CN110511889A - Salmonella AD19 plants of one plant of donkey source and its application - Google Patents
Salmonella AD19 plants of one plant of donkey source and its application Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, more particularly to salmonella AD19 plants of a breeding ass source and its application, microbial preservation number is CGMCC N0.18046, preservation date: on June 27th, 2019, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.The present invention miscarries from Shandong donkey acquisition separates 1 plant of gram negative bacilli in dead coltfoal pathological material of disease, after isolated strains purifying culture, through Morphological Identification, biochemical identification, 16Sr DNA Cloned culturing, determine that this plant separation bacterium is salmonella；What pathogenicity test results showed this plant of salmonella has certain lethality to mouse, wherein main is the miscarriage that can cause pregnant mouse.The bacterial strain can be used for preparing the inactivated vaccine for the salmonella for causing donkey to be miscarried.There is good immunogenicity by inactivated vaccine prepared by the bacterial strain, highly-safe, protecting effect is good.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to AD19 plants of a breeding ass salmonella and its application.
Background technique
Salmonellosis is one of the zoonosis being of great significance on public hygienics, cause of disease Salmonella
Enteric bacteria section, including those cause to poison by food, and lead to the bacterium of gastroenteritis, Typhoid and paratyphoid.They, which are removed, can infect people
Outside, can also infect many animals includes mammality, bird etc..Salmonellosis is the livestock and poultry group as caused by the bacterium of Salmonella
The general name of infectious disease, this disease often betide the animals such as pig, chicken, ox, wherein miscarriage detection of Salmonella is caused to be to cause the disease of horse paratyphoid
Substance, it is related with miscarriage, sepsis of the newborn and polyarthirtis.The disease easily infects primiparity dam and young coltfoal, Duo Shuo
Before miscarriage occurs, generally without apparent clinical symptoms, miscarriage generation is more unexpected, and the sramana about equus especially donkey
The domestic report of Salmonella disease is seldom.
Donkey is pregnancy period long mammal (360 days or so) and is mostly single tire, is quickly increased to meet large-scale cultivation
The needs for adding breeding stock, promote the fast development of donkey industry, purify this disease as early as possible and control the generation and prevalence of the bacterium, deter in advance
The cause of disease of disconnected donkey salmonellosis, it is necessary to separate local donkey salmonella epidemic strain, reinforce cause of disease biochemistry and Molecular Identification etc.
Research and possible application, realizing causes donkey abortion salmonella disease to effectively prevent locality.
Summary of the invention
One plant of donkey source salmonella strain AD19, microbial preservation number are CGMCC N0.18046, preservation date: 2019 6
The moon 27, classification naming: salmonella Salmonella SP., depositary institution: Chinese microorganism strain preservation conservator
Meeting common micro-organisms center, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.The bacterium is trained in salmonella color
Support culture bacterium colony in base purple, median size, neat in edge, smooth wet, circular protrusions.It is seeded to the bacterium solution of nutrient broth
It after 37 DEG C of cultures for 24 hours, is observed using Gram's staining, which is purified, and is the brevibacterium of Gram-negative, both ends blunt circle.
Above-mentioned donkey salmonella strain AD19,16S rDNA is nucleotide sequence shown in sequence table 1.
The universal primer of the PCR amplification bacterial 16 S rDNA of 16S rDNA are as follows:
F27:5 '-agagttgatcctggctcag-3 ' (SEQID No.2),
R1492:5 '-aaggaggtgatccaaccgca-3 ' (SEQID No.3).
Above-mentioned donkey salmonella strain AD19 is preparing the application in inactivated vaccine.Including by above-mentioned donkey salmonella strain
AD19 expands bacterium, formalin-inactivated, steriling test, safety verification qualification through the nutrient broth that peptone and beef extract powder are component formula
Salmonella inactivated vaccine is prepared with the adjuvant emulsion of equivalent afterwards.
