CN113755368B - Fujian chicken mycoplasma synoviae and culture medium thereof - Google Patents
Fujian chicken mycoplasma synoviae and culture medium thereof Download PDFInfo
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Abstract
The invention provides a strain of mycoplasma synoviae and a culture medium thereof. The mycoplasma synoviae strain is separated from the sick chicken adnexal joint with the mycoplasma synoviae in Fuzhou, and belongs to a typical strain in Fujian province. The strain is preserved to China center for type culture Collection, the preservation address is China, wuhan university, the preservation number is CCTCC NO: M2021210, and the preservation date is No. 3/8 in 2021. The culture medium for culturing the mycoplasma synoviae comprises the following components: brain heart leachate broth, yeast leaching powder, inactivated fetal calf serum, L-cysteine solution, arginine solution, phenol red solution and coenzyme I solution, and the pH value is 7.6-7.8. Inoculating the mycoplasma synoviae to the culture medium, placing the culture medium in an incubator at 37 ℃, standing and culturing for 36h to obtain the growth peak value of the mycoplasma synoviae, wherein the viable count of the cultured mycoplasma synoviae is not less than 10 12 CCU/ml。
Description
Technical Field
The invention belongs to the technical field of pathogen separation identification and pathogen culture, and particularly relates to mycoplasma synoviae and a culture medium thereof.
Background
Mycoplasma Synoviae (MS) is an acute or chronic infectious disease that mainly causes respiratory symptoms and infectious synovitis symptoms in chickens and turkeys, which can occur all the year round, but is more prevalent in winter and spring. Mycoplasma synoviae can directly invade chicken cartilage tissues, and causes the swelling and swelling of joints, inflammation of bursa and tendon sheaths, lameness and paralysis of chickens; the infection of the mycoplasma synoviae can also reduce the laying rate, the egg quality and the hatchability of laying hens, reduce the feed conversion rate, increase the ketone body abandonment rate, and the mycoplasma synoviae can be vertically spread, thereby bringing great difficulty to the extinction of the mycoplasma synoviae of chicken flocks. At present, the mycoplasma synoviae is widely existed in chicken farms all over the world, and the mycoplasma synoviae is easy to cross-infect with other pathogens, thus exacerbating the pathogenicity of each pathogen, increasing the death rate and causing serious economic loss to the chicken industry.
The mycoplasma of the bursa of chicken belongs to the mycoplasma of the mollicutes, has the diameter of about 0.2 to 0.4 mu m, can propagate through a bacterial filter of 0.45 mu m in a geminization mode or a binary fission method, has good effect after the dyeing of the Jiemsa, has polymorphism when observed under an electron microscope, and is mostly circular or pear-shaped; the mycoplasma synoviae has only one serotype, and the pathogenicity of different strains is different, so that the caused symptoms are different due to the tropism of the pathogens. Mycoplasma synoviae has characteristics of common mycoplasma: i.e., fermented glucose, does not hydrolyze arginine, and does not utilize urea. Mycoplasma synoviae is a prokaryotic organism without cell wall, and is insensitive to macrolide antibiotics influencing cell wall synthesis due to the lack of cell wall, such as penicillin and the like; however, they are more sensitive to environmental factors than those pathogens with cell walls, mycoplasma synoviae can survive in feathers for only 3 days, can survive in cotton fabrics for only 2 days, and can die after 12 hours in wood and straw, so mycoplasma synoviae has more complex nutritional requirements, and during artificial culture, nicotinamide adenine dinucleotide (coenzyme I) needs to be added into a culture medium, bovine or pig serum needs to be added, and the optimal culture temperature is 37 ℃.
Disclosure of Invention
The invention aims to provide a strain of mycoplasma synoviae and a culture medium thereof. The mycoplasma synoviae strain cultured and separated by using the mycoplasma synoviae culture medium has the advantages of simple and convenient culture method, short culture period and less required reagents, and has good application prospect in culturing and producing the mycoplasma synoviae.
