CN1128214C - Cloned weakening strain of chicken virus mycoplasma - Google Patents
Cloned weakening strain of chicken virus mycoplasma Download PDFInfo
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Abstract
The present invention provides a cloned low virulent strain of mycoplasma gallisepticum. The strain is formed by three times of solid cloning, acellular culture and weakening of S6 strain to over 168 generations, and a low virulent strain with immunogenicity is formed. The strain has the advantages of safety, no strong recovery, easy detection and strong immunogenicity, and can effectively prevent chronic respiratory track diseases of chicken. The bacterial strain is preserved in the Common Microorganism Center of the China Committee for Culture Collections of Microorganisms, and the preservation number is CGMCC No. 0397.
Description
Technical field:
The invention belongs to the new bacterial strain of microorganism.
Background technology:
(Mycoplasma gallisepticum is that (ChronicRespiratory Disease of Chicken, main pathogen CRD) draw attention with serious economy loss because of it is pathogenic widely chronic respiratory disease MG) to chicken virus mycoplasma.In recent years, the research of MG is from the pathogen separation of routine, serological test, and immunity and medicine control develop into molecular biology research, make people from more in depth having understood the attribute of MG in essence, and provide new technology for the control of MG infection.Isolating mycoplasma has 20 kinds more than from poultry, but harm chicken group mainly contain 3 kinds: MG, Synovial sac mycoplasma (M.Synoviae.MS), mycoplasma meleagridis (M.Meleagritis MM).MS different isolates virulence differs greatly, and major lesions is in Synovial sac, stndon sheath and joint, and what also have causes the minimizing of laying eggs.MM causes big head disease of turkeys and turkey airsacculitis.The harm maximum is that MG infects, and the symptom that it causes shows respiratory system, and major lesions is at air bag.This disease is distributed in all over the world, and mortality ratio is not high, and minority can reach 20%-30%, and sickness rate makes young chicken poor growth up to more than 90%, and the minimizing of laying eggs causes heavy losses.External many heavy chicken houses of planting have all been eliminated this disease.According to 25 the chicken house investigation in 7 cities, nineteen ninety Jiangsu Province, positive rate reaches 48.5%, and CRD has become one of four big diseases of raising chickens with Marek, fabricius bursa, the arranged side by side important threat of the minimizing syndromes (EDS-76) of laying eggs at large-scale chicken house at present.
The MG thalline is tiny, oval, the about 0.25-0.5 micron of diameter, Gram-negative, Ji's nurse Sa Albert'stain Albert and Wright's staining are painted good, the energy glucose fermentation, nutritional requirement is higher during cultivation, on solid medium, formed the translucent small colonies of smooth circle in 5-6 days, diameter 0.2-0.3 millimeter, the center has projection slightly.
The inoculation of 7 age in days chick embryo yolk sacs, part are dead after 5-7 days, and produce the characteristic pathology: the cheesy exudate of respiratory tract, abscess of joint, hepatosplenomegaly, pericarditis, hydrosarca, hepatic necrosis, continuous passage death rule more is obvious, and toxic amount is the highest in yolk sac and the chorion in embryo.
Cause of disease exists more at the infected chicken upper respiratory tract, also can be separated in lower respiratory tract, air bag, ovary, the uterine tube.Energy aggegation chicken red blood cell, infected chicken has hemagglutination inhibition antibody, so available HI diagnoses this disease.
The cause of disease resistibility is not strong, and survival is 1-3 days in 20 ℃ of chicken manures, can survive for 8 weeks for 37 ℃ in the yolk, and heat is had certain resistibility, can survive 1 hour for 45 ℃, but under the low temperature in the dust of hen house, ight soil, feed or drinking-water long-term survival.MG freeze-drying tissue can be survived 14 years for 4 ℃.General Herb of Common violet all can kill it rapidly, thaliium acetate (1: 4000) to penicillin, Xin Meisu, many glutinosins, sulfa drugs and lower concentration has resistibility, to Streptomycin sulphate and other many Broad spectrum antibioticss such as sensitivities such as terramycin, duomycin, paraxin, kantlex and erythromycin, positive chicken house uses tylosin, doxycycline, sharp proceomycin, lincomycin (Linco-spectin 100), (Gallimycin) and Stereomycin etc. all have better curative effect earlier for former (safe wonderful clever Tiamulin), high-tensile strength rice only.
