CN1928076A - Live vaccine for mycoplasma pneumonia of swine and production method thereof - Google Patents
Live vaccine for mycoplasma pneumonia of swine and production method thereof Download PDFInfo
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- CN1928076A CN1928076A CNA2006101525197A CN200610152519A CN1928076A CN 1928076 A CN1928076 A CN 1928076A CN A2006101525197 A CNA2006101525197 A CN A2006101525197A CN 200610152519 A CN200610152519 A CN 200610152519A CN 1928076 A CN1928076 A CN 1928076A
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Abstract
The present invention relates to one kind of live vaccine for pig asthma and its preparation process. The live vaccine is prepared through culturing Mycoplasma hyopneumoniae RM48 strain on Lps culture medium the present invention designs, adding protecting agent and vacuum freeze drying. The P97 gene of the Mycoplasma hyopneumoniae RM48 strain has 17 R1 (AAKPV/E) repetition sequences, and this molecular biological characteristic may be used in identifying from other bacterial strain. The live vaccine has high safety and high efficiency, and may be used to immunize pig through breast injection or nasal cavity spraying.
Description
Technical field the present invention relates to the prevention of infectious disease living vaccine of a boar, belongs to the veterinary biologics field.
The background technology mycoplasma pneumonia of swine claims porcine mycoplasmal pneumonia or the popular pneumonia of pig region again, is a kind of contact chronic respiratory transmissible disease that is caused by mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae), is prevalent in all over the world.Ill pig mainly shows as cough and asthma, growth retardation, and feed conversion rate is low, and body temperature is normal substantially.When dissected is based on pulmonary lesion, and especially with two lung lobus cardiacuses, the change of pancreas sample appears in middle leaf and sharp leaf and carnification is its feature.The sickness rate height, mortality ratio is low.Discovering in recent years, mycoplasma hyopneumoniae and porcine reproductive respiratory syndrome virus and other cause of disease polyinfection further improve the importance of its infection.Up to the present, this disease is still and causes one of the most important disease of financial loss of raising pigs.
Mycoplasma hyopneumoniae requires very harshness to the nutritional condition of substratum, is difficult to growth in general substratum, is a kind of of difficult cultivation in the animal mycoplasma.
Mycoplasma pneumonia of swine is the global problem that pig industry faces, and vaccine immunity is that prevention and control should the valid approach of disease.Yet; though the research of vaccine has just begun in the sixties in last century; but just begin to occur wishing up to the nineties; though China Veterinery Drug Inspection Office is with mycoplasma hyopneumoniae virulent strain continuous passage generation more than 700 in newborn rabbit body; make its attenuation; it is low to have cultivated a strain virulence; mycoplasma hyopneumoniae rabbitization low virulent strain (the Chinese invention patent ZL 86108515 that immunogenicity is good; the production of mycoplasma hyopneumoniae attenuated vaccine); but; because this bacterial strain for a long time at newborn rabbit interior generation, has almost lost the ability of growing in substratum, can't adapt to the production of vaccine of mass-producing under the batch production condition.
Summary of the invention
The objective of the invention is to produce prevention live vaccine for mycoplasma pneumonia of swine (claiming the mycoplasma hyopneumoniae living vaccine again) with industrial production method by using different substratum and different isolation cultivation methods to make the former mycoplasma hyopneumoniae attenuated vaccine seedling bacterial strain R790 strain that has lost energy for growth in substratum can in substratum, grow and keep good immunogenicity.
The present invention is that bacterial strain R790 strain (Chinese invention patent ZL 86108515 is produced in the mycoplasma hyopneumoniae rabbitization attenuated vaccine production that I am cultivated, the production of mycoplasma hyopneumoniae attenuated vaccine) acclimatizing culture medium has obtained substratum adaptation bacterial strain, this bacterial strain a series of evaluations have been carried out, through growth characteristics, the serological characteristic qualification result shows: this bacterial strain meets the various features of mycoplasma hyopneumoniae, be defined as mycoplasma hyopneumoniae, and it is named (this bacterial strain was delivered BeiJing ZhongGuanCun China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number: CGMCCNo.1816) on September 19th, 2006 for mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) MR48 strain.
Specific embodiments
1 breeding strain
With weak malicious R790 strain (the Chinese invention patent ZL 86108515 of the mycoplasma hyopneumoniae rabbitization that I cultivated, the production of mycoplasma hyopneumoniae attenuated vaccine) by going down to posterity repeatedly at the Lps substratum, obtain the substratum adapted strain, (this bacterial strain was delivered BeiJing ZhongGuanCun China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number: CGMCCNo.1816) on September 19th, 2006 in called after mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) MR48 strain.
The characteristic of 2 mycoplasma hyopneumoniae MR48 low virulent strains.
