CN107354114A - One plant of haemophilus paragallinarum and its application - Google Patents
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Abstract
The invention discloses one plant of haemophilus paragallinarum and its application, described haemophilus paragallinarum is haemophilus paragallinarum (Haemophilus paragallinarum) HNHpg1, deposit number：CGMCC NO：13859, preservation date：On May 26th, 2017, depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address：BeiJing, China.Haemophilus paragallinarum bacterial strain HNHpg1 in the present invention be isolated from occurring typical respiratory symptom, Head And Face swelling laying hen socket of the eye under sinus secretion, with stable biological characteristics, there is stronger pathogenicity to chick, chick morbidity can be caused dead, and there is good immunogenicity.The vaccine safety prepared using the haemophilus paragallinarum bacterial strain HNHpg1 in the present invention is reliable, has preferable protecting effect to the infective rhinitis of chicken.
Description
Technical field
The present invention relates to field of molecular biotechnology, more particularly to one plant of haemophilus paragallinarum and its application.
Background technology
Haemophilus paragallinarum (Haemophilus paragallinarum, HPg) is Pasteurella section fowl Bacillus
(Avibacterium) a kind of short and small gram-Negative bacillus, it is infectious coryza of chicken (Infectious coryza, Ic)
Pathogen, cause the acute or subacute respiratory tract infection of chicken, clinical manifestation is sinus and upper tracheal inflammation under nasal cavity, socket of the eye, is flowed
Tear, flow nose liquid, expiratory dyspnea.Diseased chicken sneezing, shake the head, facial area unilateral or bilateral oedema.It is each that this disease is distributed widely in the world
Ground, Beach first reported the disease in nineteen twenty, and subsequent De Bliekc are separated to Hpg first.China 1987 is first in Beijing
It is secondary to be separated to this cause of disease, the report of this bacterium is separated on Hebei, Liaoning, Shandong and other places in succession.
Haemophilus paragallinarum can cause laying hen egg yield to decline, meat quality of table poultry decline, Young cock group's hypoevolutism be obstructed and
Mortality increase, larger economic loss is caused to poultry husbandry, has a strong impact on the development of poultry husbandry.Haemophilus paragallinarum has more
The bacterium, can be divided into 3 serotypes of A, B, C by kind serotype according to slide agglutination test.Can be with according to indirect hemagglutination inhibition test
It is divided into the serotype of sero-group 9 of A, B, C 3.Without cross-protection between the vaccine of different sero-groups, and same sero-group
There is obvious cross-protection between middle different serotypes.This sick incidence of disease is high, and big chicken is more susceptible than chicken, and the death rate does not surpass typically
Cross 20%.Economic loss is reduced mainly due to mortality increase and egg production.In China, the Ic incidences of disease are 20%~50%, extremely
Die rate 5%~20%.The course of disease can be shown in concurrent chicken virus mycoplasma disease or animal infectious diease to extend and death and culling rate rise.Epidemic disease
It is the important prevention and control measure of the disease that seedling is immune, should according to corresponding to the serotype selection of prevalence monovalent or polyvaccine.At present, urgently
A kind of new vaccine that there is the good bacterial strain of safety, immunogenicity to prepare is needed to prevent and treat the disease.
The content of the invention
In order to solve the above problems, it is an object of the invention to provide one plant of haemophilus paragallinarum and its application, bacterial strain poison
Power is strong, and the vaccine immunity of preparation is good.
To achieve these goals, the technical solution adopted in the present invention is：
One plant of haemophilus paragallinarum, described haemophilus paragallinarum are haemophilus paragallinarum (Haemophilus
Paragallinarum) HNHpg1, deposit number：CGMCC NO：13859, preservation date：On May 26th, 2017, preservation list
Position：China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address：BeiJing, China.
Described haemophilus paragallinarum is haemophilus paragallinarum A types.
A kind of application of haemophilus paragallinarum in terms of haemophilus paragallinarum inactivated vaccine is prepared.
The preparation method of described haemophilus paragallinarum inactivated vaccine is：By described haemophilus paragallinarum successively by training
After foster, harvest and inactivation obtain vaccinogen liquid, add adjuvant and produce vaccine.
Described adjuvant is aluminum hydroxide adjuvant.
The preparation method of described haemophilus paragallinarum inactivated vaccine is：Haemophilus paragallinarum is inoculated into the training of TSB liquid
Base is supported, is placed in 37 DEG C of shaking tables and cultivates, culture is collected after 24h, determines concentration, adjusts the bacterium of haemophilus paragallinarum in culture
Number is 2 × 109CFU/mL, the formaldehyde of culture volume fraction 0.2% is added, inactivate 12h in 37 DEG C, vaccinogen liquid is made；Again
Addition and the isometric aluminum hydroxide adjuvant of vaccinogen liquid, are mixed to prepare haemophilus paragallinarum inactivated vaccine.