Because MG has only a serotype, be anti-system CRD, foreign study many vaccine strains, as the G250 strain of Japan, the CP strain of France, the F strain of the U.S., Australian Ts strain, but all have some problems.The S6 strain is the mesogen strain of MG, and domestic immunopathology test proves that also its virulence is not strong, and immunogenicity is relatively poor.
Summary of the invention:
The purpose of this invention is to provide strain chicken virus mycoplasma clone low virulent strain, this strain is caused weak to more than 168 generations by three solid clones of S6 strain acellular cultivation in back, be formed with immunogenic less-virulent strain.This strain safety is not returned by force, easily detects, and stronger immunogenicity is arranged, and can effectively prevent chronic respiratory disease.
Purpose of the present invention can reach by following measure:
Mycoplasma (Mycoplasma, M. also claim mycoplasma), the earliest by Nocard and Roux (1898) by being separated in the contagious bovine pleuropneumonia case, first mycoplasma species is that thread mycoplasma ox type subspecies (M.mycoidessubsp.bovi/M.mycoides subsp.mycoides) are separated the back because its character is difficult to define, and satisfies this big quasi-microorganism called after pleuropneumo-nia-like organisms(PPLO) (PPLOs).Middle fifties, the beginning is used this title of mycoplasma.1967, Edward and Frundt suggestion was branched away it by Schizomycetes, set up a new guiding principle separately: mantle body guiding principle (Mollicutes soften film Gammaproteobacteria, moccasin body guiding principle), and mycoplasma is different from Schizophyceae because of lacking photosynthetic pigments (chlorophyll); Lack hard cell walls and cell wall components, be different from Schizomycetes; Can carry out acellular cultivation and be different from filtrable virus.It is minimum prokaryotic organism that can self-replacation, and the genome size has only the 20-40% of typical bacteria.International bacterial system classification mantle body guiding principle classification branch of the council (ICSB-ISTM) divides four orders with mantle body guiding principle: order I: and Mycoplasmas (Order I, Mycoplasmatales); Order II: insect substance order (OrderII, Entomoplasmatales); Order III: the acholeplasma order (OrderIII, Acholoplasmatales); Order IV: anaerobism substance order (OrderIV, Anaeroplasmatales).Mycoplasmas divide into a section be Mycoplasmataceae (FamilyI, Mycoplasmataceae), section built-in two belongs to: Mycoplasma (Mycoplasma) has 120 kinds in the genus; Urine Ureaplasma (Ureaplasma) has 6 kinds in the genus, this two genus of common animal Mycoplasma.
The classification position of chicken virus mycoplasma (Mycoplasma gallisepticum) JS99 cloned weakening strain is:
Prokaryota (Procaryotae)
Tenericutes (Teneribacteria)
Mantle body guiding principle (Mollicutes)
Mycoplasmas (OrderI, Mycoplasmatales)
Mycoplasmataceae (FamilyI, Mycoplasmataceae)
Mycoplasma (Mycoplasma)
Chicken virus mycoplasma (Mycoplasma gallisepticum)
The chicken virus mycoplasma whole world so far has only a kind, and its type strain is: and PG31 (ATCC19610, NCTC10115), S6, A5969.
Chicken virus mycoplasma JS99 cloned weakening strain proves the Mycoplasma member through serial biochemical test, and it is with a kind of with chicken virus mycoplasma S6 strain that growth inhibition test detects the serotype proof.
This bacterial strain is deposited in the China Committee for Culture Collection of Microorganisms common micro-organisms center of BeiJing ZhongGuanCun on May 24th, 1999, the preservation proposal name is " chicken virus mycoplasma JS99 clone strain " (this patent name is called " cloned weakening strain of chicken virus mycoplasma "), and deposit number is: CGMCC NO.0396.This strain is caused weak to more than 168 generations by three solid clones of S6 strain acellular cultivation in back, be formed with immunogenic less-virulent strain.This strain safety is not returned by force, easily detects, and stronger immunogenicity is arranged, and can effectively prevent chronic respiratory disease.
