CN113957007B

CN113957007B - Inactivated vaccine for mycoplasma synoviae

Info

Publication number: CN113957007B
Application number: CN202111186197.9A
Authority: CN
Inventors: 陈吉龙; 李训良
Original assignee: Fujian Agriculture and Forestry University
Current assignee: Fujian Agriculture and Forestry University
Priority date: 2021-10-12
Filing date: 2021-10-12
Publication date: 2022-09-27
Anticipated expiration: 2041-10-12
Also published as: CN113957007A

Abstract

The invention discloses a chicken mycoplasma synoviae inactivated vaccine, which comprises an antigen and a vaccine adjuvant, wherein the antigen is an inactivated mycoplasma synoviae MS-FJ01 strain; the strain is separated from the sick chicken adnexa of Fuzhou with chicken bursal mycoplasma, and belongs to a typical strain in Fujian province; the strain is preserved to China center for type culture Collection, the preservation address is Wuhan, China, the preservation number is CCTCC NO: M2021210, and the preservation date is No. 3/8 in 2021; the vaccine can generate antibodies with higher level, the immunity duration is long, the morbidity of the mycoplasma synoviae after immunization is obviously reduced, and the prevalence of the mycoplasma synoviae can be effectively prevented; the inactivated vaccine of the mycoplasma synoviae MS-FJ01 prepared from the strain of the mycoplasma synoviae MS-FJ01 can effectively prevent and control the disease, improve the richness of the marketable vaccine of the chicken type and provide an alternative.

Description

Inactivated vaccine for mycoplasma synoviae

Technical Field

The invention relates to the technical field of biological products for livestock, in particular to a mycoplasma synoviae inactivated vaccine.

Background

Mycoplasma Synoviae (MS) of chicken mainly causes upper respiratory diseases, air sacculitis, bursitis, lameness, paralysis and the like of poultry. Although the mortality rate is low, it is difficult to eradicate once infected in a flock, resulting in a long-term growth retardation and a drop in egg production in the flock. And the MS can be vertically transmitted, which brings great difficulty to the extinguishing of the MS. At present, MS widely exists in chicken farms all over the world, and is easy to cross-infect with other pathogens, so that the pathogenicity of other pathogens is aggravated, and huge economic loss is caused to the chicken industry.

At present, the main measures for preventing and controlling the harm of MS are three measures of chicken farm purification, antibiotic treatment and preventive inoculation. The chicken farm purification mainly comprises the steps of eliminating sick chicken flocks and breeding chicken flocks without MS infection through various detection methods, but because the scale and the management level of the Chinese poultry farm are different, the cost of chicken farm purification is high, and the chicken flock without MS infection is difficult to establish and maintain, so the method is not basically adopted. The frequent use of antibiotics not only easily causes the drug resistance of bacterial strains, but also has the risk that the drug residue influences the food safety. Vaccination is therefore the best option for the prevention and control of MS.

Two attenuated live vaccines for preventing MS exist in the market nowadays, namely a temperature-sensitive attenuated MS-H vaccine strain and a Nicotinamide Adenine Dinucleotide (NAD) independent MS1 vaccine strain. In 1998, the MS-H vaccine strain is successfully developed in Australia, is obtained by mutating Australia field isolate with N-methyl-N9-nitro-N-nitrosoguanidine, and the safety and effectiveness of the MS-H vaccine strain are determined through experimental data. However, MS attenuated vaccines suffer from several disadvantages. It can only be applied to chicken farms without MS infection, which limits its application to a large extent. Furthermore, MS attenuated vaccines carry the risk of horizontal transmission and may be transmitted to each other in the colony house of the same chicken farm, resulting in MS infection in unvaccinated birds. In contrast, inactivated vaccines can not only overcome such limitations, but because they are non-infectious, there is no risk of cross-infection or virulence reversal to other populations.

Therefore, the development of effective inactivated vaccines has important significance for the prevention and control of MS in our country.

Disclosure of Invention

In view of the above, the present invention aims to provide a mycoplasma synoviae inactivated vaccine which is reliable in implementation, convenient in preparation, and can be used for immune prevention of mycoplasma synoviae diseases.

