CN100419073C - Porcine asthma live vaccine and its production method - Google Patents

Abstract

The invention relates to a swine asthma live vaccine and a production method thereof. This live vaccine is to use a Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) RM48 strain adapted to grow on a kind of Mycoplasma hyopneumoniae Lps culture medium designed by the present invention, add freeze-drying protection agent after cultivating, and make live vaccine by vacuum freeze-drying . There are 17 R1 (AAKPV/E) repeats in the P97 gene of this strain, which can be used to distinguish other strains. The live vaccine is safe and effective, and can be used for thoracic injection or nasal spray inoculation to immunize pigs.

Description

Porcine asthma live vaccine and its production method

technical field

The invention relates to a live vaccine for preventing infectious diseases of pigs, which belongs to the field of veterinary biological products.

Background technique

Swine panting disease, also known as swine mycoplasma pneumonia or porcine endemic pneumonia, is a contact chronic respiratory infectious disease caused by Mycoplasma hyopneumoniae, which is prevalent all over the world. Affected pigs mainly showed cough and wheezing, growth retardation, low feed conversion rate, and normal body temperature. During anatomy, the main lesions were in the lungs, especially in the heart lobes, middle lobes, and apical lobes of the two lungs, which were characterized by pancreatoid changes and fleshy changes. High morbidity and low mortality. Studies in recent years have found that Mycoplasma hyopneumoniae is mixed with porcine reproductive and respiratory syndrome virus and other pathogens, which further increases the importance of infection. So far, the disease is still one of the most important diseases causing economic losses in pig farming.

Mycoplasma hyopneumoniae has very strict requirements on the nutritional conditions of the culture medium, and it is difficult to grow in general culture medium.

Swine panting disease is a worldwide problem faced by the pig industry, and vaccination is the most effective way to prevent and control the disease. However, although the research on vaccines began in the 1960s, it was not until the 1990s that hope began to appear. Attenuated, bred a low-virulence, good immunogenic Mycoplasma hyopneumoniae rabbitized attenuated strain (Chinese invention patent ZL 86108515, production of Mycoplasma hyopneumoniae attenuated vaccine), however, due to the long-term use of this strain in sucking rabbits Passaging in vivo almost loses the ability to grow in the culture medium, and cannot adapt to large-scale vaccine production under factory conditions.

Contents of the invention

The purpose of the present invention is to make the Mycoplasma hyopneumoniae attenuated vaccine vaccine strain R790 strain that has lost the ability to grow in the medium can grow in the medium and maintain good immunity by using different medium and different methods of isolation and cultivation. Originality, produce live vaccine (also known as Mycoplasma hyopneumoniae live vaccine) to prevent swine panting disease with factory production method.

The present invention is that mycoplasma hyopneumoniae mycoplasma hyopneumoniae lathe attenuated vaccine production production bacterial strain R790 strain (Chinese invention patent ZL 86108515, the production of mycoplasma hyopneumoniae attenuated vaccine) adapted culture medium obtained the culture medium adapted bacterial strain, this bacterial strain is carried out A series of identifications were carried out, and the results of the identification of growth characteristics and serological characteristics showed that the strain conformed to the characteristics of Mycoplasma hyopneumoniae, and was determined to be Mycoplasma hyopneumoniae, and it was named Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) MR48 strain (this strain On September 19, 2006, it was sent to the General Microorganism Center of China Microorganism Culture Collection Management Committee in Zhongguancun, Beijing, and the preservation number: CGMCCNo.1816).

specific implementation plan

1 strain cultivation

The mycoplasma hyopneumoniae rabbitized attenuated R790 strain (Chinese invention patent ZL 86108515, production of mycoplasma hyopneumoniae attenuated vaccine) cultivated by me was repeatedly passaged in Lps medium to obtain a medium-adapted strain, which was named Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae). hyopneumoniae) MR48 strain (this bacterial strain has been sent to Beijing Zhongguancun China Microorganism Culture Collection Management Committee General Microbiology Center on September 19, 2006, preservation number: CGMCCNo.1816).

2 Characteristics of attenuated strain of Mycoplasma hyopneumoniae MR48.

2.1 Morphological and biochemical characteristics are ring-shaped, spherical, filamentous, rod-shaped and point-shaped polymorphic microorganisms, without cell walls, and Gram staining is negative. The serological and biochemical characteristics should conform to the characteristics of the bacteria in bacterial taxonomy.