Embodiment:
Below cultivation of the present invention and evaluation are further described:
One, acellular cultivation culture medium prescription commonly used
A.
OX-heart soup 1000ml
Niacinamide 30mg
Glucose 10g
Going out can horse serum 200ml
Phenol red 25mg
25% yeast leach liquor 40ml
PH 8.0
*Solid medium 1% agar
B
OX-heart digestion soup 1000ml
Milk protein hydrolysate 5g
NaCl 5g
25% yeast leach liquor 50-100ml
NAD(COI) 0.02g
Phenol red 0.01-0.02g
Arginine 0.1-0.2g
VITAMIN (100 *) 10ml in the Eagles subsistence level nutrient solution
Porcine blood serum 150ml
Thaliium acetate 0.25g
Penicillin 1000u/ml
PH7.2
*Solid medium adds 1% agar, and it is essential that NAD cultivates by MS
C. Gai Liang Hayflick substratum
PPLO meat soup (Difco) 70ml
Horse or porcine blood serum 15-20ml
20% yeast leach liquor 5ml
Glucose 1g
Arginine 0.2g
1% phenol red 0.1-0.2ml
Thaliium acetate (1: 80) 1ml
Penicillin 1000u/ml
D.
OX-heart soaks juice 75ml
Go out and to be good for porcine blood serum 12.5ml
Fresh yeast immersion liquid 1.3ml
1% phenol red 0.2ml
0.4% thaliium acetate 1ml
PH7.2
*OX-heart soaks juice and contains 1% glucose, 0.5%NaCl, and 0.5% polyprotein peptone (Polypeptone) preserved behind the autoclaving for 10 pounds in 10 minutes, and solid medium adds water 75ml dissolving with pplo agar 3.5g, and other adds the 15ml porcine blood serum, the immersion liquid of 1.3ml fresh yeast.
E Friis improves substratum:
Get grow up strong clean muscle of rabbit and rabbit cardiac muscle 125 grams, grind, boiled cold filtration, 1000 milliliters of supernatants 30 minutes, add 0.2% glucose (A.R.), 20% go out can health pig serum, 2.5% fresh yeast diffusion juice, all material low temperature are down proofreaied and correct its pH to 7.8 with 1N NaOH solution, after Φ 0.1 μ l minipore filter filtered and removes bacterium and mycoplasma, packing was stored in 4 ℃ of refrigerators standby.
Two, chicken virus mycoplasma JS99 cloned weakening strain authentication method
A Wright Stain microscopy: after smear, fixing, the drying, Wright's staining is put 1600 times in microscopically oil mirror, and visible dispersive has specific polymorphic thalline in a large number.
B solid bacterium colony: chicken virus mycoplasma JS99 cloned weakening strain culture does 10
6-10
10After the dilution, be inoculated in the PPLO Agar solid plate 7%CO
2Cultivate 4-8 days visible specificity solid bacterium colonies for 37 ℃, umbilical is obvious, and chicken red blood cell is had stronger blood clotting.
The present invention compared with prior art has the following advantages:
1, virulence has security a little less than, and has better immunogenicity;
2, solid bacterium colony umbilical is obvious, and blood clotting is strong, is easy to detect;
3, continuously on chicken 7 generations return and not return by force,, there is immunogenicity in 150-170 generation.
Its test-results is as follows:
One, cause weak and the safety testing result
1. materials and methods
1.1 test materials
1.1.1 substratum: prepare Friis, KM according to a conventional method
2, OX-heart soup substratum and PPLO solid medium.
1.1.2 test strain: chicken virus mycoplasma JS99 cloned weakening strain contrast bacterial classification: MG W3 street strain, this chamber is by separating in the sick chicken of CRD, and warp is made growth-inhibiting with standard serum, the agglutination inhibition test assay certificate is MG.