Beneficial effect
Being isolated from Shandong District the present invention provides one plant leads to the donkey source salmonella advantage epidemic strain of miscarriage.It miscarries from donkey
Salmonella pathogenic bacteria are only isolated in pathological material of disease, the etiology to cause donkey to be miscarried provides accurate foundation.Reality is infected in mouse
In testing, show that this plant of bacterium is general to the pathogenecity of mouse, but very strong for the cause of pregnant mouse miscarriage ability, this and this
The miscarriage of strain salmonella pregnancy period caused by donkey is consistent.The inactivated vaccine immunity that the present invention prepares the bacterium with it is good, for having
Effect, which prevents donkey miscarriage caused by local epidemic link, important value.
Detailed description of the invention
Fig. 1 is the Gram's staining form of donkey salmonella strain AD19；
Fig. 2 is pcr amplification product M:Marker；1: isolated strains AD19.
Specific embodiment
Embodiment below does not limit the present invention convenient for better understanding the present invention.Test in subordinate's embodiment
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
Routine biochemistry reagent shop is commercially available.Quantitative test in following embodiment is respectively provided with and repeats to test three times, is as a result averaged
Value.
Embodiment 1
1 materials and methods
1.1 pathological material of disease
In April, 2019, the small-sized donkey amounted to 200 donkeys the case where Shandong Donga donkey breaks out the miscarriage of extensive jenny ass,
Miscarriage jenny ass 45 is to give birth to for the first time in the pregnant age miscarriage of the 6-11 month.There is the symptoms such as body temperature raising, and phase in miscarriage jenny ass
After death.To the postmortem of the dead donkey in both ends show the diffusivity hemorrhagic inflammation of caecum and big colon, lung liver fibrosis and
The necrotic lesion of kidney and liver.Acquire the mediastinal lymph nodes of 1 dead coltfoal, the heart, lung, kidney,liver,spleen, brain, colon tissue samples into
The test such as row bacteriological detection and virus screenings.
1.2 main agents
Salmonella color culture medium, plain agar and ordinary nutritional broth bouillon are purchased from the rich limited public affairs of biology in Qingdao sea
Department；Biochemical identification pipe is purchased from the rich biological Co., Ltd in Qingdao sea；Susceptibility piece is purchased from Oxoid company.
1.3 bacterial strains are separately cultured
The pathological material of disease tissue acquired in 1.1 is placed in superclean bench, with oese take pathological material of disease organization internal and inner wall of intestine and
Content is seeded in salmonella color culture medium, is placed in 37 DEG C of incubator cultures for 24 hours, is observed its colonial morphology.Picking is aobvious
The bacterium colony that purple is shown in color culture medium, is seeded to nutrient broth medium, is put into 37 DEG C of incubator cultures for 24 hours, culture is removed from office blue
Whether Albert'stain Albert microscopy, observation strain morphology feature purify.
1.4 biochemical identification
By the strain inoculated isolated and purified to triple sugar iron agar, lysine decarboxylase test broth, indole (tryptophan meat soup), urine
Plain enzyme, potassium cyanide, potassium cyanide control and amino acid decarboxylases control, are placed in 37 DEG C of incubator cultures for 24 hours, observe its bottom and inclined-plane
Color change and production gas situation, according to GB4789.4-2010 standard judging result.
The PCR sequencing of 1.5 separation bacterium 16S rDNA
1.5.1 pcr template prepares the picking separation strains pathogenic bacteria pure culture single colonie, and ordinary nutritional broth bouillon increases bacterium
Afterwards, bacterium solution DNA is extracted with EasyPure Bacteria Genomic DNA Kit.
1.5.2 the universal primer sequence of the PCR amplification bacterial 16 S rDNA of 16S rDNA is
F27:5 '-AGAGTTGATCCTGGCTCAG-3 ', R1492:5 '-AAGGAGGTGATCCAACCGCA-3 '；It is intended to expand
Clip size is 1500 bp or so, and above-mentioned primer is synthesized by Beijing Qing Ke Biotechnology Co., Ltd；Reaction system (25 μ
L): 12.5 μ L of 2x Taq PCR Master Mix, ddH2Each (10mmol/L) 1 μ L of 9 μ L of O, upstream and downstream primer,
1.5 μ L of template；Response procedures are 95 DEG C of 5min, and 95 DEG C of denaturation 40s, 56 DEG C of annealing 40s, 72 DEG C of extension 90s, 35 are followed
Ring, 72 DEG C of extension 8min.PCR product observes result through 1% agarose gel electrophoresis.