In order to achieve the above purpose, the technology adopted by the invention is as follows:
a strain of mycoplasma synoviae is named as follows:MycoplasmasynoviaeMS-FJ 01; has been preserved to China Center for Type Culture Collection (CCTCC) with a preservation address of China, wuhan university with a preservation number of M2021210 and a preservation date of No. 3/8 in 2021.
The Fujian chicken synovial bursa mycoplasma is separated from a diseased chicken adnexal joint with the Fuzhou chicken synovial bursa mycoplasma, purified and cultured, and bacteriological identification and animal infection tests show that the pathogenic characteristics of the strain are stable.
The nucleotide sequences of a pair of specific primers for detecting the mycoplasma synoviae are as follows:
MS-JD506-1：5’-CTTCTATGCTTAAACTTTCC-3’，
MS-JD506-2：5’-TAAAGATATTACAACGACAT-3’。
the mycoplasma synoviae culture medium comprises the following components: 21 g of brain-heart leachate broth, 5.6 g of yeast leaching powder, 2 ml of 1wt% phenol red solution, 2 ml of 50 mg/ml L-cysteine solution, 8 ml of 50 mg/ml arginine solution and 0.4 ml of 250 mg/ml coenzyme I solution, fixing the volume to 900ml, adding 100ml of inactivated fetal calf serum, adding 80 ten thousand units/L of penicillin, and keeping the pH value at 7.6-7.8.
Further, 1wt% of agar powder is added into the culture medium to prepare the mycoplasma synoviae optimized solid culture medium.
The application of the mycoplasma synoviae culture medium in culturing the mycoplasma synoviae is disclosed.
The aforementioned Mycoplasma synoviaeMycoplasma synoviaeApplication of MS-FJ01 in preparing mycoplasma synoviae vaccine.
The invention has the beneficial effects that:
1. mycoplasma synoviae of chicken in the inventionMycoplasma synoviae MS-FJ01 belongs to a typical strain in Fujian province, has stable pathogenic characteristics, and can cause SPF (specific pathogen free) chickens to have typical joint swelling symptoms.
3. The chicken mycoplasma synoviae original culture medium needs few reagents, is simple and convenient in culture method and short in culture period, and can effectively inhibit the influence of infectious microbes on mycoplasma synoviae.
4. Mycoplasma synoviae (Mycoplasma gallisepticum)Mycoplasma synoviae The MS-FJ01 reaches the growth peak value after being cultured for 36h in the mycoplasma synoviae culture medium, and the viable count of the cultured mycoplasma synoviae is not less than 10 12 CCU/ml。
Description of the drawings:
FIG. 1 is a bacterial colony of Mycoplasma synoviae.
FIG. 2 is a graph showing clinical symptoms after challenge of M.synoviae to SPF chickens.
FIG. 3 is a phylogenetic tree analysis of M.synoviae 16s rRNA gene.
FIG. 4 shows Mycoplasma synoviaeMycoplasma synoviae MS-FJ01 nucleic acid electrophoresis identification chart, wherein M is Marker,1 is identification result of designed primer, and 2 is identification result of primer of cited reference.
FIG. 5 is a graph showing the growth of Mycoplasma synoviae cultured in four different formulations of Mycoplasma synoviae optimized media.
The specific implementation mode is as follows:
the experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
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
Collecting joint fluid and contents in a swollen tarsal joint cavity of a sick chicken suspected to be infected with mycoplasma synoviae in a Fuzhou chicken farm, inoculating the joint fluid and the contents in the swollen tarsal joint cavity of the sick chicken in the Fuzhou chicken farm under an aseptic operation condition, placing the joint fluid and the contents in an improved Frey's liquid culture medium for constant-temperature shaking culture at 37 ℃ for 48 hours, filtering the bacteria fluid by using a 0.45-micrometer filter when the bacteria fluid turns yellow from red, re-inoculating the bacteria fluid into the improved Frey's liquid culture medium for re-culture according to the volume ratio of the filtrate to the improved Frey's liquid culture medium of 1:10, and collecting the 2 nd generation of bacteria fluid for identifying pathogenic bacteria.