1.1.3 test chicken is provided by eastern suburb, Nanjing kind chicken house, the SPF dawn is provided by Agricultural College Affiliated to Yangzhou Univ..
1.2 bacterial classification clone
Chicken virus mycoplasma JS99 cloned weakening strain is behind multiple shape on the Friis substratum, supernatant is done 10 (6)～10 (9) dilutions (containing 0～10 (2) ccu/ml), press 0.5ml/ ware inoculation solid plate, behind 37 ℃ of anaerobic culture 36h-72h, choose single colony clone under the low power lens, after the propagation, repeat above-mentioned steps on the liquid medium within.Culture behind three time clonings is accredited as MG with standard serum.
1.3 return test continuously
Chicken virus mycoplasma JS99 cloned weakening strain F108 culture is inoculated 1 age in days parts in Beijing opera spoken in Beijing dialect chick by 0.5ml/ eye droppings, collunarium, carrying out MG to 15 age in days aseptic collection test chicken lungs, tracheae, air bag sample separates, after isolate was identified, the same method repeated, and finished three these animals of MG and returned.Isolate F126 makes 10 (3) dilutions, yolk sac inoculation 9 age in days SPF embryos, every 0.1ml for the third time.The chicken embryo is hatched the back by separating mycoplasma in chick tracheae, lung, the air bag, and isolate inoculates the SPF embryo after identifying, realizes continuous cubic regression in the chicken embryo again.Obtain at last through six regressive chicken virus mycoplasma JS99 cloned weakening strain F141 cultures of this animal.
1.4 the pathogenic test of tracheal ring
Aseptic operation is taked a Japanese instar chickling tracheae, is cut into the wide tracheal ring of 0.5mm behind the Hank ' s liquid fine laundering, is sub-packed at random in the bottle that contains pH7.5 MEM nutrient solution, adds MG dilution culture 0.1ml (10 (4) ccu/ml).37 ℃ of cultivations, every day, microscopy write down the ciliary movement percentage, and test is divided into four groups, and 4 bottles every group, every bottle 5 ring.
1.5 the pathogenic test of fryer
40 of 6 aa broiler chicken, sampling Detection HA negative antibody.Be divided into three groups at random, press 0.5ml/ eye droppings, collunarium inoculation chicken virus mycoplasma JS99 cloned weakening strain F150, chicken virus mycoplasma JS99 cloned weakening strain F144 (the 6th recurrence culture), the wild poison of W3 and MG substratum respectively, HA antibody is surveyed in blood sampling after 14 days, checks clinical symptom and air bag pathology simultaneously.
1.6 immunoreactivity test
7 age in days HA detect 30 negative of Hai Saikesi chick and are divided into 3 groups at random, every group 10, first group of left side torso bag injection chicken virus mycoplasma JS99 cloned weakening strain F153 culture 0.5ml, second group of eye droppings, collunarium chicken virus mycoplasma JS99 cloned weakening strain F153 culture 0.5ml, the 3rd group of blank, 45 ages in days are checked HA antibody, and observe clinical symptom and air bag pathology.
2. result
2.1 return test
Table one chicken virus mycoplasma JS99 cloned weakening strain F108 returns test-results six times continuously
Annotate: (1) one routine tracheae, lung, air bag sample all pollute, and separation has no resolution.
| Return number of times | The inoculum generation | Return object | MG separation rate test group control group | Airsac disease rate test group control group |
| 1 2 3 4 5 6 | F108 F113 F121 F126 F134 F136 | 1 Japanese instar chickling, 1 Japanese instar chickling, 1 Japanese instar chickling, 9 age in days SPF embryos, 9 age in days SPP embryos, 9 age in days SPP embryos | 6/6 0/3 6/6 0/3 5/6 (1) 0/3 4/4 (2)3/3 (2)5/5 | 0/6 0/3 0/6 0/3 0/6 0/3 |
(2) there is 1,2 piece of SPF embryo dead in hatching respectively.
