CN1109783A - Oral hydrophobia vaccine for dog and productive process thereof - Google Patents
Oral hydrophobia vaccine for dog and productive process thereof Download PDFInfo
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- CN1109783A CN1109783A CN 94115203 CN94115203A CN1109783A CN 1109783 A CN1109783 A CN 1109783A CN 94115203 CN94115203 CN 94115203 CN 94115203 A CN94115203 A CN 94115203A CN 1109783 A CN1109783 A CN 1109783A
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Abstract
A viral strain for oral hydrophobia vaccine for dog, its separation and culture method, and the method for large-scale industrialized production of said vaccine using the viral strain as seed are disclosed. After the vaccine is orally applied to dog, the domestic dog can effectively resist the infection of hydrophobia, so preventing large-area prevalence of hydrophobia.
Description
The present invention relates to rabies vaccine, particularly relate to the rabies vaccine of adopting oral route immunity dog, prepare the method for the Strain of this vaccine, reaching with this Strain is seed culture of viruses, produces the method for oral hydrophobia vaccine for dog.
Rabies are potent virus diseases of a kind of infecting both domestic animals and human that is caused by rabies virus, still do not have the specific treatment measure at present, and once infection morbidity, mortality rate almost reaches absolutely.
China is since the sixties in this century, and rabic sickness rate constantly rises, and the dead sum of accumulative total has reached 150,000 people over 30 years.Domestic animal (horse, cattle, sheep, pig, the Cervus nippon Temminck etc.) death that runs mad, annual in thousand.Rabies not only threaten people ' s health, threat to life, and bring for international contact, tourism development, farming and animal husbandry development and public health etc. to have a strong impact on.
A lot of in the world countries have eliminated rabies as states such as Sweden, Portugal, Britain and Japan, account for 95% of ill total number of persons but propagate the person of running mad in developing country's (containing China) by domesticated dog.Mad dog is many more, and rabies are suffered from the also corresponding increase of example.State such as Switzerland, Canada is owing to take general prevention and treatment measures such as animal inoculation vaccines, basic controlling rabic popular.According to FAO (Food and Agriculture Organization of the United Nation) (FAO) report, the whole world has 87 countries to have rabies at present, and major part belongs to developing country.The rabies death toll that these are national accounts for 99% of global rabies example sum.China is annual to be only second to India by lyssodexis and rabies death toll, occupies the second place of the world.External experience shows, and is continuous when popular in a particular locality when rabies, emphasizes with the control animal reservoir as preventive measure substantially.Someone once reported with oral vaccine immunity fox (referring to G, M, Baer et al., Develop.Biol.Standar.33:417,1976), but the research that relates to the domesticated dog oral hydrophobia vaccine does not appear in the newspapers as yet.
In China, the domesticated dog injecting pstudorabies vaccine has received that certain effect is (referring to Heilongjiang Province's Suihua area health and epidemic prevention station etc.: biological product communication 9(2): 59,1980: Guo Yide etc.: livestock contagious disease 4:21,1984), yet with the outer a large amount of immune domesticated dogs of approach (as intramuscular injection) of gastrointestinal tract, need to mobilize and organize a large amount of manpowers, and the epidemic prevention worker's that bites in practical operation example occurs repeatedly.Therefore, press for a kind of easy to use, rabies virus infection that can effectively prevent dog of exploitation, propagate, thereby effectively prevent the new semple type rabies vaccine of human rabies to cut off between dog-people rabies.
An object of the present invention is to provide a kind of method for preparing dog with oral hydrophobia attenuated live Strain, this method comprises the rabies virus that is separated to from the fresh cerebral tissue of dying from hydrophobe's corpse, in mouse brain, passed for 50～60 generations continuously, obtaining virulence obviously reduces but the good vaccine strain of immunogenicity, then this strain is transferred to and passed for 40～50 generations in the human embryonic lung diploid fibroblast strain continuously, pass back to again and passed for 2～10 generations in the mouse brain, promptly obtain oral hydrophobia vaccine for dog strain of the present invention, it is No. 22 seeds culture of viruses that this strain is named.
