CN103602637B - Vaccine strain for mycoplasma pneumonia of swine - Google Patents
Vaccine strain for mycoplasma pneumonia of swine Download PDFInfo
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Abstract
The invention discloses a strain Vaccine strain for mycoplasma pneumonia of swine, its Classification And Nomenclature is mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae), bacterial strain number is NJ, be preserved in China typical culture collection center, deposit number: CCTCC NO:M2012286, preservation date: on July 16th, 2012.Vaccine strain for mycoplasma pneumonia of swine of the present invention has certain pathogenic to pig, have good immunogenicity.
Description
Technical field
The present invention relates to a kind of Vaccine strain for mycoplasma pneumonia of swine, belong to veterinary biologics field.
Technical background
Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp) be cause porcine mycoplasmal pneumonia (Mycoplasmal pneumoniae of swine, Mps is also known as swine enzootic pneumonia) main pathogen, be also a kind of important primary cause of disease of porcine respiratory disease syndrome.Mhp main infection porcine respiratory, virulence own is strong, clinical symptom mainly based on cough and asthma, characteristics of lesion mainly the sharp leaf of lung, lobus cardiacus, middle leaf and every leaf leading edge in " meat sample " or " shrimp sample " consolidation.After Mhp infects body, cilium that is main and respiratory tract inwall sticks, cause ciliated cell's pathology and apoptosis, cilium is caused to rupture and come off, body is caused to fall ill, and growing and feed conversion rate of pig can be had a strong impact on, or destroy mucous membrane-cilium barrier, the infection (particularly to young pig) of easy other pathogenic bacterium of secondary, improve lethality rate, thus cause huge financial loss to pig industry.The main prophylactico-therapeutic measures of current porcine mycoplasmal pneumonia is vaccine and medicine, but effective medicine comparatively lacks, and therefore, the prevention and corntrol for this disease has great importance by the vaccine development carrying out this disease.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of Vaccine strain for mycoplasma pneumonia of swine, and this vaccine strain has certain pathogenic to pig, and it is high and have good immunogenicity to cultivate titre.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
One strain Vaccine strain for mycoplasma pneumonia of swine, its Classification And Nomenclature is mycoplasma hyopneumoniae (Mycoplasmahyopneumoniae), bacterial strain number is NJ, be preserved in China typical 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 Screening and Identification voluntarily in August, 2004 in pig farm, Nanjing and obtains a strain Vaccine strain for mycoplasma pneumonia of swine.
Beneficial effect: the present invention has following outstanding advantage:
1) new strains immunogenicity is good: Vaccine strain for mycoplasma pneumonia of swine of the present invention is the new strains from domestic separation and Culture seed selection, has good immune prototype, ensure that specificity and the immune efficacy of vaccine.
2) new strains production vaccine is easy: Vaccine strain for mycoplasma pneumonia of swine of the present invention is the local isolated strains through cultivating, and has the cultural characters of high titre, ensure that the high titre of antigen for vaccine, decrease enrichment step; This bacterial strain can use Thiomersalate deactivation simultaneously, and Thiomersalate itself is a kind of sanitas, carrys out deactivation and has taken into account deactivation and anticorrosion, decrease consumption, decrease the untoward reaction to animal with it; Because bacterial strain of the present invention has good immunogenicity, in the inactivated vaccine of the preparation made, immunological adjuvant composition is simple, and seedling method is easy, simplifies production technique, reduces the risk of producing and polluting.
3) vaccine safety of new strains production is efficient: Vaccine strain for mycoplasma pneumonia of swine of the present invention is the Local Isolates through cultivating; have and cultivate the feature that titre is high, immunogenicity is good; the adjuvant used is for having good immune protective through the oily adjuvant of screening acquisition; aqueous adjuvants has good security, ensure that the safety and efficiently of vaccine.
In a word, inactivated vaccine of the present invention produce easy, vaccine safety 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 other bacterial strains 16s rRNA gene similarity-rough set.
The NJ strain of Fig. 3 mycoplasma hyopneumoniae and other bacterial strains 16s rRNA phylogenetic trees.
The NJ strain of Fig. 4 mycoplasma hyopneumoniae and other bacterial strains P46 gene similarity-rough set.
The NJ strain of Fig. 5 mycoplasma hyopneumoniae and other bacterial strains P46 phylogenetic trees.
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 DEG C and cultivate pH value after 3 ~ 10 days and reduce to 6.6 ~ 7.0, slight homogeneous muddiness.Inoculate solid medium to put 37 DEG C and cultivate after 3 ~ 10 days, visible fried egg sample bacterium colony, solid medium goes down to posterity after 3 times, inoculation liquid nutrient medium, obtains mycoplasma hyopneumoniae.