Return six strain MG:F113, F121, F126, F134, F136, the F141 (result such as table one) that test has obtained different recurrence generations continuously six times that last 2 years, 3 zoogenetic infection tests, from the chicken body, all isolate MG, but do not cause the air bag pathology, the preliminary chicken virus mycoplasma JS99 cloned weakening strain F108 that shows, F113, F121 is safe to chick.The 6th time recurrence strain isolated F144 also proves there is no through the pathogenic test of fryer and returns strong phenomenon (seeing Table three).This test shows, the above generation culture of chicken virus mycoplasma JS99 cloned weakening strain F108 can be got rid of it during natural infection and return strong possibility in the chicken group.
2.2 different strain tracheal ringes are cultivated pathogenic test
By table two as seen, tracheal ring ciliary movement in the MEM substratum kept 9 days, proved that the tracheal ring cultivation is reliable.To the restraining effect of ciliary movement, street strain is the strongest, and weak malicious cubic regression strain is taken second place, chicken virus mycoplasma JS99 cloned weakening strain F137 minimum.Both differences of back are little, but compare obvious difference with the former.
2.3 the pathogenic test of fryer
By table three as seen, chicken virus mycoplasma JS99 cloned weakening strain F150 heavy dose (10 (8)～10 (9) ccu) infects chick and does not cause clinical symptom and air bag pathology, still very safe through the recurrence strain after the propagation of this animal three sub-levels and three vertical transmissions continuously, and street strain can not only cause slight clinical symptom, also has tangible air bag pathology.20 age in days HA antibody tests show that also chicken virus mycoplasma JS99 cloned weakening strain F150 has stronger immunogenicity.
The different strains of table two are cultivated effect on ciliary movement to tracheal ring
Annotate: the long-pending aggregate-value of (1) ciliary movement ratio and motion fate.
| Infection strain different ratios ciliary movement fate 50% 25% 10% | The ciliary movement total points (1) | The total fate of ciliary movement |
| Cloned weakening F137 05 (2)2 cloned weakening F121 (3)005 MEM of 042 W3 street strains contrast 243 | 1.45 1.20 0.50 2.30 | 7 6 5 9 |
(2) expression can be observed average every ring (10 ring cumulative mean) in continuous 5 days 25% above ciliary movement.
Return strain isolated (3) 3 times
Table three fryer pathogenicity test results
Annotate: the standards of grading of (1) air bag pathology :-, air bag is transparent; +, slight muddy; ++, the thick spot of the big yellow-white of minority small rice grain; The cheesy thing of +++, strip and block yellow-white.Air bag pathology both sides have ++ or a side has ++ and+above test chicken is judged to the chicken of causing a disease.Return culture (2) six times
| Inoculation content HA antibody positive/test chicken 6 ages in days 20 ages in days | Clinical manifestation | The air bag pathology (1) - + ++ +++ |
| Cloned weakening F150 0/5 10/10 cloned weakening F144 (2)0/5 10/10 W, 3 wild poison 0,/10 8/10 blanks 0/5 1/10 | Normal slight expiratory dyspnea is normal | 10 8 2 2 1 6 1 10 |
2.4 immunogenicity test
Left side torso bag inoculation and eye droppings, collunarium infected chicken, 45 age in days HA check and all are strong positive (10/10,10/10) that do not have tangible clinical symptom, the air bag pathology all is negative.Blank HA8/10 feminine gender.The result shows that MG cloned weakening F153 has stronger immunogenicity, also shows that it is a safety, low strain simultaneously.
Two, immunogenicity test-results
1 materials and methods
1.1 test materials
1.1.1 a test strain chicken poison strain body JS99 cloned weakening strain F156, virulent strain MG W3, virulent strain MGHS2, weak poison of NDV IV system and the weak poison of IBV H52.
1.1.2 the Experimental Animal Center development of transparent plastics shield retaining Beijing Agricultural University, shield retaining is placed in constant temperature, the airfiltering uv sterilisation room through the Peracetic Acid sterilization during test.
1.1.3 ammoniacal liquor 25～28%, the AR level, chemical building material factory of Nanjing chemical company produces.