1.5.3 sequencing compares single purpose band PCR product of analysis, send Hua Da gene limited liability company to be sequenced, sequence
It is classified as shown in SEQID No.1.Sequencing result is passed through into Blast(https: //blast.ncbi.nlm.nih.gov/) In
It is compared in Gen-Bank.
1.7 mouse pathogenicities
1.7.1 experimental animal SPF kunming mice, 42, weight about 20g, half male and half female, the pregnant mouse in the Kunming SPF 12 is purchased from mountain
Eastern university's Experimental Animal Center.
1.7.2 mouse is randomly divided into 7 groups by measurement LD50, wherein 6 tests, 1 control group, every group 6, male and female
Fifty-fifty, bacterium solution is according to 1.8 × 109-1.8×104CFU/mL.Using intraperitoneal injection, with the capacity such as the bacterium solution of culture 8h not isoconcentration
Mice Inoculated, only, control group injects same amount of normal saline to 0.2mL/.After attacking poison, 10d is observed continuously.
1.7.3 it calculates according in 1.7.2 as a result, pregnant mouse is divided into 3 groups, wherein 2 tests, each 5；1 control group, 2
Only；Selection 1.8 × 109With 1.8 × 108Two gradients detect this plant of salmonella and cause miscarriage performance.
1.7.4 dissect mouse, recycling bacterium and PCR identification is attacked against each other dead mouse after poison, and dissect observes each tissue internal organs
Lesion and abortion mouse embryo.Liver, spleen and the abortion mouse embryonic vaccination salmonella color culture medium of mouse are taken, recycling is thin
Bacterium, kit extract DNA of bacteria.PCR, which is detected, whether there is salmonella in internal organs and abortion mouse.
1.8 mouse immuning test
1.8.1 the bacterium of separation amplification is added into 0.4% formalin-inactivated, 36~48 h, centrifugation discards clear.Precipitating is with the PBS(10mmol that sterilizes
PH7) it is diluted to 1 × 106CFU/mL.The Fu Shi of qualified inactivated bacterial liquid and equivalent is complete after steriling test and safety verification
Full adjuvant and Fu Shi Freund's incomplete adjuvant emulsification preparation immunogene.
1.8.2 the mouse 12 of 15g weight is only divided into 2 groups, wherein 6 only subcutaneous with the 0 .3 mL/ of inactivation antigen of preparation
Injection, another 6 are only subcutaneously injected with 0.9% physiological saline 0.3mL/ and compare, and each group carries out 2 after after the first exemption 20 days
Exempt from, 2, which exempt from rear 15d, carries out immunoprotection experiment according to 1.7.
Embodiment 2
2 results and analysis
2.1 strain culturing result
The bacterium is named as AD19, and deposit number (CGMCC N0.18046) cultivates bacterium colony in purple in salmonella color culture medium
Color, median size, neat in edge, smooth wet, circular protrusions.After being seeded to 37 DEG C of bacterium solution cultures for 24 hours of nutrient broth, utilize
Gram's staining observation, the bacterium are purified, and are the brevibacterium (Fig. 1) of Gram-negative, both ends blunt circle.
2.2 biochemical results
The PCR amplification of 2.3 bacterial strain 16S rDNA is analyzed
There is apparent purpose band (Fig. 2) at the place 1500bp or so in AD19 bacterial strain 16S rDNA pcr amplification product.To the strain
It separates bacterium and carries out 16S rDNA PCR sequencing, sequence is compared through Blast, the results show that the 16S rDNA sequence of separation bacterium is
Salmonella.