2 qualifications
The mycoplasma hyopneumoniae strain of separation is put 37 DEG C to 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 DEG C of cultivations after 3 ~ 10 days, visible fried egg sample bacterium colony.Meet the cultural characters of mycoplasma hyopneumoniae.
The mycoplasma hyopneumoniae 16s rRNA reported according to Genbank and P46 gene order design primer respectively, and after pcr amplification, order-checking, the sequence of mensuration carries out ClustalW comparison with the whole genome sequence announced.
For 16s rRNA gene, NJ strain and GenBank report that the similarity of other bacterial strains is 99.7% ~ 100.0%, and similarity-rough set is 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-rough set is 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 illustrates that mycoplasma hyopneumoniae NJ strain is mycoplasma hyopneumoniae, and different from other bacterial strains.
Through cultural characters, PCR and order-checking qualification (sequence is shown in sequence table SEQ ID No:1), be separated and obtain mycoplasma hyopneumoniae NJ strain.
3 strain passages
Be separated the mycoplasma hyopneumoniae NJ strain obtained, through liquid Secondary Culture, carry 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 prepare vaccine, then the healthy susceptible pig of each intramuscular injection immunity 7 ~ 15 age in days, booster immunization again after 14 days; After head exempts from 35, together with the contrast pig that condition is identical, organize strong malicious intratracheal injection to attack poison with mycoplasma hyopneumoniae Js, attack poison and cut open inspection, Computation immunity group porcine mycoplasmal pneumonia tuberculosis varying index decrement after 28 days.Vaccine immunity group porcine mycoplasmal pneumonia tuberculosis varying index decrement indifference prepared by each generation bacterial strain of result.Thus obtained have 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 selects mycoplasma hyopneumoniae NJ strain, and this bacterial strain is by our isolation identification, and go down to posterity after cultivating and obtain, this bacterial strain send China typical 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, not easy coloring, 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 DEG C 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 DEG C 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 unit, 0.4% phenol red 3.5mL is 7.4 ~ 7.6 with 10g/L NaOH adjust pH.
Solid culture is filled a prescription: add Noble Agar in liquid medium within.
2.3 serological characteristic metabolic inhibition tests are identified, are inoculated in the liquid nutrient medium being added with anti-mycoplasma hyopneumoniae serum, cultivate 10, nondiscoloration compared with contrasting with substratum at 37 DEG C.
2.4 virulence are by strain culture through the healthy susceptible 5mL of intratracheal injection 7 ~ 15 age in days, and cut open inspection after 28 days, typical porcine mycoplasmal pneumonia pathology appears in visible Pigs Inoculated lung.
2.5 immunogenicities make vaccine by the present invention, the healthy susceptible pig of intramuscular injection immunity 7 ~ 15 age in days, booster immunization again after 14 days; After head exempts from 35, together with the contrast pig that condition is identical, organize strong malicious intratracheal injection to attack poison with mycoplasma hyopneumoniae Js, attack poison and cut open inspection after 28 days, visible immune swine significantly can reduce porcine mycoplasmal pneumonia tuberculosis and become.
The preparation of 3 production bacterial classifications
Get mycoplasma hyopneumoniae NJ strain freeze-drying lactobacillus, after liquid nutrient medium dilution, inoculation solid medium, cultivate 3 ~ 10 at 37 DEG C, the bacterium colony that growth selection is good, purely after the assay was approved, as first order seed.Get first order seed inoculation liquid nutrient medium again, at 37 DEG C, cultivate pH value after 3 ~ 7 days reduce to 6.6 ~ 7.0 results, through purely after the assay was approved, as secondary seed.
The 4 seedlings preparation of bacterium liquid
Get secondary seed inoculation liquid nutrient medium, cultivate 3 ~ 7 for 37 DEG C, after culture variable color, carry out enlarged culturing, subculture was no more than for 6 generations, gathers in the crops, purely check and assay according to the regulation of Chinese veterinary pharmacopoeia when pH value reduces to 6.6 ~ 7.0.
The deactivation of 5 antigens
The seedling be up to the standards bacterium liquid is added the Thiomersalate that final concentration is 0.005%, and 37 DEG C are carried out deactivation.Getting inactivated samples 1mL joins in the liquid nutrient medium of 50mL, puts 37 DEG C of cultivations, observes 5, then gets 0.5mL and be inoculated in 4.5mL liquid nutrient medium, and put 37 DEG C 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 PBS supplies.
6.1 injection white oils join seedling and injection white oil 94 parts (weight ratio) is got in mixing, Si Ben-806 parts, add in oil phase tank, stir, through 121 DEG C of sterilizings 30 minutes, be cooled to room temperature for subsequent use.Get the bacterium liquid 96 parts that deactivation is up to the standards, tween-80 4 parts, add in aqueous phase tank, stirring and evenly mixing is for subsequent use.Oil phase 2 ~ 3 parts is added in emulsion tank, first stirring at low speed 30 minutes, more slowly add 1 part of aqueous phase, continue to stir, carry out emulsification with high-speed shearing machine, before emulsification terminates, add final concentration is 0.005%(g/mL) Thiomersalate solution, make water-in-oil emulsion.