1.1.4 test chicken with raise condition SPF Leghorn, provide kind of an egg by academy of agricultural sciences, Shandong poultry institute, behind the sterilization eggshell, hatching 20d, Peracetic Acid is sterilized and is moved into hatching in the shield retaining behind the eggshell; Go out shell in the 48h and well-grown chick makes test chicken, shield retaining is raised, raising condition: 25～29 ℃ of temperature, humidity 60～75%; Feed is that common compound is distributed into pouch, every bag of 1～2kg, Co 60 irradiation sterilizations of 250md dosage; Drinking-water is the autoclaved tap water of steam.
1.2 the concentration of inducible factor ammonia is selected test
After closing the shield retaining blower fan, in shield retaining sterilization add 2,4,6,8 in the plate respectively, 10ml ammoniacal liquor, behind the volatilization 15min, add a cover the taking-up plate, measure volatile quantity, and calculate ammonia concentration in the shield retaining.Ammonia concentration [1/1,000 000 (massfraction)] in the shield retaining=ammoniacal liquor volatile quantity (ml) * 100 shield retaining volume (m
3)
Keep 2h, observe the reaction of 6 SPF chickens.
1.3 7,0/1,000 000 (massfraction) ammonia environment is to chicken virus mycoplasma artificial onset's influence
10 of 20 age in days test chickens are divided into 3 groups, first group 4, measure MG and attack poison after 7,0/1,000 000 (massfraction) ammonia is post-stimulatory pathogenic; Second group 4, measure MG and directly attack pathogenic behind the poison; The 3rd group 2, do not attack the poison contrast through the ammonia stimulation.
Attack malicious method: MG W3F 11 and HS2F5 24h culture is mixed by 1: 1, attack malicious chicken eye droppings, collunarium 0.2ml, left torso bag injection 0.2ml.Ammonia stress test 6h after attacking poison carries out, and inhales the 6ml ammonia soln and volatilizees in plate, and ammonia concentration reaches 7,0/1,000 000 (massfractions) in the surplus 5.6ml behind the 15min, shield retaining, keeps 2h, carries out every day 2 times, continuously 4d.Attack poison back 12 days, and slaughtered test chicken, cut open inspection and record air bag pathology.
1.4 the weak poison of NDV, IBV is induced CRD artificial onset test
10 of 20 age in days SPF chickens are divided into three groups, and first group of 4 collunarium inoculated 10 part NDV and the weak malicious seedling of IBV, carries out the MG artificial challenge behind the 10min; Second group 4, only attack poison as MG; The weak malicious seedling of the 3rd group of 2 collunariums inoculation 10 part NDV, IBV is not attacked the poison contrast, attacks poison back 12d and slaughters test chicken, cuts open inspection record air bag pathology.
1.5 the weak poison of NDV, IBV induces the combined action with the ammonia environmental stimulus that the CRD artificial onset is influenced test
30 13 of age in days SPF chickens are divided into three groups, and first group 7 are carried out the weak poison of NDV, IBV and induce (with 1.4) and MG artificial challenge, and stimulate down at 70/1000,000 (massfraction) ammonia environment; Second group 4, only attack poison as MG; The 3rd group 2 are carried out that the weak poison of NDV, IBV is induced and ammonia stimulates, and do not attack the poison contrast, morbidity check together 1.3.
1.6 chicken virus mycoplasma JS99 cloned weakening strain F156 culture immunogenicity test
Chicken virus mycoplasma JS99 cloned weakening strain F156 24h culture (10 (9)～10 (11) ccu) is inoculated 3 age in days SPF chickens by 0.5ml/ eye droppings, collunarium, day by day observe to face after the immunity and examine performance, and in 10d, 20d, the 30d sample of drawing blood, (9) do MG HA and HI detection to serum sample according to a conventional method, the immune chicken in blood sampling back, contrast chicken change another shield retaining over to behind the mark respectively in batches, attack poison by 1.5 first group of methods, day by day observe clinical symptom after attacking poison, compel behind the 12d to kill, dissect record air bag pathology.