2.2 Culture characteristics It grows well in the liquid medium, and after 4-10 days of culture at 37°C, the medium drops by more than 0.5 pH value, showing slight turbidity. The live bacterial titer of Mycoplasma hyopneumoniae in the culture can reach between 10 ⁸ and 10 ⁹ CCU/ml (CCU-color change unit). After inoculating Lps solid medium and culturing at 37°C for 7 to 14 days, fried egg-like colonies can be seen.

Lps liquid medium formula (according to 1000ml): PPLO 10.0～25.0g, NaCl 2.0～5.0g, KCl 0.2～1.0g, MgSO ₄ 0.1～0.4g, KH ₂ PO ₄ 0.2～0.6g, Na ₂ HPO ₄ * 12H ₂ O 2.0～8.0g, glucose 5.0～10.0g, hydrolyzed milk protein 1.0～5.0g, 2.5% thallium acetate 5～10ml, 25% yeast extract 100～200ml, 10×MEM 10～50ml, beef heart soup or lung Soup 100-200ml, FeSO ₄ 0.001-0.01g, phenol red 1% 0.5-2.0ml, add distilled water to 800ml, filter and sterilize, add fresh pig serum 100ml and fresh horse serum 100ml, adjust pH to 7.3-7.6 for later use; Add 1.5% agar to the liquid medium to become Lps solid medium.

2.3 Serological characteristics

2.3.1 Identified by metabolic inhibition test In the liquid culture medium added with Mycoplasma hyopneumoniae antiserum, after 15 days of constant temperature cultivation at 37°C, the pH value did not drop, showing specific inhibition of glucose metabolism.

2.3.2 Identification by Growth Inhibition Test No colonies appeared on the surface of the solid culture medium added with antiserum of Mycoplasma hyopneumoniae at 37°C for 10-20 days, showing specific growth inhibition of Mycoplasma hyopneumoniae.

2.4 Safety inspection

2.4.1 The strains were collected from mice for security inspection and diluted with normal saline to 10 ⁷ CCU/ml, subcutaneously injected into 3 clean-grade mice with a body weight of 18-22g, 0.4ml/mouse, observed for 10 days, and all of them were healthy and alive.

2.4.2 Safety inspection of pigs For each batch of cultures, 10 times the immune dose (5×10 ⁶ CCU) was used to inoculate 3 healthy susceptible pigs aged 30 to 50 days in the chest cavity, and 3 blank controls were set up, and the observation was carried out for 20 days. All the test pigs had no clinical symptoms, and the pigs' lungs were normal after autopsy, and there was no pathological change of swine panting disease.

2.5 Immunogenicity test The immunization methods (nasal spray and intrapulmonary injection) were divided into 9 groups for immunization, and a non-immunization control group was set at the same time. 55 days after immunization, a total of 30 pigs were challenged with the virulent F65 strain of Mycoplasma hyopneumoniae together with control pigs, and all were slaughtered 30 days after the challenge to check for lung lesions. The test results showed that the protective effects of intrapulmonary injection route and intranasal spray immunization in pigs were 90% and 55%, respectively.

2.6 Molecular biological identification (determination of P97 gene sequence) The P97 gene sequence of the strain was determined according to the method of Ding Fang et al. The Mycoplasma hyopneumoniae MR48 strain retains the characteristics of the Mycoplasma hyopneumoniae R659 strain, that is, the R1 (AAKPV/E) repeat sequence of the P97 gene of the MR48 strain and the R659 strain is also 17, which is obviously different from other strains. strain.

3. Vaccine preparation The seed solution of Mycoplasma hyopneumoniae MR48 strain cultured in Lps liquid medium was inoculated in Lps liquid medium at a ratio of 1:10 (volume ratio). Store at 37°C for 3 to 6 days, and the culture will drop by more than 0.5 pH value. After passing the pure inspection, expand the culture in the same way. Up to the amount required for seedling production (the number of subcultures should not exceed 6 generations), add the freeze-dried protective agent in equal proportions, mix thoroughly, and quantitatively dispense according to the prescribed portion, containing no less than 10 ⁸ CCU/ml of viable bacteria. After freeze-drying in a vacuum, a porcine wheezing disease live vaccine is prepared.