In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:

an inactivated vaccine of mycoplasma synoviae (MS-FJ 01) comprising an antigen and a vaccine adjuvant, wherein the antigen is inactivated mycoplasma synoviae (MS-FJ 01); the Latin name of the Mycoplasma synoviae is Mycoplasma synoviae, and the Mycoplasma synoviae MS-FJ01 is preserved in China center for type culture Collection, wherein the preservation address is Wuhan, the preservation number is CCTCC NO: M2021210, and the preservation date is 2021, 3 and 8 days.

As a possible embodiment, further, the mycoplasma synoviae MS-FJ01 was inactivated with 0.2% formaldehyde.

As a possible embodiment, further, the mycoplasma synoviae MS-FJ01 is cultured in a mycoplasma synoviae medium prepared by a method comprising:

5.6g of yeast extract powder, 21g of brain-heart extract broth, 8mL of 50mg/mL arginine solution, 2mL of 50mg/mL L-cysteine solution, 2mL of 1 wt% phenol red solution and 0.4mL of 250mg/mL coenzyme I solution are dissolved by deionized water and subjected to constant volume to 900mL, then 80 ten thousand units/L of penicillin and 100mL of inactivated fetal calf serum are added, the pH is adjusted to 7.6-7.8, and the mixture is filtered by using a 0.22 mu m filter to a sterile container to complete the preparation.

Based on the vaccine scheme, the invention also provides a preparation method of the mycoplasma synoviae inactivated vaccine, which comprises the following steps:

1) adding mycoplasma synoviae MS-FJ01 into a mycoplasma synoviae culture medium with a pH value of 7.6-7.8 according to a ratio of 1:10, culturing at a constant temperature of 37 ℃ to a logarithmic phase at an oscillation speed of 180r/min to enable bacterial liquid to change from red to yellow, collecting the bacterial liquid as seed liquid, and storing at 4 ℃;

2) centrifuging the bacterial liquid prepared in the step 1) by using a high-speed centrifuge, concentrating the bacterial liquid by 50 times, and inactivating the bacterial liquid immediately after concentration;

3) bacterial liquid inactivation: adding 10% formaldehyde solution into the concentrated bacterial liquid obtained in the step 2) to enable the final concentration of formaldehyde to be 0.2%, fully and uniformly mixing, and inactivating at 37 ℃ for 24 hours at a constant temperature under an oscillation speed of 180 r/min;

4) adding the inactivated mycoplasma synoviae MS-FJ01 bacterial liquid obtained in the step 3) into a vaccine adjuvant according to the proportion of 1:2.81 to obtain a mixture, emulsifying the mixture by using a shear type dispersion emulsifying machine (HR-500), gradually increasing the speed from 3000rpm/min to 15000rpm/min, and continuously emulsifying for 30min to finish the preparation of the vaccine.

Preferably, the content of the bacterial antigen of the vaccine prepared in the step 4) is not less than 1 x 10 ¹² CCU/mL。

Based on the vaccine scheme, the invention also provides an inactivated avian vaccine, which comprises the inactivated vaccine for mycoplasma synoviae.

By adopting the technical scheme, compared with the prior art, the invention has the beneficial effects that:

1. the vaccine provided by the scheme of the invention can stimulate chicken flocks to generate higher antibody level and has long duration, the incidence of mycoplasma synoviae after immunization is obviously reduced, and the prevalence of the mycoplasma synoviae can be effectively prevented.

2. The inactivated vaccine of the mycoplasma synoviae MS-FJ01 prepared by the strain can effectively prevent and control the disease, and can improve the richness of the marketable vaccine and provide an alternative.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph showing the clinical symptoms of M.synoviae MS-FJ01 after challenge to SPF chickens.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. It is to be noted that the following examples are only illustrative of the present invention, and do not limit the scope of the present invention. Similarly, the following examples are only some but not all examples of the present invention, and all other examples obtained by those skilled in the art without any inventive work are within the scope of the present invention.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; meanwhile, materials and reagents used in the following examples are commercially available unless otherwise specified.

Example 1 isolation and identification of Mycoplasma synoviae

Isolation of Mycoplasma synoviae 1

Collecting the contents of swollen tarsal joints of sick chickens suspected to be infected with mycoplasma synoviae in Fuzhou chicken farms, inoculating the contents into an improved Frey's liquid culture medium under an aseptic operation condition, placing the medium in an incubator at 37 ℃ for constant-temperature shaking culture for 48 hours, filtering the medium by using a 0.45-micrometer filter when bacterial liquid turns from red to yellow, inoculating the filtrate into the improved Frey's liquid culture medium in a ratio of 1:10 for culture, and taking the 2 nd generation bacterial liquid for identification of pathogenic bacteria.