2.1 form and biochemical characteristic are ring-type, spherical, thread, the shaft-like and polymorphic microorganism of point-like, acellular wall, and Gram-negative, serology and biochemical characteristic should meet the characteristic of this bacterium in the systematic bacteriology.
2.2 present good growth in the cultural characters liquid medium within, cultivate that substratum descends more than 0.5 pH value after 4～10 days for 37 ℃, present slight muddiness.Mycoplasma hyopneumoniae viable bacteria titre can reach 10 in the culture
8～10
9Between the CCU/ml (CCU---colour-change unit).Inoculation Lps solid medium is cultivated after 7～14 days visible fried egg sample bacterium colony for 37 ℃.
Lps liquid culture based formulas (by 1000ml): PPLO 10.0～25.0g, NaCl 2.0～5.0g, KCl 0.2～1.0g, MgSO
40.1～0.4g, KH
2PO
40.2～0.6g, Na
2HPO
4* 12H
2O 2.0～8.0g, glucose 5.0～10.0g, lactoalbumin hydrolysate 1.0～5.0g, 2.5% Yi Suan , 5～10ml, 25% yeast leach liquor, 100～200ml, 10 * MEM, 10～50ml, OX-heart soup or lung soup 100～200ml, FeSO40.001～0.01g, phenol red 1%0.5～2.0ml add distilled water to 800ml, add fresh pig serum 100ml and fresh horse serum 100ml after the filtration sterilization, transfer pH to 7.3～7.6 standby; The agar of adding 1.5% becomes the Lps solid medium in the liquid nutrient medium.
2.3 serological characteristic
2.3.1 identify in being added with the sero-fast liquid nutrient medium of mycoplasma hyopneumoniae that with metabolic inhibition test through 37 ℃ of constant temperature culture 15 days, pH value did not descend, and presents special glucose metabolism inhibition.
2.3.2 identify with growth inhibition test to be added with the sero-fast solid culture primary surface of mycoplasma hyopneumoniae, through 37 ℃ of constant temperature culture 10～20 days, asepticly drop out now, present special mycoplasma hyopneumoniae growth-inhibiting.
2.4 safety examination
2.4.1 the mouse safety check is got bacterial classification and is diluted to 10 with physiological saline
7CCU/ml, the subcutaneous injection body weight is 3 of 18～22g cleaning level mouse, 0.4ml/ only observed 10, all strong living.
2.4.2 every batch of culture of pig safety verification is with 10 times immunizing dose (5 * 10
6CCU), the healthy susceptible pig of pleural inoculation 30～50 ages in days is 3 respectively, establishes 3 of blanks, observes 20.Test pig does not all have clinical symptom, and it is normal to cut open inspection back observation pig lung, no mycoplasma pneumonia of swine pathological change.
2.5 it is that 9 groups are carried out immunity that immunogenicity check immunization ways (nasal spray and pulmonary injection) etc. is divided into, and establishes not immune control group simultaneously.Back 55 days of immunity is adopted the strong malicious F65 of mycoplasma hyopneumoniae to involve in a criminal case to amount to 30 first with the contrast pig and is attacked poison, attacks poison and all slaughters in back 30 days, checks pulmonary lesion.Test-results shows that the protection effect of thoracic cavity pulmonary injection approach and nose internal spraying immunization pig is respectively 90% and 55%.
2.6 molecular biology identification (mensuration of P97 gene order) is measured this bacterial strain P97 gene order according to people's such as Ding Fang method (" journal of animal science and veterinary medicine " 2004.Vol.35 No6.698-701), the result shows, mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) MR48 strain has kept the characteristic of mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) R659 strain, that is: R1 (AAKPV/E) tumor-necrosis factor glycoproteins of the P97 gene of MR48 strain and R659 strain is similarly 17, significantly is different from other bacterial strains.
3. vaccine production is inoculated in the mycoplasma hyopneumoniae MR48 strain bacterium seed liquor of Lps liquid nutrient medium cultivation in the Lps liquid nutrient medium with 1: 10 (volumetric ratio).Deposit 37 ℃ and cultivated 3～6 down, culture descends more than 0.5 pH value, purely after the assay was approved, and enlarged culturing in the same way again.Until the required amount (subculture was no more than for 6 generations) of seedling, equal proportion adds lyophilized vaccine, thorough mixing, and head part quantitatively packing in accordance with regulations contains viable bacteria and is no less than 10
8CCU/ml forms live vaccine for mycoplasma pneumonia of swine after vacuum freezedrying.
The quality standard of 4 mycoplasma hyopneumoniae living vaccines (MR48 strain)
4.1 measuring, colour-change unit (CCU) presses CCU measuring method mensuration, every part bacterium of freeze-dried vaccine number 〉=5 * 10
6CCU.