(II) identification of chicken mycoplasma synoviae
1. Morphological identification of thallus
Smear the separated 2 nd generation bacterium liquid, and make microscopic examination after giemsa staining, and the observed thallus morphology is characterized by spherical or club type.
2. Colony morphology observation
Inoculating the filtered strain to modified Frey's solidOn a volume medium and placed in a volume fraction of 5% CO 2 The cells were cultured in an incubator at 37 ℃ for 7 days, and observed under a microscope to find fine, smooth and compact microcolonies, which appeared as "fried eggs" and were semi-recessed in the medium (see FIG. 1).
3. L-shaped identification of thallus
Inoculating the separated 2 nd generation bacterium liquid into an improved Frey's liquid culture medium without penicillin and thallium acetate, continuously subculturing for 10 times, inoculating the culture bacterium liquid into an improved Frey's solid culture medium, culturing at a constant temperature of 37 ℃, observing colony forms and performing smear microscopy. The results show that the isolated bacteria are negative for the L-form of the bacteria.
4. Adsorption test of chicken red blood cells
Preparing 1wt% chicken erythrocyte suspension, taking 5 mL suspension on the surface of an improved Frey's solid culture medium cultured with the separating bacteria, incubating for 20 min at room temperature, discarding the suspension, washing for 3 times by using normal saline, and observing the erythrocyte adsorption condition under a low power microscope. The surface of the colony can be observed to have red blood cells adsorbed under a low power microscope.
3. Biochemical identification
Preparing 90ml of a basic culture medium by using 2.1g of brain-heart leachate broth (microorganism cyclokava), 10vol% of pig serum, 1.12g of yeast leaching powder and 1wt% of phenol red, respectively preparing 10wt% of glucose, lactose, arginine and urea, and respectively adding 10ml of the 10wt% of solutions into the 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 from a basic culture medium, adding 2wt% of triphenyltetrazolium chloride to prepare a tetrazolium reduction test culture medium, and then filtering and sterilizing. The isolated 2 nd generation bacterial liquid was cultured in heart infusion broth to which 10vol% pig serum was added for 24 hours, and 1ml 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.
6. Identification of Artificial infections
0.5 mL of the separated generation 2 bacterial liquid is taken, and is injected into SPF chicks by dropping nose and eyes and injecting into foot pads, and after 40 days, the infected chickens have obvious clinical symptoms such as lameness, paralysis, arthrocele, cheese-like sediments at keel positions and the like (see figure 2).
7. Evolutionary tree analysis of 16s rRNA Gene
Amplifying the 16s rRNA gene of the mycoplasma synoviae strain, sequencing the amplified gene sequence, and comparing the sequence with other known mycoplasma synoviae strains and performing phylogenetic tree analysis. As shown in fig. 3, the mycoplasma synoviae strain has higher homology with asian mycoplasma synoviae strain.
7. Molecular biological identification
According to the whole 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 are MS-JD506-1:5 'CTTCTATGCTTAAACTTTCC-3', MS-JD506-2:5'-TAAAGATATTAC-AACGACAT-3', the expected size of the target fragment amplified by the two primers is 506 bp. And synthesizing a pair of primers with the target fragment size of 208bp according to the second version of mycoplasma science compiled by Wushimur, wherein the primer pair comprises MS-208-F:5 'GAAGCAAAATAGTGATATCA-3', MS-208-R:5' GTCGTCCGAAGTTAACAA-. The isolate was PCR amplified with M.synoviae specific primers, as shown in FIG. 4, and M.synoviae strains amplified specific fragments of about 506 bp and 208 bp.
The identification results show that the separated mycoplasma synoviae strains all accord with the characteristics of the mycoplasma synoviae.
(III) preservation of Mycoplasma synoviae
Culturing the mycoplasma synoviae to a stable period, collecting bacterial liquid of 60-100ml, centrifugally collecting mycoplasma synoviae bacterial precipitates by using an ultra-high speed centrifuge, suspending the precipitates by using 18% skim milk, freeze-drying and storing by using a freeze-dryer, and sending the precipitates to a China Center for Type Culture Collection (CCTCC) for storage. The mycoplasma synoviae is classified and named as:MycoplasmasynoviaeMS-FJ01, the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation address is China, wuhan university, the preservation number is CCTCC NO: M2021210, and the preservation date is No. 3/8 in 2021.