2 identification of Mycoplasma synoviae

2.1 morphological identification of cells

Taking 2 nd generation bacterium liquid smear, and observing by microscopic examination after Giemsa staining, wherein the shape of the thallus is characterized by a spherical shape or a club shape.

2.2 colony morphology Observation

Inoculating the filtered bacterial solution on an improved Frey's solid culture medium, and placing the culture medium at 37 ℃ in 5% CO ₂ Culturing in incubator for 7 days, observing visible tiny, smooth and compact microcolony under low power lens, expressing as "fried egg", and semi-sinking in culture medium.

2.3 identification of L-form of the bacterium

Inoculating the separated generation 2 bacterial liquid into an improved Frey liquid culture medium without penicillin and thallium acetate, continuously subculturing for 10 times, then inoculating into an improved Frey solid culture medium, culturing at a constant temperature of 37 ℃, observing colony morphology and performing smear microscopy. The results show that the isolated bacteria are negative for the L-form of the bacteria.

2.4 Chicken erythrocyte adsorption test

Adding 5mL of 1 wt% chicken erythrocyte suspension to the surface of an improved Frey's solid culture medium cultured with the isolate, incubating for 20min at room temperature, discarding the suspension, washing for 3 times by PBS, and observing that the surface of a bacterial colony has erythrocyte adsorption under a low power microscope.

2.5 Biochemical identification

Preparing 90mL of basal medium by using 2.1g of brain-heart leachate broth (microorganism of Kyork) and 10 vol% of pig serum, 1.12g of yeast extract powder and 1 wt% of phenol red; respectively preparing 10 wt% of solutions of glucose, lactose, arginine and urea, and respectively adding 10mL of the solutions into a basic culture medium; the pH of the glucose decomposition medium was adjusted to 7.6, and the pH of the medium for the arginine and urea decomposition tests was adjusted to 7.0. Removing phenol red in the basic culture medium, adding 2 wt% of triphenyltetrazolium chloride to prepare a tetrazolium reduction test culture medium, and then filtering and sterilizing. The isolated 2 nd generation bacterial liquid was added to 10 vol% pig serum in heart infusion broth and cultured for 24h, and 1mL of the culture was inoculated into the detection medium. The result shows that the strain can decompose glucose and lactose; reducing tetrazole; arginine and urea are not decomposed.

2.6 identification of Artificial infection

0.5mL of the separated generation 2 bacterial liquid is taken, and inoculated into SPF chickens of 3-5 days old through eye dropping and foot pad injection, after 25 days, the infected chickens have obvious clinical symptoms of mental depression, limping, paralysis, knee joint inflammation and swelling, cheese-like sediments in keels and air sacs and the like (see figure 1).

Evolutionary tree analysis of 2.716 s rRNA Gene

Amplifying 16s rRNA gene of the mycoplasma synoviae strain, sequencing, and performing evolutionary tree topological structure analysis with other known mycoplasma synoviae strains. The results show that the strain of mycoplasma synoviae has higher homology with the strain of mycoplasma synoviae of Asian chicken.

2.8 molecular biological identification

According to the complete sequence of the mycoplasma synoviae gene registered in GenBank, a pair of specific primers are designed and synthesized by using Primer 5 software, wherein the specific primers respectively comprise

MS-JD506-1：5’-CTTCTATGCTTAAACTTTCC-3’；

MS-JD506-2：5’-TAAAGATATTAC-AACGACAT-3’；

The size of the target fragment amplified from the two primers was expected to be 506bp, and a pair of primers of 208bp in size was synthesized according to the second edition of Mycoplasma, compiled by Wushizhu, ed,

MS-208-F：5’-GAAGCAAAATAGTGATATCA-3’；

MS-208-R：5’-GTCGTCTCCGAAGTTAACAA-3’。

the isolate is amplified by PCR with mycoplasma synoviae specific primers, and specific fragments of about 506bp and 208bp can be amplified.

The identification results show that the separated mycoplasma synoviae strains all accord with the characteristics of the mycoplasma synoviae.