2.4 bacterial strain pathogenicity test results
2.4.1 this plant of salmonella of bacterial strain LD50 measurement result can with infecting mouse, be inoculated with 36h after, inoculum density be 1.8 ×
109There is disease symptom at first in the test group mouse of CUF/mL, and slightly disorderly, apathetic, loss of appetite is right for morbidity mouse coat
The infection conditions of mouse are shown in Table 1.According to Reed-Muench method, calculate AD19 to the LD50 value of mouse be 2.29 ×
107CFU/mL。
Infection lethal cases of the 1 various dose salmonella of table to mouse
2.4.2 bacterial strain causes pregnant mouse miscarriage this plant of salmonella of test result that pregnant mouse can be caused to miscarry, to pregnant mouse
Abortion situation is shown in Table 2.When inoculation metering is 1.8 × 109, it is inoculated with pregnant mouse and occurs disease symptom after 4h, pregnant mouse is complete in 5-11h
Portion's miscarriage, abortion ratio 100% show that this plant of salmonella can cause the miscarriage of pregnant mouse.
Table 2
2.3 dissect mouse, recycling Bacteria Identification result
Dissect is carried out to infecting mouse, finds metainfective mouse spleen enlargement, enteron aisle swelling；From the liver of mouse, spleen and stream
Renewed vaccination salmonella color culture medium in mice embryonic is produced, 37 DEG C of cultures grow purple bacterium colony for 24 hours, in culture medium, pass through
Gram stain microscopy is consistent with the injection strain morphology of mouse before.Other pathogenic microorganisms are not detected from pathological material of disease,
It is considered that this plant of salmonella AD19 is the pathogenic bacteria for causing donkey to be miscarried.
2.4 inactivated vaccine immune effects
AD19 causes mouse depressed spirit, expiratory dyspnea, dead and miscarriage occur through alimentary canal infection, and infection leads to mouse spleen
There is enlargement, necrosis.AD19 infection causes pregnant mouse to miscarry, and researches show that salmonella infections, and pregnant donkey can be caused to miscarry, but
Have no the report that salmonella causes mouse to be miscarried, reason needs further to be studied with mechanism.As a result negative control group is whole
It miscarries, and with the bacterial strain after ordinary nutritional meat soup culture increases bacterium (36 ± 1 DEG C of cultures are no less than 6h), the inactivated bacteria of preparation
Seedling (0 .4% formaldehyde), the serum antibody titer after being immunized twice are 1:5.2 × 104, immune group do not occur miscarriage symptom,
The immune protective efficiency of immune group is up to 100%.
Sequence table
<110>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricul
Salmonella AD19 plants of<120>one plants of donkey sources and its application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1356
<212> DNA
<213>donkey salmonella strain AD19
<400> 1
tacttctttt gcaacccact cccatggtgt gacgggcggt gtgtacaagg cccgggaacg 60
tattcaccgt ggcattctga tccacgatta ctagcgattc cgacttcatg gagtcgagtt 120
gcagactcca atccggacta cgacgcactt tatgaggtcc gcttgctctc gcgaggtcgc 180
ttctctttgt atgcgccatt gtagcacgtg tgtagccctg gtcgtaaggg ccatgatgac 240
ttgacgtcat ccccaccttc ctccagttta tcactggcag tctcctttga gttcccggcc 300
taaccgctgg caacaaagga taagggttgc gctcgttgcg ggacttaacc caacatttca 360
caacacgagc tgacgacagc catgcagcac ctgtctcaca gttcccgaag gcaccaatcc 420
atctctggaa agttctgtgg atgtcaagac caggtaaggt tcttcgcgtt gcatcgaatt 480
aaaccacatg ctccaccgct tgtgcgggcc cccgtcaatt catttgagtt ttaaccttgc 540
ggccgtactc cccaggcggt ctacttaacg cgttagctcc ggaagccacg cctcaagggc 600
acaacctcca agtagacatc gtttacggcg tggactacca gggtatctaa tcctgtttgc 660
tccccacgct ttcgcacctg agcgtcagtc tttgtccagg gggccgcctt cgccaccggt 720
attcctccag atctctacgc atttcaccgc tacacctgga attctacccc cctctacaag 780
