CN103602637A - Vaccine strain for mycoplasma pneumonia of swine - Google Patents
Vaccine strain for mycoplasma pneumonia of swine Download PDFInfo
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- CN103602637A CN103602637A CN201310608564.9A CN201310608564A CN103602637A CN 103602637 A CN103602637 A CN 103602637A CN 201310608564 A CN201310608564 A CN 201310608564A CN 103602637 A CN103602637 A CN 103602637A
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Abstract
The invention discloses a vaccine strain for mycoplasma pneumonia of a swine. The classified name of the vaccine strain is Mycoplasma hyopneumoniae, wherein the number of the bacterial strain is NJ, the vaccine strain is preserved in China Center for Type Culture Collection with the preservation number: CCTCC NO: M2012286, and the preserving date is 16th, July, 2012. The vaccine strain for mycoplasma pneumonia of the swine disclosed by the invention has certain pathogenicity to swine and good immunogenicity.
Description
Technical field
The present invention relates to a kind of porcine mycoplasmal pneumonia vaccine strain, belong to veterinary biologics field.
Technical background
Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp) be to cause porcine mycoplasmal pneumonia (Mycoplasmal pneumoniae of swine, Mps claims again swine enzootic pneumonia) main pathogen, be also a kind of important primary cause of disease of porcine respiratory disease syndromes.Mhp main infection porcine respiratory, virulence own is not strong, clinical symptom mainly take cough and asthma as main, characteristics of lesion is mainly sharp leaf, lobus cardiacus, the middle leaf of lung and is " meat sample " or " shrimp sample " consolidation every leaf leading edge.After Mhp infects body, cilium main and respiratory tract inwall sticks, cause ciliated cell's pathology and apoptosis, cause cilium fracture and come off, cause body morbidity, and can have a strong impact on growing and feed conversion rate of pig, or destroy mucous membrane-cilium barrier, the easily infection (particularly to young pig) of other pathogenic bacterium of secondary, improve lethality rate, thereby cause huge financial loss to pig industry.At present the main prophylactico-therapeutic measures of porcine mycoplasmal pneumonia is vaccine and medicine, but effectively medicine lacks, and therefore, carrying out this sick vaccine development will have great importance for the prevention of this disease and control.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of porcine mycoplasmal pneumonia vaccine strain, and this vaccine strain has certain pathogenic to pig, cultivates titre high and have a good immunogenicity.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
One strain porcine mycoplasmal pneumonia vaccine strain, its Classification And Nomenclature is mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae), bacterial strain number is NJ, be preserved in Chinese Typical Representative culture collection center, address: China. Wuhan. Wuhan University, postcode 430072, deposit number: CCTCC NO:M2012286, preservation date: on July 16th, 2012.This bacterial strain is that contriver gathers voluntarily Screening and Identification and obtains a strain porcine mycoplasmal pneumonia vaccine strain in August, 2004 in pig farm, Nanjing.
Beneficial effect: the present invention has advantages of following outstanding:
1) new bacterial strain immunogenicity is good: porcine mycoplasmal pneumonia vaccine strain of the present invention is the new bacterial strain from domestic separation and Culture seed selection, has good immune prototype, has guaranteed specificity and the immune efficacy of vaccine.
2) new bacterial strain production vaccine is easy: porcine mycoplasmal pneumonia vaccine strain of the present invention is the local isolated strains through cultivating, and has the cultural characters of high titre, has guaranteed the high titre of antigen for vaccine, has reduced enrichment step; This bacterial strain can be used Thiomersalate deactivation simultaneously, and Thiomersalate itself is a kind of sanitas, with it, comes deactivation to take into account deactivation and anticorrosion, has reduced consumption, has reduced the untoward reaction to animal; Because bacterial strain of the present invention has good immunogenicity, in the inactivated vaccine of the preparation making, immunological adjuvant composition is simple, and seedling method is easy, has simplified production technique, has reduced and has produced the risk of polluting.
3) vaccine safety that new bacterial strain is produced is efficient: porcine mycoplasmal pneumonia vaccine strain of the present invention is the Local Isolates through cultivating; there is the feature that titre is high, immunogenicity is good of cultivating; the adjuvant using is for having good immune protective through screening the oily adjuvant obtaining; water-based adjuvant has good security, has guaranteed the safety and efficiently of vaccine.