Another object of the present invention provides the rabies virus strain alive of a strain attenuation, and this strain is deposited in Chinese typical culture collection center (CCTCC) in 19 dates, its preservation registration number CCTCC NO
A further object of the present invention provides a kind of with the seed culture of viruses of No. 22 seeds culture of viruses of the present invention as oral hydrophobia vaccine for dog, the method for this vaccine of large-scale production.This method comprises and will seed culture of viruses being inoculated in the mouse brain after the dilution, culturing in vivo 5-7 days, treat mice typical case morbidity after, dissect with aseptic formality and to get brain, grind to form the brain suspension, add the due care agent after, vacuum lyophilization.
A further object of the present invention provides a kind of oral route immunity dog, makes it to avoid catching rabic immunization method.This method comprises sneaks into the immune dog of quilt that feed in the semiliquid diet is in starvation with the dog of the present invention of prevention rabies effective dose with rabies vaccine.
The preparation of I, oral hydrophobia vaccine for dog strain
Dissect typical hydrophobe's corpse, get cerebral tissue and be ground into tissue homogenate, and be diluent, make 10% brain suspension with 2% Ox blood serum distilled water with aseptic formality.Get supernatant 0.03ml intracerebral injection mice, treat typical case morbidity after, dissect and get brain, make the brain suspension after, the intracerebral injection mice passed for 50～60 generations so continuously once more.After the white mice intracranial inoculation to the incubation period that occurs between the classical symptom because the passage number purpose increases, corresponding shortening incubation period, the 1st～5 on behalf of 11～14 days, when reaching for the 46th～53 generation, shortens to 5～6 days incubation period.The subcutaneous vaccination virulence is along with passage number in the brain increases, and is decremented to approximately 1.50 by about 4.20, obtains finally that virulence is stabilized in 5.50 to 6.34(LogLD50 in the brain) weaken strain.
In order further to cultivate the strain that is fit to the preparation oral hydrophobia vaccine for dog, with above-mentioned mouse brain organize endoadaptation 51～53 generation strain, be transferred to human embryonic lung diploid fibroblast strain KMB-17(China Medical Sciences Academy Medical Biology Institute, Kunming), after reaching for the 47th generation, return and passed for 2～5 generations in the mouse brain tissue, promptly obtaining naming is the oral hydrophobia vaccine for dog seed culture of viruses of No. 22 seeds culture of viruses.
With No. 22 seeds culture of viruses that obtain as stated above, make 5% mouse brain suspension, give 1 once oral 4ml of monthly age doggie, oral before serum antibody negative, oral back 1 month, the antibody male rotary rate reached 90%, antibody titer reaches 1: 16～1: 32.
The suitability for industrialized production of II, oral hydrophobia vaccine for dog
At first get seed culture of viruses No. 22,, become 1: 200, intracranial inoculation body weight 11～13 gram mices, every suitable 5000LD50 virus quantity of inoculum concentration 0.03ml(with 2% calf serum distilled water diluting as the seed culture of viruses seed liquor), culturing in vivo 5～7 days.The mice that select to occur typical rabies after incubation period with 1 ‰ bromo geramine washing by soaking 3 times, carries out the fur sterilization, is no less than 15 minutes at every turn.Dissect mice with aseptic formality then and get brain, with the speed that per minute 10,000 changes the germfree mouse brain is ground to form homogenate, add 2% Ox blood serum distilled water then, make 5% mouse brain suspension with electronic tissue grinder.Sampling titration virus titer requires the interior LogLD50/0.03ml of mouse brain to be not less than 6.0.Adding 50%(V/V) skimmed milk is as freeze drying protectant, and packing is after the lyophilisation finished product, in-10 ℃ of preservations, 1 year effect duration.
III, oral immunity mode
In the vaccine (freeze-dried products) of above-mentioned suitability for industrialized production, add the 4ml sterile purified water, after treating the full and uniform suspension of vaccine, pour into and boil and refrigerative pasty state or semiliquid diet, as stirring in maize gruel or the rice congee, feed domesticated dog after making the abundant mixing of vaccine and food, and must once feed.For fear of can not once eating up, can suitably cause starvation for domesticated dog feeding vaccine the previous day, once feed the Seedling success to reach.