4 Quality Standards for Mycoplasma Hyopneumoniae Live Vaccine (MR48 Strain)

4.1 Determination of color change unit (CCU) Measured according to the CCU determination method, the number of bacteria per head of freeze-dried seedlings is ≥5×10 ⁶ CCU.

4.2 Safety inspection

4.2.1 Mice Use 3 mice weighing 18-22 g, restore the vaccine to its original volume with normal saline, inoculate each subcutaneously with 0.4 ml, and observe for 10 days. All should be healthy.

4.2.2 For each batch of pig cultures, use 10 times the immune dose, that is, take 15 bottles (2 heads/bottle) of the vaccine and dilute it with normal saline to a total volume of 15ml, and inoculate 3 healthy susceptible pigs aged 30 to 50 days in the chest cavity. , 3ml per head, and 3 blank control heads were set at the same time, and observed for 20 days. The pigs for security inspection should have no clinical symptoms. Otherwise, it should be checked again.

4.3 Efficacy test (immune immune response) Use 5 big-eared white rabbits with a body weight of 1.5-2.0kg, take 3 bottles of vaccine (2 heads/bottle) and dilute the vaccine with normal saline to a total volume of 12ml, and each big-eared white rabbit Inject 2ml into each lung, collect blood on the 30th day, and use the indirect hemagglutination method to check the serum agglutination antibody, at least 4 should be positive (hemagglutination value ≥ 10++), otherwise, retest once.

4.4 The immunization period is 6 months.

4.5 Usage and dosage Inject into the right chest cavity or intranasally spray, inoculate 1 dose per pig.

Advantages of the present invention:

A Mycoplasma hyopneumoniae RM48 strain involved in the present invention is adapted by repeated subculture on a kind of Mycoplasma hyopneumoniae vaccine medium designed by the present invention, and can be industrialized For live vaccine production, the mycoplasma strain retains the immunogenicity and safety of the original strain, and its molecular biological characteristic of 17 R1 (AAKPV/E) repeats in the P97 gene of the strain can be used to distinguish other strains. The Mycoplasma hyopneumoniae live vaccine produced by the strain can be injected into the chest cavity or vaccinated by nasal spray inoculation.

Claims (4)

1. a strain is used to produce the mycoplasma hyopneumoniae bacterial strain of live vaccine for mycoplasma pneumonia of swine, it is characterized in that this strain mycoplasma hyopneumoniae 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: CGMCC No.1816 on September 19th, 2006.

2. a live vaccine for mycoplasma pneumonia of swine is characterized in that this living vaccine contains MR48 strain mycoplasma hyopneumoniae and the lyophilized vaccine that preserving number is CGMCC No.1816.

3. a kind of live vaccine for mycoplasma pneumonia of swine as claimed in claim 2 is characterized in that this living vaccine is with thoracic cavity injection or with nasal spray inoculation method immune swine.

4. the production method of a live vaccine for mycoplasma pneumonia of swine is characterized in that:

1) the seedling bacterial strain of this vaccine is that preserving number is the mycoplasma hyopneumoniae MR48 strain of CGMCC No.1816;

2) the seedling substratum is the Lps liquid nutrient medium, and its prescription is counted by 1000ml: PPLO 10.0～25.0g, NaCl2.0～5.0g, KCl 0.2～1.0g, MgSO ₄0.1～0.4g, KH ₂PO ₄0.2～0.6g, Na ₂HPO ₄12H ₂O 2.0～8.0g, glucose 5.0～10.0g, lactoalbumin hydrolysate 1.0～5.0g, 2.5% thallium acetate, 5～10ml, 25% yeast leach liquor, 100～200ml, 10 * MEM, 10～50ml, OX-heart soup or lung soup 100～200ml, FeSO ₄0.001～0.01g, 1% phenol red 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;

3) the bacterium seed liquor is inoculated in the Lps liquid nutrient medium with 1: 10 by volumetric ratio; cultivated 3～6 down in 37 ℃; culture descends more than 0.5 pH value; be up to the standards purely, enlarged culturing in the same way again is 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.