actcaagcct gccagtttcg aatgcagttc ccaggttgag cccggggatt tcacatccga 840
cttgacagac cgcctgcgtg cgctttacgc ccagtaattc cgattaacgc ttgcaccctc 900
cgtattaccg cggctgctgg cacggagtta gccggtgctt cttctgcggg taacgtcaat 960
tgctgcggtt attaaccaca acaccttcct ccccgctgaa agtactttac aacccgaagg 1020
ccttcttcat acacgcggca tggctgcatc aggcttgcgc ccattgtgca atattcccca 1080
ctgctgcctc ccgtaggagt ctggaccgtg tctcagttcc agtgtggctg gtcatcctct 1140
cagaccagct agggatcgtc gccttggtga gccgttacct caccaactag ctaatcccat 1200
ctgggcacat ctgatggcaa gaggcccgaa ggtccccctc tttggtcttg cgacgttatg 1260
cggtattagc caccgtttcc agtagttatc cccctccatc aggcagtttc ccagacatta 1320
ctcacccgtc cgccactcgt cagcgaagca gcaagc 1356
<210> 2
<211> 19
<212> DNA
<213>artificial synthesized
<400> 2
agagttgatc ctggctcag 19
<210> 3
<211> 20
<212> DNA
<213>artificial synthesized
<400> 3
aaggaggtga tccaaccgca 20
Claims (3)
1. one plant of donkey salmonella strain AD19, which is characterized in that microbial preservation number be CGMCC N0.18046, preservation date:
On June 27th, 2019, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. donkey salmonella strain AD19 according to claim 1, it is characterised in that its 16S rDNA is SEQID No.1 institute
The nucleotide sequence shown.
3. in the method for the donkey abortion salmonella fire extinguishing vaccine of donkey source salmonella strain AD19 described in claim 1 preparation,
Including donkey salmonella strain AD19 described in claim 1 is expanded through the nutrient broth that peptone and beef extract powder are component formula
Bacterium, formalin-inactivated, steriling test prepare salmonella inactivated vaccine with the adjuvant emulsion of equivalent after safety verification is qualified.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111100817A (en) * | 2019-11-26 | 2020-05-05 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Equine abortion salmonella equine origin strain and application thereof in preparation of inactivated vaccine for equine abortion salmonella |
| CN111374094A (en) * | 2020-03-02 | 2020-07-07 | 扬州大学 | Method for establishing non-pregnant mouse uterine infection model caused by subcutaneous administration of donkey-derived salmonella abortus |
| CN113881589A (en) * | 2021-09-09 | 2022-01-04 | 山东省农业科学院畜牧兽医研究所 | Klebsiella pneumoniae KP21 strain and application thereof |
| CN114712528A (en) * | 2022-04-11 | 2022-07-08 | 江苏靶标生物医药研究所有限公司 | Method for evaluating toxicity of oncolytic bacteria on mice and application of method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111100817A (en) * | 2019-11-26 | 2020-05-05 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Equine abortion salmonella equine origin strain and application thereof in preparation of inactivated vaccine for equine abortion salmonella |
| CN111100817B (en) * | 2019-11-26 | 2021-07-16 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Equine abortion salmonella equine origin strain and application thereof in preparation of inactivated vaccine for equine abortion salmonella |
| CN111374094A (en) * | 2020-03-02 | 2020-07-07 | 扬州大学 | Method for establishing non-pregnant mouse uterine infection model caused by subcutaneous administration of donkey-derived salmonella abortus |
| CN111374094B (en) * | 2020-03-02 | 2022-04-19 | 扬州大学 | Method for establishing non-pregnant mouse uterine infection model caused by subcutaneous administration of donkey-derived salmonella abortus |
| CN113881589A (en) * | 2021-09-09 | 2022-01-04 | 山东省农业科学院畜牧兽医研究所 | Klebsiella pneumoniae KP21 strain and application thereof |
| CN114712528A (en) * | 2022-04-11 | 2022-07-08 | 江苏靶标生物医药研究所有限公司 | Method for evaluating toxicity of oncolytic bacteria on mice and application of method |
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| Publication number | Publication date |
|---|---|
| CN110511889B (en) | 2021-05-18 |
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