States such as external Canada, the Vulpes of development is used oral hydrophobia vaccine, and the vaccine envelope is fused to plastic sheet, inserts in the smoked chicken head, or embeds in the sausage as bait.Be to be fit to the Chinese countryside practical situation, must to seek a kind of not only convenient but also economic feeding mode, we finally are defined as every dog and feed 4ml(1.3 hundred million LD50 through groping repeatedly) dosage and mix food and be best mode.Behind the clothes Seedling 1 month, the seroconversion rate of serum antibody of domesticated dog can reach more than 90%, 1～6 month antibody titer (GMT) remained on 1: 42～1: 76, still reached more than 1: 34 in back 15 months to immunity.
Up to now, we have used the oral hydrophobia vaccine for dog by the inventive method suitability for industrialized production, (Putian, Fujian Province county, Guangxi Zhuang Autonomous Region Yulin county, Nongan County, Jilin Province) was total to 19196 of oral immunity domesticated dogs in 1985～1986 years continuous 2 years in Chinese rubies epidemiology area, observed to carry out epidemiological effect.Wherein immune group comprises 2 townshiies, and matched group comprises 2 townshiies.Immunity township behind oral immunity 83 days, because of an external mad dog scurries into, 7 of the oral immunity dogs of biting, observe 1 year 7 be snapped dog and any rabies all do not occur, rabies took place and caused death in biting in the same period not 1 of immune swine afterwards.All immune township domesticated dogs were observed after immunity 1 year, did not see that rabies take place.On the contrary, the contrast township then finds to catch 20 of rabies domesticated dogs.Continue to observe 10 months, 37 of mad dogs appear in the contrast township.Above-mentioned observed result shows with oral hydrophobia vaccine for dog of the present invention can significantly reduce tame rabies sickness rate for the domesticated dog immunity.
The safety of IV, oral hydrophobia vaccine for dog
Above-mentioned EPDML effective observation, in order to check the influence of the different regional conditions of this vaccine to vaccine effect, the area of effect observation comprises the Jilin Province, northeast in Chinese rubies epidemiology district and Guangxi, the Fujian of South China.Add up to and observe 19425 domesticated dogs except proving its epidemiological effect, also do not find unsafe problems.Take the domesticated dog of oral vaccine of the present invention, do not fasten under the situation of freely putting in a suitable place to breed not enclosing, clothes Seedling dog is with during domestic animals such as other dog, pig, sheep, poultry (chicken, duck, goose) frequently contact, as robs the food of eating pig etc., do not find the non-clothes Seedling of vaccine strain contact infection animal.To newborn dog of nascent 1 monthly age, behind the oral vaccine, 7 newborn dogs were observed 6 months, and any rabies is not found in all safety survivals of all observed newborn dogs.10 adult domesticated dogs were wiped away from the oral cavity with cotton behind oral vaccine and get mucosal sites secretions in 1,2,3 day, were not separated to virus.
In sum, use oral hydrophobia vaccine for dog of the present invention, in Chinese rubies epidemiology area, show through 1985～1987 years 3 years observed results, can reduce rabies sickness rate between local dog, thereby blocking-up dog → people infects rabies, this is to controlling rabic popular will playing an important role.
Embodiment:
1, in March, 1985, to be diluted to the 1:200(diluent after the aseptic formality grinding: 2% Ox blood serum sterile purified water) inoculation body weight 11～13 gram mices were 495, intracranial inoculation amount 0.03ml with No. 22 seed culture of viruses Mus brains.Selected disease symptom typical case, dying mice in 5～7 days, get brain with aseptic formality, gather in the crops 450 in aseptic Mus brain altogether, electronic tissue grinder grinds the back and adds diluent 1800ml, add aseptic skimmed milk 1800ml again, be sub-packed in the penicillin bottle, every bottled 4ml, be divided into and adorn 900 bottles, be i.e. 900 part oral hydrophobia vaccine for dog.Virus titer mensuration is carried out in sampling, and the result is LogLD50 6.5/0.03ml.Lot number 8503.