In a word, that inactivated vaccine of the present invention is produced is easy, vaccine safety is efficient, can meet preferably current pig farm in the urgent need to, be easy to apply on a large scale, there are wide market outlook and larger economical, societal benefits.
Accompanying drawing explanation
Fig. 1 porcine mycoplasmal pneumonia inactivated vaccine (NJ strain) preparation flow figure.
The NJ strain of Fig. 2 mycoplasma hyopneumoniae and the comparison of other bacterial strains 16s rRNA gene similarity.
The NJ strain of Fig. 3 mycoplasma hyopneumoniae and other bacterial strains 16s rRNA gene evolution tree.
The NJ strain of Fig. 4 mycoplasma hyopneumoniae and the comparison of other bacterial strains P46 gene similarity.
The NJ strain of Fig. 5 mycoplasma hyopneumoniae and other bacterial strains P46 gene evolution tree.
Embodiment
The following examples will further illustrate the present invention, and not limit the scope of the invention.
Embodiment 1: the seed selection of mycoplasma hyopneumoniae NJ strain
1 strains separation is cultivated
After sick lung tissue is ground, add in liquid nutrient medium (pH value 7.4～7.6), put 37 ℃ and cultivate pH value after 3～10 days and reduce to 6.6～7.0, slight homogeneous muddiness.Inoculate solid medium and put 37 ℃ of cultivations after 3～10 days, visible fried egg sample bacterium colony goes down to posterity after 3 times on solid medium, and inoculation liquid nutrient medium, obtains mycoplasma hyopneumoniae.
2 identify
Separated mycoplasma hyopneumoniae strain is put to 37 ℃ and cultivate pH value after 3～10 days and reduce to 6.6～7.0, slight homogeneous muddiness, carries out smear, and dyeing microscopic examination is shown in typical mycoplasma hyopneumoniae thalline.Inoculate solid medium and put 37 ℃ of cultivations after 3～10 days, visible fried egg sample bacterium colony.The cultural characters that meets mycoplasma hyopneumoniae.
According to mycoplasma hyopneumoniae 16s rRNA and the P46 gene order of Genbank report, design respectively primer, after pcr amplification, order-checking, the sequence of mensuration is carried out ClustalW with the whole genome sequence of having announced and is compared.
For 16s rRNA gene, NJ strain and GenBank report that the similarity of other bacterial strains is 99.7%～100.0%, and similarity is relatively shown in Fig. 2, and evolutionary tree as shown in Figure 3, be shown in shown in sequence table SEQ ID No:1 by NJ strain 16s rRNA sequence.For P46 gene, NJ strain and GenBank report that the similarity of other bacterial strains is 98.7%～99.9%, and similarity is relatively shown in Fig. 4, and evolutionary tree as shown in Figure 5, be shown in shown in sequence table SEQ ID No:2 by NJ strain P46 sequence.This explanation mycoplasma hyopneumoniae NJ strain is mycoplasma hyopneumoniae, and different from other bacterial strains.
Through cultural characters, PCR and order-checking, identify (sequence is shown in sequence table SEQ ID No:1), separation has obtained mycoplasma hyopneumoniae NJ strain.
3 strain passages
The separated mycoplasma hyopneumoniae NJ strain obtaining, through the liquid cultivation of going down to posterity, carries out assay every 3 generations.Result content is from 10
5cCU/mL rises to 10
10cCU/mL.
4 immunogenicity determinings
The different generations of mycoplasma hyopneumoniae NJ strain are prepared vaccine, healthy susceptible pigs of each intramuscular injection immunity 7～15 ages in days then, booster immunization again after 14 days; Head exempted from after 35 days, together with the identical contrast pig of condition, with mycoplasma hyopneumoniae Js, organized strong malicious intratracheal injection to attack poison, attacked poison and cutd open inspection after 28 days, calculated immune group porcine mycoplasmal pneumonia tuberculosis varying index decrement.Vaccine immunity group porcine mycoplasmal pneumonia tuberculosis varying index decrement indifference prepared by each generation bacterial strain of result.Thus obtained have that to cultivate titre high, the mycoplasma hyopneumoniae NJ strain that immunogenicity is good.
Embodiment 2: the preparation method of porcine mycoplasmal pneumonia inactivated vaccine (NJ strain).
