）Clinical symptoms：After crop intracapsular injection conidiospore suspension, chicken feeding is reduced, and is lost weight, crop enlargement.
2）Substantially pathological changes：5 days after injection, crop has big quantity of fluid, and mucosa has cheesy thing, forms pseudomembrane（Fig. 1）, and 5
Modeling success after it, the 7th day, 9 days, 11 days put to death chicken crop symptom with the 5th day execution chicken crop pathological changes it is identical.
3）Separation and Culture：In potato agar culture medium, separation strains bacterium colony is circle, and milky, the back side do not have pigment
It is calm（Fig. 2）.Under the microscope, thalline is not of uniform size, is circular or the yeast-like funguses of ellipse, Candida albicans as shown in Figure 3
Bacterium Gram’s staining photo.
4）Histopathology：Chicken stomach horny layer is substantially thickened under the microscope, and horny layer has obvious inflammatory cell infiltration,
There are a large amount of red microbiological contamination silks（Fig. 4）.
9th, A groups blank group experimental result
Blank group chicken stomach is normal, pathological changes does not occur, as shown in Figure 5.
Claims (4)
1. a kind of method that candida albicans infection chicken model is set up, it is characterised in that：Comprise the following steps：A, Candida albicans
The preparation of strain conidiospore suspension：The Candida albicans bacterial strain being clinically separated is seeded in potato agar culture medium, in temperature
At 24 ~ 31 DEG C, cultivate 2.5 ~ 4 days；Candida albicans bacterium colony is rinsed with PBS again, Candida albicans conidium is collected
Or the flushing liquor of mycelia, 5000 turns/min centrifugations 5 minutes；Abandon supernatant, collect precipitation, grinding；It is diluted to PBS white
The conidial concentration of color beads bacterial strain is 0.5 × 106~2×106/ mL, makes conidiospore suspension standby；
B, formation model：Shell age chickling is just gone out, has observed 7, during 7 age in days, the above-mentioned conidiospore suspension 1mL of crop intracapsular injection will
Inoculation chickling chicken coop raising, free water and feeding 4 ~ 9 days, modeling success.
2. a kind of method that candida albicans infection chicken model is set up according to claim 1, it is characterised in that：The PBS
The concentration of buffer is 0.01mol/L.
3. a kind of method that candida albicans infection chicken model is set up according to claim 1, it is characterised in that：The white
The conidial concentration of beads bacterial strain is 1 × 106 /mL。
4. a kind of method that candida albicans infection chicken model is set up according to claim 1, it is characterised in that：The a steps
In rapid, Candida albicans bacterial strain is seeded in the temperature in potato agar culture medium for, at 28 DEG C, incubation time is 3 days.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1443075A (en) * | 2000-06-19 | 2003-09-17 | 坎迪瓦克斯有限公司 | Compositions and methods for treatment of candidiasis |
| CN101078011A (en) * | 2007-02-05 | 2007-11-28 | 大连大学 | Candida albicans infection mould and construction method thereof |
| CN104189020A (en) * | 2014-08-11 | 2014-12-10 | 中山大学附属第一医院 | Construction method of lung candida albicans infected mouse model after transplantation |
-
2016
- 2016-11-15 CN CN201611003951.XA patent/CN106635849A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1443075A (en) * | 2000-06-19 | 2003-09-17 | 坎迪瓦克斯有限公司 | Compositions and methods for treatment of candidiasis |
| CN101078011A (en) * | 2007-02-05 | 2007-11-28 | 大连大学 | Candida albicans infection mould and construction method thereof |
| CN104189020A (en) * | 2014-08-11 | 2014-12-10 | 中山大学附属第一医院 | Construction method of lung candida albicans infected mouse model after transplantation |
Non-Patent Citations (4)
| Title |
|---|
| JACOBSEN ID 等: "Pathogenesis of Candida albicans infections in the alternative chorio-allantoic membrane chicken embryo model resembles systemic murine infections", 《PLOS ONE》 * |
| 孙敬方 主编: "《动物实验方法学》", 30 November 2001, 人民卫生出版社 * |
| 杜发娅 等: "侵袭性白念珠菌小鼠感染模型的建立与评估", 《西南国防医药》 * |
| 闫金坤 等: "鸡白色念珠菌五种染色方法的比较研究", 《中国兽医科学》 * |
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Application publication date: 20170510 |
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| RJ01 | Rejection of invention patent application after publication |