Beneficial effects of the present invention：
1st, the haemophilus paragallinarum bacterial strain HNHpg1 in the present invention is isolated from that typical respiratory symptom, Head And Face swelling occurs
Laying hen socket of the eye under sinus secretion, when being separated on TSA solid mediums without other bacterial growths, only haemophilus paragallinarum is given birth to
Long, viral titer is high in TSB fluid nutrient mediums, up to 109More than CFU/mL.
2nd, the haemophilus paragallinarum bacterial strain HNHpg1 in the present invention has stable biological characteristics, has to chick stronger
Pathogenicity, chick morbidity can be caused dead, and there is good immunogenicity.
3rd, the vaccine safety prepared using the haemophilus paragallinarum bacterial strain HNHpg1 in the present invention is reliable, to the infectiousness of chicken
Rhinitis has preferable protecting effect.
Brief description of the drawings
Fig. 1 is bacterial strain HNHpg1 colonial morphologies.
Fig. 2 is form under bacterial strain HNHpg1 thalline 100 × microscopes of Gram's staining.
Fig. 3 is bacterial strain HNHpg1 PCR qualification results, and the Marker in figure is DL 2000bp, is followed successively by from top to bottom
2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp；1 is negative control, and 2 be primer P1, P2 to haemophilus paragallinarum A
The amplification of type vaccine strain C-Hpg-8 strains (CVCC254)；3 be the amplification of primer P1, P2 to bacterial strain HNHpg1.From figure
In it can be seen that, it was demonstrated that amplified fragments are purpose fragment, meet desired design size 860bp.
Embodiment
The embodiment of the present invention is described in further detail with reference to embodiments.
The separation and identification of embodiment 1, haemophilus paragallinarum
1.1 pathological material of diseases gather
Pathological material of disease comes from occurs expiratory dyspnea, head swelling in April, 2017 in the large-scale laying hen field in Henan Province Weihui City
Laying hen.
The preparation of 1.2 culture mediums
TSB fluid nutrient mediums：By TSB gravy powders (Tryptic Soy broth, pancreas peptone soybean broth) 30g, it is dissolved in
In 1000mL ultra-pure waters, 115 DEG C of sterilizing 15min, add hyclone 50mL, aseptic quality fraction 1%NAD (Nicotinamide
Adenine dinucleotide, NADH, cozymase) 1mL；
TSA solid mediums：By TSA agar powders (Tryptic Soy Agar, tryptose soya agar) 40g, it is dissolved in
In 1000mL ultra-pure waters, 115 DEG C of sterilizing 15min, add hyclone 50mL, aseptic quality fraction 1%NAD 1mL (as needed
It is added), saved backup in 4 DEG C.
1.3 bacteriums are separately cultured
Sinus secretion under the socket of the eye of aseptic collection diseased chicken, it is inoculated in containing on TSA solid mediums, in 37 DEG C of constant incubators
Cultivate 36h, grow consistent tip-like size, water white transparency, it is smooth, moistening, diameter be 1~2mm bacterium colony (see figure
1).Purify and be inoculated on the TSA solid mediums without NAD, can not be grown on the TSA culture mediums without NAD.Gram
Dyeing, the bacterium be Gram-negative bacillus pumilis, has a variety of different forms, from it is spherical, shaft-like or grow thread thalline (see
Fig. 2), it is named as bacterial strain HNHpg1.
The identification of 1.4 bacterial strains
1.4.1 primer is designed
According to a pair of universal primers of sequences Design of haemophilus paragallinarum 23S rRNA genes, for haemophilus paragallinarum
Amplification, primer sequence are as follows：
P1：5’-GTAACTATAACGGTCCTAAG-3’(SEQ ID NO.1)
P2：5’-CCCGCTTAGATGCTTTCAGC-3’(SEQ ID NO.2)
It is expected that amplified fragments size is 860bp.
1.4.2PCR identification
Operating procedure according to DNA extraction kit extracts bacterial strain HNHpg1 DNA, and spectrophotometric determination concentration is
100 μ g/mL, enter performing PCR identification.Meanwhile set haemophilus paragallinarum A type vaccine strain C-Hpg-8 strains (CVCC254) right for the positive
According to.