2 results
2.1 the reaction of SPF chicken is observed under the different ammonia concentration environment
The results are shown in Table 1.By table 1 as seen, 3,0/1,000 000～5,0/1,000 000 (massfraction) ammonia concentration can not cause lasting tangible stress, and 8,5/1,000 000 above ammonia concentration can cause serious injury chicken, even shock, death, 7,0/1,000 000 ammonia concentration can be used as suitable stressed condition.
The stress reaction of test chicken under the different ammonia concentration of table 1
The reaction of ammonia concentration (1/1,000 000 (massfraction)) test chicken
Closed order at 30 o'clock, and drank water, search for food, movable normal
Closed order at 50 o'clock, peck cage, diet is normal substantially
70 photophobia, normally closed eye, idol has cough, squats sometimes, and diet reduces
85 photophobia are got rid of head, cough, and the later stage is extruded into a pile, can not struggle
100 eyelids close, expiratory dyspnea, shock
2.2 7,0/1,000 000 (massfraction) ammonia environment is to MG artificial onset's influence
The results are shown in Table 2.By table 2 as seen, simple ammonia stimulates can not cause test chicken clinical symptom and air bag pathology, attack poison with MG merely and also only cause slight air bag pathology, and the test chicken major part is fallen ill (3/4) after increasing the ammonia stress, and show certain symptom.
2.3 the weak poison of NDV, IBV is induced CRD artificial onset influence
The results are shown in Table 3.By table 3 as seen, poison is induced a little less than NDV, IBV, MG artificial challenge's pathogenic obvious enhancing, and 2/4 can be judged to the morbidity chicken, and attack rarely seen 3/4 test chicken of poison merely slight air bag pathology is arranged, and does not attack poison contrast chicken air bag and does not have the pathology variation
Table 2 7,0/1,000 000 (massfraction) ammonia environment is to MG artificial onset's influence
| Group | The test chicken number of elements | Content of the test | Attack the toxication shape | The air bag pathology * - + ++ +++ |
| 1 2 3 | 4 4 2 | Attack poison, 7,0/1,000 000 (massfraction) ammonia stress only be attacked poison and do not attacked poison, and 7,0/1,000 000 (massfraction) ammonia stress | The slight expiratory dyspnea of 2/4 performance is normal | 5 1 3 1 3 2 |
*The judgment criteria of air bag pathology :-air bag is transparent; + slight muddy; ++ the thick spot of minority sesame size yellow-white; +++strip and block yellow-white has in junket sample thing air bag pathology both sides ++ or a side has ++ and+above test chicken is judged to the morbidity chicken.
2.4 weak poison is induced and the influence of ammonia environmental stimulus combined action to the MG artificial challenge
The weak poison of table 3 NDV, IBV is induced the influence to the CRD artificial onset
| Group | Test chicken | Content of the test | The air bag pathology * |
| 1 2 3 | Number of elements 442 | The weak poison of NDV, IBV is induced, and MG attacks malicious MG and attacks the weak poison inoculation of malicious NDV, IBV | - + ++ +++ 1 1 2 1 3 2 |
Two kinds of inducements of table 4 are to MG artificial challenge's influence
| Group | The test chicken number of elements | Content of the test is attacked poison back symptom | The air bag pathology-+++ +++ |
| 1 2 3 | 7 4 2 | The weak poison of NDV, IBV is induced, 2/7 performance expiratory dyspnea MG attacks poison, ammonia stress be attacked the weak poison inoculation of malicious no abnormal NDV, IBV by MG, and normal ammonia stress | 6 1 2 2 2 |
By table 4 as seen, after poison was induced a little less than NDV, the IBV, MG attacked malicious chicken through the ammonia environmental stimulus, morbidity (7/7) fully behind the 12d, thus set up MG artificial challenge's animal model.