1 strain culturing
Bacterial strain is selected mycoplasma hyopneumoniae NJ strain, and this bacterial strain, by our isolation identification, goes down to posterity and cultivates rear acquisition, and this bacterial strain send Chinese Typical Representative culture collection center (CCTCC) on July 16th, 2012, and preserving number is: CCTCCM2012286.
2 strain characteristics
2.1 forms and this bacterium of biochemical characteristic are the polymorphic microorganism of Gram-negative, have ring-type, spherical, point-like, shaft-like and the two poles of the earth shape, easy coloring not, acellular wall.Biochemical characteristic should meet the characteristic of this bacterium in systematic bacteriology.
In 2.2 cultural characters liquid medium withins (pH value 7.4～7.6), put 37 ℃ and cultivate pH value after 3～7 days and reduce to 6.6～7.0, slight homogeneous muddiness, viable bacteria titre can reach 10
10more than CCU/mL.Inoculation solid medium, puts 37 ℃ and cultivates after 3～10 days, visible fried egg sample bacterium colony.
Liquid culture based formulas is: EaglesShi liquid 1000mL, lactoalbumin hydrolysate 10.0g, porcine blood serum 200～400mL, fresh yeast diffusion juice 10～20mL, phosphate buffered saline buffer 600mL, penicillin 200～1,000 ten thousand units, 0.4% phenol red 3.5mL is 7.4～7.6 with 10g/L NaOH adjust pH.
Solid culture formula: add Noble Agar in liquid medium within.
2.3 serological characteristics are identified with metabolic inhibition test, are inoculated in the liquid nutrient medium that is added with anti-mycoplasma hyopneumoniae serum, cultivate 10 at 37 ℃, contrast and compare nondiscoloration with substratum.
2.4 virulence through the healthy susceptible 5mL of intratracheal injection 7～15 ages in days, are cutd open inspection after 28 days by strain culture, and typical porcine mycoplasmal pneumonia pathology appears in Pigs Inoculated lung as seen.
2.5 immunogenicities are made vaccine by the present invention, the healthy susceptible pigs of intramuscular injection immunity 7～15 ages in days, booster immunization again after 14 days; Head exempted from after 35 days, together with the identical contrast pig of condition, with mycoplasma hyopneumoniae Js, organized strong malicious intratracheal injection to attack poison, attacked poison and cutd open inspection after 28 days, and visible immune swine can significantly reduce porcine mycoplasmal pneumonia tuberculosis and become.
3 produce the preparation of bacterial classification
Get mycoplasma hyopneumoniae NJ strain freeze-drying lactobacillus, with after liquid nutrient medium dilution, inoculation solid medium is cultivated 3～10 at 37 ℃, selects well-grown bacterium colony, purely after the assay was approved, and as first order seed.Get again first order seed inoculation liquid nutrient medium, at 37 ℃, cultivate pH value after 3～7 days and reduce to 6.6～7.0 results, through purely after the assay was approved, as secondary seed.
The preparation of bacterium liquid for 4 seedlings
Get secondary seed inoculation liquid nutrient medium, cultivate 3～7 for 37 ℃, carry out enlarged culturing after culture variable color, subculture was no more than for 6 generations, until pH value, reduced to 6.6～7.0 o'clock results, according to the regulation of Chinese veterinary pharmacopoeia, purely checked and assay.
The deactivation of 5 antigens
It is 0.005% Thiomersalate that the seedling being up to the standards is added to final concentration with bacterium liquid, and 37 ℃ are carried out deactivation.Get deactivation sample 1mL and join in the liquid nutrient medium of 50mL, put 37 ℃ of cultivations, observe 5, then get 0.5mL and be inoculated in 4.5mL liquid nutrient medium, put 37 ℃ of cultivations, observe 10, nondiscoloration as equal in twice cultivation, is judged to deactivation and is up to the standards.
6 join seedling and mix
Every part is no less than 2.86 * 10 containing mycoplasma hyopneumoniae before deactivation
8cCU, joins seedling ratio and is determined by adjuvant used, and antigen insufficient section is supplied with PBS.
6.1 injection white oils join seedling and 94 parts of injection white oils (weight ratio) are got in mixing, Si Ben-806 part, add in oil phase tank, stir, and through 121 ℃ of sterilizings 30 minutes, are cooled to room temperature standby.Get 96 parts of the bacterium liquid that deactivation is up to the standards, 4 parts of tween-80s, add in water tank, and stirring and evenly mixing is standby.2～3 parts of oil phases are added in emulsion tank, first stirring at low speed 30 minutes, more slowly add 1 part of water, continue to stir, with high-speed shearing machine, carry out emulsification, before emulsification finishes, adding final concentration is 0.005%(g/mL) Thiomersalate solution, make water-in-oil emulsion.