Pcr amplification reaction system is 25 μ L：The μ L of 10 × buffer solution, 2.5 μ L, 2.5mM dNTPs 0.5,10 μM/L is general to be drawn
Thing P1, P2 each the μ L of 1 μ L, 5U/ μ L rTaq 1, the μ L of 100 μ g/mL DNA profilings 1, add ddH2O to 25 μ L.
Reaction condition：95 DEG C of pre-degeneration 5min, into circulation：95 DEG C of 1min, 57 DEG C of 1min, 72 DEG C of 30s, totally 35 are followed
Ring, last 72 DEG C of extensions 10min, 4 DEG C of preservations of PCR primer.
Amplified production carries out electrophoresis respectively, the 10 μ L of sample-adding per hole, 1% agarose gel electrophoresis, and result is observed under ultraviolet light
(see Fig. 3).As a result 860bp fragments, sequencing and haemophilus paragallinarum A type vaccine strain C-Hpg-8 strains (CVCC254) are amplified
99.6% is homologous, and sequencing result is as follows, it was demonstrated that bacterial strain HNHpg1 is haemophilus paragallinarum (Haemophilus
paragallinarum)。
GTAACTAATAACGGTCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCGACCTGCACGAATGGCATAATGATGGC
CAGGCTGTCTCCACCCGAGACTCAGTGAAATTGAAATCGCCGTGAAGATGCGGTGTACCCGCGGCTAGACGGAAAGA
CCCCGTGAACCTTTACTATAGCTTGACACTGAACCTTGAATTTTGATGTGTAGGATAGGTGGGAGACTATGAAGCGG
TAACGCCAGTTATCGTGGAGTCGTTGTTGAAATACCACCCTTTAACGTTTGATGTTCTAACGAAGCGCCTGAAACGG
GTGTTCGGACAGTGTCTGGTGGGTAGTTTGACTGGGGCGGTCTCCTCCCAAAGTGTAACGGAGGAGCACGAAGGTTT
GCTAATGACGGTCGGACATCGTCAGGTTAGTGCAATGGTATAAGCAAGCTTAACTGCGAGACAGACAAGTCGAGCAG
GTGCGAAAGCAGGTCATAGTGATCCGGTGGTTCTGAATGGAAGGGCCATCGCTCAACGGATAAAAGGTACTCCGGGG
ATAACAGGCTGATACCGCCCAAGAGTTCATATCGACGGCGGTGTTTGGCACCTCGATGTCGGCTCATCACATCCTGG
GGCTGAAGTAGGTCCCAAGGGTTATGGCTGTTTCGCCATTTAAAGTGGTACGCGAGCTGGGTTTAGAACGTCGTTGA
GACAGTTCGGTCCCTATCTGCCGTGGGCGTTGGAGAATTGAGAGGGGGCTGCTCCTAGTACGAGAGGACCGGAGTGG
ACGCATCACTGGTGTACCAGTTGTCTCGCCAGAGGCACTGCTGGGGTAGCTAATTGCGGATGAGATAAGTGCTGAAA
GCATCTAAGCGGG(SEQ ID NO.3)
1.4.3 Serotype Identification
With slide agglutination test method, the HNHpg1 separation strains and A, B, the standard male of c-type haemophilus paragallinarum of purifying are taken
Property serum carry out agglutination test, the standard positive serum of separation strains and A type haemophilus paragallinarums occurs aggegation in 3~5min and showed
As, with B, c-type Haemohpilus paragallinaram standard positive serum without agglutinating reaction.Proof HNHpg1 is haemophilus paragallinarum A types.
1.5 virulence test
Experimental animal is 42 age in days chicken 20.Experimental animal is divided into two groups, control group 10, test group 10.Will
HNHpg1 is inoculated in TSB fluid nutrient mediums, after 37 DEG C of shaking table culture 24h, determines cell concentration, continuous 10 times of dilution spread solids
Flat board, count plate under low power lens, culture stoste cell concentration is calculated, is adjusted to 108CFU/mL, test group animal pass through nose
Interior inoculation 0.1mL haemophilus paragallinarum HNHpg1 liquid cultures, the TSB that control animals are sterilized by intranasal vaccination 0.1mL
Fluid nutrient medium.Continuous Observation 2 weeks after injection, situations such as observing and record its morbidity number and death toll.