2.5 chicken virus mycoplasma JS99 cloned weakening strain immunogenicity determining
The results are shown in Table 5.Test group 1.3,2.4,3.4 not immune not challenge test contrast chickens do not show visual respiratory symptom in entire test, Serological testing feminine gender and air bag pathology negative findings prove this batch SPF Leghorn reliable in quality.Its yolk antibody of the preceding extraction of SPF egg incubation makes HA and the HI detection also is negative.MG S6 cloned weakening strain has proved its security by returning test, tracheal ring cultivation ciliary movement damage test and animal experiment.This is tested all immune chickens and is not all showed any expiratory dyspnea, and the air bag pathology is also not obvious, has further proved this conclusion.Test chicken serum HA, HI detected result all conform to except that 2.1 groups substantially, begin to have antibody to occur behind the test chicken immunization 20d, and be positive most of (7/8) behind the 30d.By 1.2,2.2,3.2 results, utilize NDV, IBV to induce, and it is certain to carry out the strong malicious artificial challenge's effect of MG under the hormesis of ammonia environment, guaranteed that so immune chicken attacks the reliability of malicious protection ratio.Immune group and control group are attacked poison back 3～4d all the slight expiratory dyspnea of performance, and 10d all recovers.And 1.1,2.1,3.1 show evident difference with 1.2,2.2,3.2 groups of test chicken pathological anatomy air bag pathology results respectively; the many slight feculences of immunity chicken air bag; immunity back 10d, 20d, 30d attack malicious protection ratio and reach 80%, 75%, 87.5% respectively; immune effect proves that MG S6 cloned weakening strain has good immunogenicity in the recent period.
Table 5. chicken virus mycoplasma JS99 cloned weakening strain F156 culture immunity SPF Leghorn 10d, 20d, 30d attack the mensuration of malicious protection ratio
| Group | Poison is attacked in the content of the test immunity | The chicken number | The immunity fate | Seroprevalence HA HI | The air bag pathology-+++ +++ | Attack malicious protection ratio |
| 1.1 1 1.2 1.3 2.1 2 2.2 2.3 2.4 3.1 3 3.2 3.3 3.4 | + + - + - - + + - + + - - - + + - + + - - - | 5 3 3 4 3 2 2 8 7 2 2 | 10 20 20 30 30 | 0/5 0/5 0/3 0/3 0/3 0/3 4/4 1/4 0/3 0/3 1/2 0/2 0/2 0/2 8/8 7/8 0/7 0/7 0/2 0/2 0/2 0/2 | 3 1 1 1 2 3 2 1 1 1 2 2 2 7 1 6 1 1 1 2 | 80％ 75％ 87.5％ |
Claims (1)
1. chicken virus mycoplasma (Mycoplasma gallisepticum) JS99 cloned weakening strain, this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is: CGMCC NO.0396.
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| CN1128214C true CN1128214C (en) | 2003-11-19 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101864387A (en) * | 2010-05-14 | 2010-10-20 | 杨季芳 | Marine crustacean mycoplasma culture medium and separation and purification method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103074246B (en) * | 2012-08-31 | 2015-09-02 | 南京天邦生物科技有限公司 | A kind of low serum high-efficient culture chicken virus mycoplasma low virulent strain substratum and preparation method thereof |
| CN114940955B (en) * | 2022-05-10 | 2023-08-11 | 兆丰华生物科技(南京)有限公司 | Mycoplasma gallisepticum attenuated vaccine strain and application thereof |
| CN114752541B (en) * | 2022-06-16 | 2022-09-13 | 佛山科学技术学院 | Construction method and application of chicken model infected by mycoplasma gallisepticum |
-
1999
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Non-Patent Citations (2)
| Title |
|---|
| 中国兽医医学报VOL.19,NO3 1999.05.01 宁宜宝"鸡败血霉形体弱毒F株对鸡的致病性和免疫效力测定" * |
| 中国兽医学报18（5） 1998.09.01 鸡败血霉形体SC克隆质弱株免疫原性实验 中国兽医学报18（5） * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101864387A (en) * | 2010-05-14 | 2010-10-20 | 杨季芳 | Marine crustacean mycoplasma culture medium and separation and purification method thereof |
| CN101864387B (en) * | 2010-05-14 | 2011-12-14 | 杨季芳 | Marine crustacean mycoplasma culture medium and separation and purification method thereof |
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| Publication number | Publication date |
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| CN1244581A (en) | 2000-02-16 |
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