Joining seedling and mixing and to join to specifications seedling and mixing 6.2ISA206VG(SEPPIC), ISA201VG(SEPPIC), Gel01ST(SEPPIC), ISA11RVG(SEPPIC).As ISA201VG, by antigen and adjuvant weight ratio 50:50, join seedling, adjuvant is preheated to 31 ℃, with low-shearing power, stir, maintain 30 ℃, add antigen (water) in adjuvant, stir homogeneous.As ISA11R VG, in the ratio proportioning of adjuvant and antigen weight ratio 15:85, with low shear force agitator, stir homogeneous in antigen water being added to adjuvant under room temperature or lower temperature.As Gel01ST, adjuvant is joined seedling by 5%～20%, stirs homogeneous in antigen water being added to adjuvant under room temperature or lower temperature with low shear force agitator.
7 inspection after constructions
7.1 proterties should meet affiliated formulation.As should be water-in-oil-type with injection white oil, as should be oil-in-water water-in type again with ISA201VG, as should be oil-in-water-type with ISA11R VG, as should be water aqua type with Gel01ST.
7.2 stability are drawn vaccine 10mL and are added centrifuge tube, and with 3000r/min centrifugal 15 minutes, the water that separate out at the pipe end should be no more than 0.5mL.
7.3 viscositys are tested by existing < < Chinese veterinary pharmacopoeia > > appendix, are no more than 200cP.
7.4 steriling tests are tested by existing < < Chinese veterinary pharmacopoeia > > appendix, asepsis growth.
7.5 safety verifications are with 5 of the healthy susceptible pigs of 7～15 ages in days, and each intramuscular injection vaccine 2mL, observed after 14 days, except occurring that one crosses gonosome temperature rise, other parts and systemic adverse reactions that the vaccinate of having no way of causes.
7.6 efficacy tests, with 15 of the healthy susceptible pigs of 7～15 ages in days, are divided into 3 groups, are respectively and attack malicious control group, normal healthy controls group and vaccine immunity group, 5 every group.1 part of each intramuscular injection vaccine of vaccine immunity group, after 14 days, then 1 part of each intramuscular injection vaccine.Head exempts from latter 35 days, together with attacking 5 of poison contrast pigs, with the strong poison of mycoplasma hyopneumoniae Js strain tissue of normal saline dilution, attacks poison, every 5mL through intratracheal injection.Attack poison after 28 days, cut open all test pig of inspection, the left sharp leaf of observed and recorded lung tissue, left lobus cardiacus, left lobus diaphragmaticus, right sharp leaf, right lobus cardiacus, right lobus diaphragmaticus, accessory lobes pathology percentage, be designated as 1 by 0～25%; 26～50% are designated as 2; 51～75% are designated as 3; 76～100% are designated as 4, add up each test swine disease tuberculosis varying index (being up to 28).Tuberculosis varying index decrement=(attacking malicious control group tuberculosis varying index mean value-immune group tuberculosis varying index mean value)/(attacking malicious control group tuberculosis varying index mean value-normal healthy controls group tuberculosis varying index mean value) * 100%, calculates porcine mycoplasmal pneumonia vaccine immunity group thumps varying index decrement by above-mentioned formula.
The 7.7 Thiomersalate determination of residual amount are measured by existing < < Chinese veterinary pharmacopoeia > > appendix, should meet the regulation of veterinary biologics general rule.
1 part of every intramuscular injection of 8 intramuscular injection piglets.
Embodiment 3: vaccine.
1 vaccine
The oily adjuvant of porcine mycoplasmal pneumonia inactivated vaccine (NJ strain), porcine mycoplasmal pneumonia inactivated vaccine (NJ strain) ISA11RVG, porcine mycoplasmal pneumonia inactivated vaccine (NJ strain) Gel01ST, the porcine mycoplasmal pneumonia inactivated vaccine that abroad certain company produces.
Strong poison for 2 checks
The strong poison of mycoplasma hyopneumoniae Js strain tissue, is prepared by Jiangsu Province Agriculture Science Institute veterinary institute.