3 preservation of Mycoplasma synoviae

Culturing the mycoplasma synoviae to logarithmic phase (the bacterial liquid is changed from red to yellow), collecting 60-100 mL bacterial liquid, centrifugally collecting bacterial precipitates by using an ultra-high speed centrifuge, and sending the bacterial precipitates to a China Center for Type Culture Collection (CCTCC) for preservation. The mycoplasma synoviae is classified and named as: m. synoviae MS-FJ01, Latin name: mycoplasma synoviae, preserved in China Center for Type Culture Collection (CCTCC), with the preservation address of Wuhan, China, the preservation number of CCTCC NO: M2021210, and the preservation date of No. 3/8 in 2021.

In this embodiment, the modified Frey's liquid culture medium and the modified Frey's solid culture medium are commercially available products, and thus the details thereof are not repeated.

Example 2 preparation of inactivated vaccine against Mycoplasma synoviae

2.1 preparation of seed liquid for production

Adding the mycoplasma synoviae MS-FJ01 into a mycoplasma synoviae culture medium according to the ratio of 1:10, culturing at a constant temperature of 37 ℃ to a logarithmic phase (bacterial liquid changes from red to yellow) at the oscillation speed of 180r/min, collecting the bacterial liquid as seed liquid, and storing at 4 ℃.

The preparation method of the mycoplasma synoviae culture medium comprises the following steps: 5.6g of yeast extract powder, 21g of brain-heart extract broth, 8mL of 50mg/mL arginine solution, 2mL of 50mg/mL L-cysteine solution, 2mL of 1 wt% phenol red solution and 0.4mL of 250mg/mL coenzyme I solution are dissolved by deionized water and subjected to constant volume to 900mL, then 80 ten thousand units/L of penicillin and 100mL of inactivated fetal calf serum are added, the pH is adjusted to 7.6-7.8, and the mixture is filtered by using a 0.22 mu m filter to a sterile container to complete the preparation.

2.2 amplification culture of the Miao-Miao bacterial liquid

Adding the seed solution into a mycoplasma synoviae culture medium according to the proportion of 1:10, culturing at the constant temperature of 37 ℃ to a logarithmic phase (the bacterial solution is changed from red to yellow) at the oscillation speed of 180r/min, and collecting the bacterial solution. Mycoplasma activity was calculated by CCU as follows: diluting the collected mycoplasma synoviae bacterial liquid in logarithmic growth phase to 10 times in a series of multiple ratios ^-24 In total, 24 tubes were used and 6 replicates were made. Incubating at 37 deg.C for 7 days at a shaking speed of 180r/min, observing the color change of the viable count tube, and recording the maximum dilution of the culture medium from red to yellow (i.e., if the 7 th tube changes color to the last tube, the CCU is 10% ⁷ /mL), the final viable bacteria amount of the MS-FJ01 strain is more than 10 ¹² CCU/mL。

2.3 concentration and inactivation of the Miao-Miao bacterial liquid

Resuspending the bacterial precipitation by using PBS through a high-speed centrifugation method (15000g centrifugation for 30min), concentrating the vaccine-making bacterial solution by 50 times, adding 10% formaldehyde solution into the concentrated bacterial solution to ensure that the final concentration of the formaldehyde is 0.2%, fully mixing, and inactivating at the constant temperature of 37 ℃ for 24 hours at the oscillation speed of 180 r/min.

2.4 inactivation assay

Adding the inactivated bacteria liquid into a mycoplasma synoviae culture medium according to the ratio of 1:10, observing for 14 days, and observing whether the culture medium has color change. The result shows that the inactivated bacterial liquid does not grow after being cultured for 14 days, the color of the culture medium does not change from red to yellow, and the result shows that the final concentration of the formaldehyde is 0.2 percent, the oscillation speed is 180r/min, and the constant temperature of 37 ℃ for 24 hours can fully inactivate the thalli.

2.5 vaccine preparation

Adding inactivated mycoplasma synoviae bacterial liquid into vaccine adjuvant at a ratio of 1:2.81, emulsifying the inactivated vaccine with shear type dispersing emulsifying machine (HR-500), gradually increasing the speed from 3000rpm/min to 15000rpm/min, and continuously emulsifying for 30min to complete the preparation of the vaccine (the content of thallus antigen is more than 1 × 10) ¹² CCU/mL)。

Wherein the vaccine adjuvant is MONTANIDE ISA 71VG from SEPPIC Sepetideae (Shanghai) specialty Chemicals, Inc., but it is not limited thereto.