2, carried out the domesticated dog oral immunity at Guangxi Zhuang Autonomous Region Yulin City on June 18th, 1985.2 townshiies of immunity, 2 townshiies of controlled observation.Immunity totally 4456 of domesticated dogs (containing 8503 batches), 3080 of domesticated dogs are observed in the contrast township.Immunity township behind oral immunity 83 days, an external mad dog scurries into, 7 of the immune dogs of biting are observed and any symptom do not occurred in 1 year, 1 of the pig of biting simultaneously, after the dog disease of going mad.The immunity township is in back 1 year of immunity, and no rabies take place.11 of mad dogs, 9 take place in back 1 year of immunity, the minority in immune township immunity dog and controlled observation altogether.Illustrate that oral immunity plays the effect of reduction rabies sickness rate to dog.
Claims (7)
1, the method for preparing the oral hydrophobia vaccine for dog seed culture of viruses, this method comprises from the fresh cerebral tissue of dying from hydrophobe's corpse and is separated to rabies virus, by passing for 50～60 generations in the mouse brain continuously, then this strain is transferred to continuous passage to 40 among the human embryonic lung diploid fibroblast strain KMB-17～50 generations, passed back to again and passed for 2～10 generations in the mouse brain.
2, the dog of the called after " No. 22 seeds culture of viruses " that makes by the described method of claim 1 of a strain is with oral hydrophobia attenuated live Strain, and its preservation registration number is CCTCC NO
3, use the oral hydrophobia Strain according to the dog of claim 1, virulence is stabilized in LogLD50 〉=6.0 in its brain.
4, produce the method for oral hydrophobia vaccine for dog; this method comprises and being inoculated in the mouse brain after will diluting with the oral hydrophobia seed culture of viruses by the dog of the described method of claim 1 preparation; culturing in vivo 5～7 days; mice typical case morbidity back is dissected and is got brain; grind to form the brain suspension, add the lyophilization of due care agent final vacuum.
5, according to the method for claim 4, wherein the virus titer of gained mouse brain tissue suspension is that 50% infective dose is not less than 6.0/0.03ml in the mouse brain.
6, according to the method for claim 4, wherein said protective agent is 50%(V/V) skimmed milk.
7, oral administration route of administration immunity dog makes it to avoid catching rabic method.This method comprises sneaks into the dog by the preparation of the method described in the claim 4 to 6 of prevention rabies effective dose in the semiliquid diet with rabies vaccine, and feed is in the method immunity domesticated dog of starvation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 94115203 CN1070381C (en) | 1994-09-21 | 1994-09-21 | Oral hydrophobia vaccine for dog and productive process thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 94115203 CN1070381C (en) | 1994-09-21 | 1994-09-21 | Oral hydrophobia vaccine for dog and productive process thereof |
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| Publication Number | Publication Date |
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| CN1109783A true CN1109783A (en) | 1995-10-11 |
| CN1070381C CN1070381C (en) | 2001-09-05 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 94115203 Expired - Fee Related CN1070381C (en) | 1994-09-21 | 1994-09-21 | Oral hydrophobia vaccine for dog and productive process thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100333793C (en) * | 2005-06-28 | 2007-08-29 | 崔栋 | Diploid-cell rabies vaccine and purified rabies vaccine, freeze-drying preparation and water injection thereof |
| CN105561293A (en) * | 2015-12-29 | 2016-05-11 | 北京中联康生物科技股份有限公司 | Immunopotentiator and preparation method and application thereof |
-
1994
- 1994-09-21 CN CN 94115203 patent/CN1070381C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100333793C (en) * | 2005-06-28 | 2007-08-29 | 崔栋 | Diploid-cell rabies vaccine and purified rabies vaccine, freeze-drying preparation and water injection thereof |
| CN105561293A (en) * | 2015-12-29 | 2016-05-11 | 北京中联康生物科技股份有限公司 | Immunopotentiator and preparation method and application thereof |
| CN105561293B (en) * | 2015-12-29 | 2019-10-15 | 北京中联康生物科技股份有限公司 | Immunopotentiator and its preparation method and application |
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| Publication number | Publication date |
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| CN1070381C (en) | 2001-09-05 |
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