4.2 safety verification
4.2.1 3 of the mouse of mouse usefulness body weight 18～22g return to original volume with physiological saline with vaccine, each subcutaneous vaccination 0.4ml observed 10.Should all be good for and live.
4.2.2 every batch of culture of pig, is promptly got 15 bottles of (2 part/bottles) vaccines with 10 times immunizing dose and is diluted to cumulative volume 15ml with physiological saline, the healthy susceptible pig of pleural inoculation 30～50 ages in days is 3 respectively, and every 3ml establishes 3 of blanks simultaneously, observes 20.The safety check pig should not have any clinical symptom.Otherwise Ying Chongjian once.
4.3 5 of the large ear rabbits of efficacy test (exempting from the body immune response) usefulness body weight 1.5～2.0kg, get 3 bottles of vaccines (2 part/bottles) vaccine and be diluted to cumulative volume 12ml with physiological saline, every each pulmonary injection 2ml of large ear rabbit, blood sampling on the 30th, use the indirect hemagglutination method. check the serum agglutinating antibody, should at least 4 positive (the blood clotting valency 〉=10++), otherwise, should heavily examine 1 time.
4.4 duration of immunity is 6 months.
4.5 usage and injection of consumption right thoracic cavity or nose internal spraying, 1 part of every pig inoculation.
Advantage of the present invention:
A strain mycoplasma hyopneumoniae RM48 strain involved in the present invention is to be adapted to through repeatedly going down to posterity on the designed a kind of i (mycoplasma hyopneumoniae) vaccine culture medium of the present invention by the weak malicious R790 strain of mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) rabbitization, and can carry out industrialized live vaccine production, this mycoplasma bacterial strain has kept immunogenicity and the security of former bacterial strain, and R1 (AAKPV/E) repetitive sequence of its this bacterium P97 gene is that 17 these molecular biological characteristic can be used for distinguishing other bacterial strains. The mycoplasma hyopneumoniae live vaccine of this bacterial strain production both can be injected with the thoracic cavity, also available nasal spray inocalation method immune swine.
Claims (4)
1 one strains are used to produce the mycoplasma hyopneumoniae bacterial strain of live vaccine for mycoplasma pneumonia of swine, it is characterized in that this mycoplasma hyopneumoniae is to produce bacterial strain R790 strain by live vaccine for mycoplasma pneumonia of swine to adapt to the substratum adaptation bacterial strain that the designed Lps substratum of the present invention obtains, this bacterial strain a series of evaluations have been carried out, through growth characteristics, the serological characteristic qualification result shows: this bacterial strain has kept the various features of mycoplasma hyopneumoniae R659 strain, and the R1 tumor-necrosis factor glycoproteins of this bacterium P97 gene and R790 strain are similarly 17, called after MR48 strain, this bacterial strain has been delivered BeiJing ZhongGuanCun China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number: CGMCCNo.1816 on September 19th, 2006.
2 one kinds of live vaccine for mycoplasma pneumonia of swine is characterized in that this living vaccine is made up of MR48 strain mycoplasma hyopneumoniae and lyophilized vaccine, and this seedling both can be injected with the thoracic cavity, also available nasal spray inoculation method immune swine.
The production method of 3 one kinds of live vaccine for mycoplasma pneumonia of swine; it is characterized in that mycoplasma hyopneumoniae MR48 strain bacterium seed liquor is inoculated in the Lps liquid nutrient medium with 1: 10 (volumetric ratio); cultivated 3～6 down in 37 ℃, culture descends more than 0.5 pH value, purely after the assay was approved; enlarged culturing in the same way again; until the required amount of seedling, equal proportion adds lyophilized vaccine, thorough mixing; head part quantitatively packing in accordance with regulations contains viable bacteria and is no less than 10
8CCU/ml forms live vaccine for mycoplasma pneumonia of swine after vacuum freezedrying.
The production method of the described live vaccine for mycoplasma pneumonia of swine of 4 claims 3 is characterized in that Lps liquid culture based formulas is by 1000ml: PPLO 10.0～25.0g, NaCl 2.0～5.0g, KCl 0.2～1.0g, MgSO
40.1～0.4g, KH
2PO
40.2～0.6g, Na
2HPO
4* 12H
2O 2.0～8.0g, glucose 5.0～10.0g, lactoalbumin hydrolysate 1.0～5.0g, 2.5% Yi Suan , 5～10ml, 25% yeast leach liquor, 100～200ml, 10 * MEM, 10～50ml, OX-heart soup or lung soup 100～200ml, FeSO40.001～0.01g, phenol red 1%0.5～2.0ml add distilled water to 800ml, add fresh pig serum 100ml and fresh horse serum 100ml after the filtration sterilization, transfer pH to 7.3～7.6 standby; The agar of adding 1.5% becomes the Lps solid medium in the liquid nutrient medium.