Example 2 preparation of Mycoplasma synoviae Medium
Preparation of different formula mycoplasma synoviae liquid culture medium
1. Taking brain-heart leaching liquid broth, yeast leaching powder, phenol red solution, L-cysteine solution, arginine solution and coenzyme I solution, mixing and dissolving, and fixing the volume to 900ml by using deionized water.
2. The pH of the culture solution was adjusted to 7.6 to 7.8 with hydrochloric acid.
3. The preparation is carried out aseptically by adding 100ml of inactivated fetal bovine serum to the above medium and filtering the medium using a 0.22 μm bacterial filter into a new sterile container and finally adding 80 ten thousand units/L of penicillin.
4 kinds of mycoplasma synoviae liquid culture media with different component proportions are prepared, the specific scheme is shown in table 1, and therefore the optimal scheme for culturing the mycoplasma synoviae culture medium is screened out.
Table 1: component ratio of mycoplasma synoviae liquid culture medium
(II) determination of growth curve of chicken mycoplasma synoviae
Separating the isolated Mycoplasma synoviaeMycoplasma synoviaeThe MS-FJ01 bacterial liquid is prepared according to the following steps of 1:10 volume ratio of the culture medium is inoculated in the 4 culture mediums, the culture medium is placed in an incubator at 37 ℃ for static culture, 1ml of bacterial liquid is extracted every 12 hours, and an ultraviolet spectrophotometer is used for drawing a growth curve of the mycoplasma synoviae under the excitation light of 620 nm. The growth curve results of the liquid culture medium of different schemes show that: the mycoplasma synoviae in the solution four medium reached the peak growth after 36h and then entered the stationary phase, while the mycoplasma synoviae in the solution one and the solution three medium hardly grew (see fig. 5). This may be due to the effect of too high a yeast extract meal on the growth of M.synoviae.
(III) viable count of Mycoplasma synoviae
Separating the isolated Mycoplasma synoviaeMycoplasma synoviaeMS-FJ01 bacterial liquid is inoculated in the 4 liquid culture mediums according to the volume ratio of 1. The viable bacteria counting results of liquid culture mediums of different schemes show that: viable count of the mycoplasma synoviae in the culture medium of the second scheme is not lower than 10 at 24h, 36h and 48h 12 The viable count results of the mycoplasma synoviae in CCU/ml and the four culture mediums of the scheme are all lower than 10 9 CCU/ml。
Since the mycoplasma synoviae in the schemes one and three does not grow, and the culture effect of the liquid culture medium in the scheme four is relatively poor due to the fact that the nutrient substances are too low, the scheme two-liquid culture medium is finally selected as the optimal mycoplasma synoviae liquid culture medium.
In conclusion, the mycoplasma synoviae strain of the inventionMycoplasma synoviaeThe MS-FJ01 has stable pathogenic characteristics and can be used as an advantageous strain for preparing the inactivated vaccine of the mycoplasma synoviae.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> Fujian chicken mycoplasma synoviae and culture medium thereof
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> MS-JD506-1
<400> 1
cttctatgct taaactttcc 20
<210> 2
<211> 20
<212> DNA
<213> MS-JD506-2
<400> 2
taaagatatt acaacgacat 20
<210> 3
<211> 20
<212> DNA
<213> MS-208-F
<400> 3
gaagcaaaat agtgatatca 20
<210> 4
<211> 20
<212> DNA
<213> MS-208-R
<400> 4
gtcgtctccg aagttaacaa 20
Claims (1)
1. A mycoplasma synoviae (A)Mycoplasma synoviae) MS-FJ01, characterized in that, it is preserved in China center for type culture Collection, the preservation address is Wuhan, china, the preservation number is CCTCC NO: M2021210, the preservation date is 2021 year, 3 months and 8 days.
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