3 inspection of finished products

3.1 appearance: 5mL of finished vaccine is put into a clean glass tube, and no impurities, no color change and the like of the vaccine can be observed.

3.2 dosage form: and (5) sucking the finished vaccine and dripping the vaccine on the surface of cold water, and observing that the vaccine does not diffuse.

3.3 stability: and (3) taking the finished vaccine into a centrifugal tube, centrifuging at 3000rpm/min for 15min, and observing that the vaccine is not layered.

3.4 sterility test: the test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and no bacteria grow.

3.5 pure test: the inspection is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the regulations are met.

3.6 and measuring the residual quantity of formaldehyde: the results are determined according to the appendix of the existing Chinese veterinary pharmacopoeia, and the results accord with the regulations of the general rules of biological products for livestock.

3.7 safety inspection: the finished vaccine is injected subcutaneously into 10 SPF (specific pathogen free) chickens of 0.5mL per chicken at the age of 14 days through the neck, and the immunized SPF chickens are observed for 14 days without local or systemic adverse reactions caused by vaccine injection and without abnormality in drinking water, food intake and mental conditions.

3.8 efficacy test: SPF chickens 14 days old were randomly divided into 2 groups, 10 in group 1 and 20 in group 2. Group 1 was given vaccine by cervical subcutaneous injection and group 2 was not immunized (control). Blood was collected from all SPF chickens every one week after immunization, and serum was separated, for a total of 4 weeks after immunization. After 4 weeks of immunization, 10 SPF chickens in group 1 and 2 were randomly selected by dropping nose and eyes to inoculate 0.5mL of Mycoplasma synoviae (thallus content at 1X 10) ¹² CCU/mL) with 10 remaining in group 2 as blank control. After the virus attack, pharyngeal swabs of the chickens are collected for PCR detection in 3 days and 5 days respectively, and the growth state and the clinical performance of the chickens are observed for 30 days. The antibody titer of mycoplasma synoviae is measured by an ELISA method, and the S/P value is calculated. The results show that the antibody level of the immunized chicken is increased, and the results are shown in the table 1.

TABLE 1 vaccine potency test results after immunization

The above description is only a part of the embodiments of the present invention, and not intended to limit the scope of the present invention, and all equivalent devices or equivalent processes performed by the contents of the present specification and the attached drawings, or directly or indirectly applied to other related technical fields, are all included in the scope of the present invention.

Claims

1. An inactivated vaccine of Mycoplasma synoviae, which is characterized in that the inactivated vaccine comprises inactivated Mycoplasma synoviae (A), (B), (CMycoplasma synoviae) MS-FJ01 and vaccine adjuvant, wherein the mycoplasma synoviae MS-FJ01 is preserved in China center for type culture Collection with the preservation address of Wuhan, China, the preservation number of CCTCC NO: M2021210, and the preservation date of 2021 years, 3 months and 8 days.

2. The inactivated vaccine of mycoplasma synoviae of claim 1, wherein the mycoplasma synoviae MS-FJ01 is inactivated with a final concentration of 0.2% formaldehyde.

3. The method for preparing the inactivated vaccine against M.synoviae according to claim 1 or 2, comprising the steps of:

1) adding the mycoplasma synoviae MS-FJ01 into a mycoplasma synoviae culture medium with the pH value of 7.6-7.8 according to the proportion of 1:10, culturing at the constant temperature of 37 ℃ to a logarithmic phase at the oscillation speed of 180r/min to enable bacterial liquid to change from red to yellow, collecting the bacterial liquid as seed liquid, and storing at the temperature of 4 ℃;

4) adding the inactivated mycoplasma synoviae MS-FJ01 bacterial liquid in the step 3) into a vaccine adjuvant according to the proportion of 1:2.81 to prepare a mixture, emulsifying the mixture by using a shear type dispersing and emulsifying machine HR-500, gradually increasing the speed from 3000rpm/min to 15000rpm/min, and continuously emulsifying for 30min to finish the preparation of the vaccine.

4. The method for preparing the inactivated vaccine against M.synoviae according to claim 3, wherein the content of the bacterial antigen of the vaccine prepared in step 4) is not less than 1X 10 ¹² CCU/mL。

5. An inactivated avian vaccine, which is characterized in that: which comprises the inactivated vaccine of M.synoviae according to claim 1 or 2.