3 test pig
30 of the healthy susceptible pigs of 7～15 ages in days, derive from Jiangsu Province agriculture and animal husbandry tomorrow Science and Technology Ltd..
4 test design
With the oily adjuvant of porcine mycoplasmal pneumonia inactivated vaccine (NJ strain), porcine mycoplasmal pneumonia inactivated vaccine (NJ strain) ISA11RVG, porcine mycoplasmal pneumonia inactivated vaccine (NJ strain) Gel01ST, each 1 batch of import porcine mycoplasmal pneumonia inactivated vaccine, the every batch of vaccine is with 5 of 7～15 age in days piglets, the vaccine of every intramuscular injection 1mL, after 14 days with Isodose booster immunization once.Set up pig that condition is identical to attack each 5 of malicious control group and normal healthy controls groups simultaneously.Vaccine immunity group head exempts to observe clinical response in latter 28 days.After head exempts from 35 days, vaccine immunity group and attack malicious control group test pig and carry out challenge test, attacked poison and cuts open inspection after 28 days, and statistics pulmonary lesion is scored, and calculates tuberculosis varying index decrement.
5 clinical responses are observed
Test pig is observed 28 after vaccinate, and the test pig of 3 immune group all occurs after immunity that one crosses heat pyrexia as a result, but all recovers normal in immunity in latter three days.Have no other abnormal clinical response.
6 effect comparison tests
In test pig is attacked after malicious 28 days, observe and find there is 4 appearance cough in various degree in 5 first taps poison control group pigs, respectively have 1～2 to occur slightly cough in 3 immune group, normal healthy controls has no cough and waits porcine mycoplasmal pneumonia characteristic symptom.Attack poison and cut open inspection after 28 days, each organizes tuberculosis varying index, calculates tuberculosis varying index decrement, and effect comparison test the results are shown in Table 1.Have result visible, the immune group tuberculosis varying index of 3 different adjuvants of porcine mycoplasmal pneumonia inactivated vaccine (NJ) is all significantly lower than attacking malicious control group and import vaccine group.
The similar vaccine potency comparison test of table 1 result
Note: a～d represents to compare between two between every group, and alphabetical identical person represents that difference is not remarkable, and alphabetical different persons represent significant difference (p<0.05).
Claims (1)
1. a strain porcine mycoplasmal pneumonia vaccine strain, its Classification And Nomenclature is mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae), and bacterial strain number is NJ, has been preserved in Chinese Typical Representative culture collection center, deposit number: CCTCC NO:M2012286, preservation date: on July 16th, 2012.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104513804A (en) * | 2015-01-27 | 2015-04-15 | 新疆天康畜牧生物技术股份有限公司 | Vaccine strain for mycoplasmal pneumonia of swine |
| CN112143666A (en) * | 2020-09-07 | 2020-12-29 | 华中农业大学 | Mycoplasma hyopneumoniae low virulent strain and application thereof in preparation of low virulent vaccine |
| CN117305192A (en) * | 2023-12-01 | 2023-12-29 | 北京瑞阳瑞泰生物科技有限公司 | Mycoplasma hyopneumoniae RT02 strain, vaccine composition and application thereof |
| CN117330764A (en) * | 2023-12-01 | 2024-01-02 | 北京瑞阳瑞泰生物科技有限公司 | Veterinary vaccine efficacy test method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104513804A (en) * | 2015-01-27 | 2015-04-15 | 新疆天康畜牧生物技术股份有限公司 | Vaccine strain for mycoplasmal pneumonia of swine |
| CN112143666A (en) * | 2020-09-07 | 2020-12-29 | 华中农业大学 | Mycoplasma hyopneumoniae low virulent strain and application thereof in preparation of low virulent vaccine |
| CN112143666B (en) * | 2020-09-07 | 2023-02-21 | 华中农业大学 | Mycoplasma hyopneumoniae low virulent strain and application thereof in preparation of low virulent vaccine |
| CN117305192A (en) * | 2023-12-01 | 2023-12-29 | 北京瑞阳瑞泰生物科技有限公司 | Mycoplasma hyopneumoniae RT02 strain, vaccine composition and application thereof |
| CN117330764A (en) * | 2023-12-01 | 2024-01-02 | 北京瑞阳瑞泰生物科技有限公司 | Veterinary vaccine efficacy test method |
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