Test group animal shows mouth breathing after haemophilus paragallinarum HNHpg1 24h are inoculated with, and flows clear nose liquid, gradually
Sinus swelling under unilateral or both sides face, eyelid, socket of the eye, conjunctiva inflammation, nose have the cheesy secretion accumulation of glutinous purulence, foul smelling
Taste.The appearance opacity of the cornea blindness of course of disease length, hat color is pale, and spirit is depressed, and feather is fluffy, and necking down is hung down wing.Some inserts head
Under wing, squat in an a corner, appetite is useless exhausted, drinks water once in a while.The obvious oedema of wattle, inflammation of upper respiratory tract spread to tracheae and lungs
When, expiratory dyspnea and noise is presented.Dead in 3~5d, a small number of courses of disease are dragged to l~2 week, and useless food of finally blinding dies of exhaustion.3d
Dead 3 afterwards, dead 5 after 7d.Survival 2, which is grown, is obstructed.Control animals body temperature between experimental period is normal and without bright
Aobvious respiratory symptom.Cutd open after off-test and kill test group animal, while cutd open and kill 10 control animals.Pathological material of disease is gathered, is carried out
Haemophilus paragallinarum separates.Test group cut open inspection nasal cavity and sinus mucosa are in acute catarrhal inflammation, and congested swelling, surface is covered with greatly
Mucus is measured, has diffusate or caseous necrosis thing in nasal sinus.Pinkeye, conjunctival congestion, swelling, face's subcutaneous dropsy.
Obvious pathological change does not occur for control group cut open inspection, and 10 parts of pathological material of diseases of control group are not separated to haemophilus paragallinarum, experiment
Haemophilus paragallinarum is separated in 10 parts of pathological material of diseases of group, PCR qualification results are consistent with HNHpg1.
Embodiment 2, the preparation of vaccine, security and potency test
The preparation of 2.1 vaccines
Haemophilus paragallinarum bacterial strain HNHpg1 is inoculated into TSB fluid nutrient mediums, is placed in 37 DEG C of shaking tables and cultivates, after 24h
Culture is collected, determines cell concentration, the bacterium number for adjusting haemophilus paragallinarum in culture is 2 × 109CFU/mL, per 1000mL
Culture adds 2mL formalins (mass fraction 37%), inactivates 12h in 37 DEG C, vaccinogen liquid is made；Add and vaccinogen liquid
Isometric aluminum hydroxide adjuvant (Sigma companies, F5881), is mixed to prepare haemophilus paragallinarum inactivated vaccine, the pair in vaccine
The bacterium number of haemophilus gallinarum is about 1 × 109CFU/mL。
The safety testing of 2.2 vaccines：
42 age in days healthy chicken 20 is chosen, is divided into 4 groups, every group 5.3 groups of vaccine group, every group of difference neck are subcutaneously injected
This vaccine of 0.5mL, 1mL, 2mL；2mL sterile salines are subcutaneously injected in 1 group of control group, neck.Clinical manifestation is observed, altogether observation
2 weeks.During observation, the chicken of vaccine group and control group breathing, appetite, the state of mind during whole observation is all normal, and be averaged body
Temperature rise is no more than 1 DEG C, has no the adverse reactions such as lassitude, feeding reduction, runny nose, expiratory dyspnea, eyelid swelling.Illustrate this
The inactivated vaccine safety of invention.
The potency test of 2.3 vaccines
45 age in days healthy chicken 100 is chosen, is divided into 5 groups, every group 20.Specially：Vaccine 1-4 groups：Every group is injected respectively
This vaccine 0.1mL, 0.2mL, 0.3mL and 0.5mL, the 5th group is non-immune group, injects 0.5mL sterile salines.Vaccine group and
The non-age in days two of immune group chicken 120 exempts to inject the vaccine and sterile saline of Isodose.Two exempt from rear 30d with 107CFU's
HNHpg1 takes the malicious mode of attacking of nasal injection to carry out attacking poison.Attack after poison and in 14d, observe the clinical symptoms of all chickens.Attacking poison
Afterwards after 14d, cut open inspection is carried out to all chickens, it is determined that attacking malicious protective rate, it is thermophilic to gather the secondary chicken of the organ such as sinus secretion, lungs progress under socket of the eye
Blood bacillus is separately cultured.
Vaccine group has obvious difference after attacking poison with non-immune group chicken, and after attacking poison, non-immune group starts to face for second day
Bed symptom, runny nose, coughs, breathes, mouth breathing, subtracting the maximum phase of an eclipse to going on a hunger strike, and starts dead, death altogether after 2 weeks occur after attacking malicious 2d
16, all morbidities.Cut open inspection result：Nasal cavity and sinus mucosa are in acute catarrhal inflammation, have diffusate or cheesy in nasal sinus
Sphacelus.And in vaccine group, there are 4 clinical symptoms and case change occur in 20 chickens of 0.1mL vaccine groups；0.2mL vaccines
There are 2 clinical symptoms and case change occur in 20 chickens of group；20 chickens of 0.3mL and 0.5mL vaccine groups do not fall ill.Exempt from
Epidemic disease is attacked malicious protection situation and see the table below.