6.2ISA206VG(SEPPIC), ISA201VG(SEPPIC), Gel01ST(SEPPIC), ISA11RVG(SEPPIC) join seedling and mixing carry out to specifications joining seedling and mixing.As ISA201VG, join seedling by antigen and adjuvant weight ratio 50:50, adjuvant is preheated to 31 DEG C, stir with low-shearing power, maintain 30 DEG C, add antigen (aqueous phase) in adjuvant, stir homogeneous.As ISA11R VG, by the proportions of adjuvant and antigen weight ratio 15:85, under room temperature or lower temperature, antigen aqueous phase is added in adjuvant and stir homogeneous with low shear force agitator.As Gel01ST, adjuvant joins seedling by 5% ~ 20%, is added by antigen aqueous phase in adjuvant and stir homogeneous with low shear force agitator under room temperature or lower temperature.
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, with 3000r/min centrifugal 15 minutes, and the aqueous phase of separating out at the bottom of pipe should be no more than 0.5mL.
7.3 viscositys are tested by existing " Chinese veterinary pharmacopoeia " annex, are no more than 200cP.
7.4 steriling tests are tested by existing " Chinese veterinary pharmacopoeia " annex, asepsis growth.
7.5 safety verifications are with the healthy susceptible pig 5 of 7 ~ 15 ages in days, and each intramuscular injection vaccine 2mL, observed after 14 days, except the transient fervescence of appearance, vaccinate of having no way of cause other locally and systemic adverse reactions.
7.6 efficacy tests, with the healthy susceptible pig 15 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, often organize 5.The each intramuscular injection vaccine of vaccine immunity group 1 part, after 14 days, more each intramuscular injection vaccine 1 part.Head exempts from latter 35 days, together with attacking poison contrast pig 5, with the strong poison of the 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%, by above-mentioned formulae discovery porcine mycoplasmal pneumonia vaccine immunity group thumps varying index decrement.
The 7.7 Thiomersalate determination of residual amount measure by existing " Chinese veterinary pharmacopoeia " annex, should meet the regulation of veterinary biologics general rule.
8 intramuscular injection piglets every intramuscular injection, 1 part.
Embodiment 3: vaccine.
1 vaccine
Porcine mycoplasmal pneumonia inactivated vaccine (NJ strain) oily adjuvant, porcine mycoplasmal pneumonia inactivated vaccine (NJ strain) ISA11RVG, porcine mycoplasmal pneumonia inactivated vaccine (NJ strain) Gel01ST, the porcine mycoplasmal pneumonia inactivated vaccine of certain company production abroad.
The strong poison of 2 inspections
The strong poison of mycoplasma hyopneumoniae Js strain tissue, is prepared by Jiangsu Province Agriculture Science Institute veterinary institute.
3 test pig
The healthy susceptible pig 30 of 7 ~ 15 ages in days, derives from agriculture and animal husbandry tomorrow Science and Technology Ltd. of Jiangsu Province.
4 test design
With porcine mycoplasmal pneumonia inactivated vaccine (NJ strain) oily adjuvant, 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, often criticize vaccine with 7 ~ 15 age in days piglet 5, the vaccine of every intramuscular injection 1mL, after 14 days with Isodose booster immunization once.Set up the identical pig of condition to attack malicious control group and each 5 of normal healthy controls group 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, attack poison and cut open inspection after 28 days, statistics pulmonary lesion was scored, and calculates tuberculosis varying index decrement.
5 clinical responses are observed
Test pig observes 28 after vaccinate, and the test pig of 3 immune group all occurs transient heating after immunity as a result, but all recovers normal in latter three days of immunity.Have no the reaction of other abnormal clinical.
6 effect comparison tests
Test pig is attacked in after malicious 28 days, and observe in discovery 5 first tap poison control group pig and have 4 appearance coughs in various degree, 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 group tuberculosis varying index, calculate tuberculosis varying index decrement, 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 remarkable in attacking malicious control group and import vaccine group.
Table 1 similar vaccine potency comparison test result
Note: a ~ d represents and often compares between two between group, and alphabetical identical person represents that difference is not remarkable, and alphabetical different person represents significant difference (p<0.05).
Claims (1)
1. a strain Vaccine strain for mycoplasma pneumonia of swine, its Classification And Nomenclature is mycoplasma hyopneumoniae (Mycoplasmahyopneumoniae), and bacterial strain number is NJ, is preserved in China typical culture collection center, deposit number: CCTCCNO:M2012286, preservation date: on July 16th, 2012.
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| 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 |
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