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Cited By (6)
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| CN102154167A (en) * | 2011-01-05 | 2011-08-17 | 北京大北农科技集团股份有限公司 | Mycoplasma hyopneumoniae culture medium and preparation method thereof |
| CN102258776A (en) * | 2011-07-07 | 2011-11-30 | 普莱柯生物工程股份有限公司 | Combined inactivated vaccine against mycoplasma hyopneumoniae (MHP) and mycoplasma hyorhinis and preparation method thereof |
| CN102458461A (en) * | 2009-04-24 | 2012-05-16 | 贝林格尔.英格海姆维特梅迪卡有限公司 | Vaccines comprising attenuated mycoplasma bovis strains and method for attenuation |
| CN103555641A (en) * | 2013-11-19 | 2014-02-05 | 浙江美保龙生物技术有限公司 | Mycoplasma hyopneumoniae culture medium and preparation method thereof |
| CN104894009A (en) * | 2015-05-15 | 2015-09-09 | 北京中海生物科技有限公司 | Mycoplasma hyopneumoniae strain and application thereof |
| CN106282300A (en) * | 2015-05-26 | 2017-01-04 | 临沂大学 | A kind of chicken virus mycoplasma detection method |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1005089B (en) * | 1986-12-15 | 1989-09-06 | 中国兽药监察所 | Method for producing mycoplasma hyopneumoniae lmh vaccine |
| JP3187419B2 (en) * | 1990-05-29 | 2001-07-11 | アメリカン・サイアナミド・カンパニー | Porcine pneumonia vaccine and production method thereof |
| CN1100871C (en) * | 1999-06-25 | 2003-02-05 | 江苏省农业科学院畜牧兽医研究所 | Cloned weakening strain of swine pneumonia mycoplasma |
| TWI238721B (en) * | 2003-06-20 | 2005-09-01 | Animal Technology Inst Taiwan | Swine enzootic vaccine, its producing method and usage |
| CN1942204A (en) * | 2004-04-26 | 2007-04-04 | 株式会社中央疫苗研究所 | Inactivated mixed vaccine for porcine respiratory disease and the method of manufacturing thereof |
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| CN102458461A (en) * | 2009-04-24 | 2012-05-16 | 贝林格尔.英格海姆维特梅迪卡有限公司 | Vaccines comprising attenuated mycoplasma bovis strains and method for attenuation |
| CN102458461B (en) * | 2009-04-24 | 2017-03-08 | 贝林格尔.英格海姆维特梅迪卡有限公司 | The method comprising the vaccine of the M. bovis strain and attenuation being attenuated |
| CN102154167A (en) * | 2011-01-05 | 2011-08-17 | 北京大北农科技集团股份有限公司 | Mycoplasma hyopneumoniae culture medium and preparation method thereof |
| CN102154167B (en) * | 2011-01-05 | 2013-07-10 | 北京大北农科技集团股份有限公司 | Mycoplasma hyopneumoniae culture medium and preparation method thereof |
| CN102258776A (en) * | 2011-07-07 | 2011-11-30 | 普莱柯生物工程股份有限公司 | Combined inactivated vaccine against mycoplasma hyopneumoniae (MHP) and mycoplasma hyorhinis and preparation method thereof |
| CN102258776B (en) * | 2011-07-07 | 2013-07-10 | 普莱柯生物工程股份有限公司 | Combined inactivated vaccine against mycoplasma hyopneumoniae (MHP) and mycoplasma hyorhinis and preparation method thereof |
| CN103555641A (en) * | 2013-11-19 | 2014-02-05 | 浙江美保龙生物技术有限公司 | Mycoplasma hyopneumoniae culture medium and preparation method thereof |
| CN103555641B (en) * | 2013-11-19 | 2015-08-05 | 浙江美保龙生物技术有限公司 | A kind of mycoplasma hyopneumoniae culture medium and preparation method thereof |
| CN104894009A (en) * | 2015-05-15 | 2015-09-09 | 北京中海生物科技有限公司 | Mycoplasma hyopneumoniae strain and application thereof |
| CN104894009B (en) * | 2015-05-15 | 2018-02-09 | 北京中海生物科技有限公司 | One plant of mycoplasma hyopneumoniae strain and its application |
| CN106282300A (en) * | 2015-05-26 | 2017-01-04 | 临沂大学 | A kind of chicken virus mycoplasma detection method |
| CN106282300B (en) * | 2015-05-26 | 2019-12-24 | 临沂大学 | Mycoplasma gallisepticum detection method |
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