Note："-" represents inapplicable.
As can be seen from the above table, the minimum immunoprotection dosage of this vaccine is 0.1mL, and bacteria containing amount is 1 × 109CFU/mL。
Illustrating the inactivated vaccine of the present invention has good protecting effect.
The optimal embodiment of the present invention is the foregoing is only, for those skilled in the art, the present invention can have
Various modifications and variations.Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., all should
Within protection scope of the present invention.
SEQUENCE LISTING
<110>Animal Busbandry ＆. Veterinary Medicine Inst., Henan Prov. Academy of Agriculture
<120>One plant of haemophilus paragallinarum and its application
<160> 3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
gtaactataa cggtcctaag 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
cccgcttaga tgctttcagc 20
<210> 3
<211> 860
<212> DNA
<213>Haemophilus paragallinarum（Haemophilus paragallinarum）
<400> 3
gtaactaata acggtcctaa ggtagcgaaa ttccttgtcg ggtaagttcc gacctgcacg 60
aatggcataa tgatggccag gctgtctcca cccgagactc agtgaaattg aaatcgccgt 120
gaagatgcgg tgtacccgcg gctagacgga aagaccccgt gaacctttac tatagcttga 180
cactgaacct tgaattttga tgtgtaggat aggtgggaga ctatgaagcg gtaacgccag 240
ttatcgtgga gtcgttgttg aaataccacc ctttaacgtt tgatgttcta acgaagcgcc 300
tgaaacgggt gttcggacag tgtctggtgg gtagtttgac tggggcggtc tcctcccaaa 360
gtgtaacgga ggagcacgaa ggtttgctaa tgacggtcgg acatcgtcag gttagtgcaa 420
tggtataagc aagcttaact gcgagacaga caagtcgagc aggtgcgaaa gcaggtcata 480
gtgatccggt ggttctgaat ggaagggcca tcgctcaacg gataaaaggt actccgggga 540
taacaggctg ataccgccca agagttcata tcgacggcgg tgtttggcac ctcgatgtcg 600
gctcatcaca tcctggggct gaagtaggtc ccaagggtta tggctgtttc gccatttaaa 660
gtggtacgcg agctgggttt agaacgtcgt tgagacagtt cggtccctat ctgccgtggg 720
cgttggagaa ttgagagggg gctgctccta gtacgagagg accggagtgg acgcatcact 780
ggtgtaccag ttgtctcgcc agaggcactg ctggggtagc taattgcgga tgagataagt 840
gctgaaagca tctaagcggg 860
Claims (6)
1. one plant of haemophilus paragallinarum, it is characterised in that：Described haemophilus paragallinarum is haemophilus paragallinarum
(Haemophilus paragallinarum) HNHpg1, deposit number：CGMCC NO：13859, preservation date：In May, 2017
26 days, depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address：BeiJing, China.
2. haemophilus paragallinarum according to claim 1, it is characterised in that：Described haemophilus paragallinarum is that secondary chicken is bloodthirsty
Bacillus A types.
A kind of 3. haemophilus paragallinarum as claimed in claim 1 or 2 answering in terms of haemophilus paragallinarum inactivated vaccine is prepared
With.
4. application according to claim 3, it is characterised in that the preparation method of described haemophilus paragallinarum inactivated vaccine
For：By described haemophilus paragallinarum successively after cultivating, harvesting and inactivation obtains vaccinogen liquid, add adjuvant and produce epidemic disease
Seedling.
5. application according to claim 4, it is characterised in that described adjuvant is aluminum hydroxide adjuvant.
6. application according to claim 5, it is characterised in that the preparation method of described haemophilus paragallinarum inactivated vaccine
For：Haemophilus paragallinarum is inoculated into TSB fluid nutrient mediums, is placed in 37 DEG C of shaking tables and cultivates, culture is collected after 24h, is determined
Concentration, the bacterium number for adjusting haemophilus paragallinarum in culture are 2 × 109CFU/mL, add the first of culture volume fraction 0.2%
Aldehyde solution, 12h is inactivated in 37 DEG C, vaccinogen liquid is made；Add the aluminum hydroxide adjuvant isometric with vaccinogen liquid, mixing system
Obtain haemophilus paragallinarum inactivated vaccine.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108977399A (en) * | 2018-09-13 | 2018-12-11 | 陕西理工大学 | One plant of Bacillus foecalis alkaligenes and its application |
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