Mycoplasma hyopneumoniae culture medium capable of remarkably improving strain titer and application of mycoplasma hyopneumoniae culture medium

Abstract

The invention provides a mycoplasma hyopneumoniae culture medium capable of remarkably improving strain titer and application of the mycoplasma hyopneumoniae culture medium, and belongs to the field of veterinary biological products. The mycoplasma hyopneumoniae culture medium is obtained by adding L-asparagine, myo-inositol, arachidonic acid, urea, histamine, sodium pyruvate, valproic acid, glyceric acid, lysophosphatidic acid, L-citrulline, L-glutamine and L-leucine into a KM2 culture medium containing Bama miniature pig serum. According to the culture medium disclosed by the invention, the growth speed and the viable bacterium titer of the mycoplasma hyopneumoniae can be remarkably improved under the condition of low serum concentration, and the production cost is reduced.

Description

Technical Field

[0001] The invention belongs to the field of veterinary biological products, and particularly relates to a Mycoplasma hyopneumoniae culture medium capable of significantly improving strain titer and shortening color change time, and a preparation method thereof. Background Art

[0002] Mycoplasma pneumonia, commonly known as swine asthma, is a chronic respiratory infectious disease caused by Mycoplasma hyopneumoniae (Mhp). It is one of the most common and difficult to purify important epidemic diseases in pigs. At present, the main measures to prevent and control Mycoplasma pneumonia infection are the use of antibiotics and vaccines. However, since Mycoplasma hyopneumoniae lacks a cell wall, antibiotics that work by destroying the cell wall are ineffective against it. In addition, due to the increasing prevalence of drug resistance, the role of vaccines in the prevention and treatment of Mycoplasma pneumonia is particularly critical. my country has successfully developed a variety of vaccines for Mycoplasma pneumonia, which have played an important role in prevention and control. However, due to the high nutritional requirements, slow growth and low bacterial density of Mycoplasma hyopneumoniae, this has increased the difficulty of studying its biology and pathogenic mechanism, and has also seriously affected the production efficiency and promotion of vaccines.

[0003] Mycoplasma hyopneumoniae has limited biosynthesis and metabolic capacity, and has strict requirements on nutrients. Pig serum needs to be added during in vitro culture, and pig serum plays a key role in the growth and reproduction of Mycoplasma hyopneumoniae. The existing culture method still relies on the addition of high-concentration pig serum. However, there are the following problems with the use of pig serum: First, the source and batch differences can significantly affect the growth rate and titer of Mycoplasma hyopneumoniae. Secondly, pathogen-negative pig serum, especially pig respiratory pathogen-negative serum, is expensive and difficult to source, resulting in high vaccine production costs, affecting the promotion and application of Mycoplasma hyopneumoniae single vaccines and combined vaccines. How to efficiently culture Mycoplasma hyopneumoniae at low serum concentrations is still an international problem that needs to be solved urgently. Summary of the invention

[0004] The object of the present invention is to provide a Mycoplasma hyopneumoniae culture medium which can significantly improve the titer of the strain, and can significantly improve the growth rate and viable bacterial titer of Mycoplasma hyopneumoniae at a low serum concentration, thereby reducing the production cost.

[0005] The purpose of the present invention is achieved by the following technical solutions:

[0006] A Mycoplasma hyopneumoniae culture medium capable of significantly improving the titer of a strain is obtained by adding L-asparagine, myo-inositol, arachidonic acid, urea, histamine, sodium pyruvate, valproic acid, glyceric acid, lysophosphatidic acid, L-citrulline, L-glutamine and L-leucine to a KM2 culture medium containing Bama Xiang pig serum.

[0007] In the present invention, the volume percentage concentration of Bama Xiang pig serum in the culture medium is 8%-20%.

[0008] In the present invention, the volume percentage concentration of Bama Xiang pig serum in the culture medium is 8%-12%.

[0009] In the present invention, the culture medium contains 3.4-4.2 mM valproic acid, 2.2-2.8 mM glyceric acid, 3.6-4.4 mM lysophosphatidic acid, 3.3-4.1 mM L-citrulline, 3.6-4.4 mM L-glutamine, and 3.2-4.0 mM L-leucine.

[0010] In the present invention, the culture medium contains 18-220 μM L-asparagine, 9-110 mM myo-inositol, 90-440 mM arachidonic acid, 180-440 μM urea, 18-110 mM sodium pyruvate, and 90-550 nM histamine.

[0011] In the present invention, the culture medium contains 18-22 μM L-asparagine, 9-11 mM myo-inositol, 90-110 mM arachidonic acid, 180-220 μM urea, 18-22 mM sodium pyruvate, and 90-110 nM histamine.

[0012] In the present invention, the culture medium contains 180-220 μM L-asparagine, 90-110 mM myo-inositol, 360-440 mM arachidonic acid, 360-440 μM urea, 90-110 mM sodium pyruvate, and 450-550 nM histamine.

[0013] The present invention also provides application of the culture medium in culturing Mycoplasma hyopneumoniae.

[0014] The culture medium of the present invention has clear components, screens out the best pig breed Bama Xiang pig serum that promotes the growth of Mycoplasma hyopneumoniae strains, and finally obtains a Mycoplasma hyopneumoniae high-titer culture medium that has a promoting effect on both strong and weak virulence strains, has clear components and significantly reduces serum concentration, provides high-titer mycoplasma for the preparation of Mycoplasma hyopneumoniae live vaccines and the production of inactivated subunit vaccines, promotes the healthy development of the pig industry, and has great theoretical significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The titers of the highly virulent strain LH of Mycoplasma hyopneumoniae were cultured in blue bottles of various culture media for 48 hours, where * indicates significant difference, p<0.05; *** indicates extremely significant difference, p<0.01.

[0016] Figure 2Titers of the virulent strain 168 of Mycoplasma hyopneumoniae in static culture in blue bottles of various culture media for 48 hours, where * indicates significant difference, p<0.05; *** indicates extremely significant difference, p<0.01.

[0017] Figure 3 The titers of the weakly virulent strain J of Mycoplasma hyopneumoniae after static culture in blue bottles of various culture media for 48 hours, where * indicates significant difference, p<0.05; *** indicates extremely significant difference, p<0.01.

[0018] Figure 4 The titers of the weakly virulent strain 168L of Mycoplasma hyopneumoniae were cultured in blue bottles of various culture media for 48 hours, where * indicates significant difference, p<0.05; *** indicates extremely significant difference, p<0.01. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only for illustrative purposes and in no way limit the scope of protection of the present invention.

[0020] The Mycoplasma hyopneumoniae strain used in the present invention comes from:

[0021] Mycoplasma hyopneumoniaepathogenic 168 strain: Strain 168 is a highly virulent strain isolated and cultured from pigs, showing typical symptoms of porcine mycoplasma pneumonia and is disclosed in the following literature: Liu, W., Xiao, S., Li, M., Guo, S., Li, S., Luo, R., et al. (2013). Comparative genomic analyses of Mycoplasma hyopneumoniaepathogenic 168 strain and its highpassaged attenuated strain. BMC Genomics 14: 80. doi: 10.1186 / 1471-2164-14-80.

[0022] Mycoplasma hyopneumoniae strain LH: It is a highly virulent strain, which is published in GenBank and has a NCBI GenBank sequence number of CP079799.

[0023] Mycoplasma hyopneumoniae strain 168L: It is a low-virulence strain produced by continuous passage of Mhp strain 168 to the 380th generation and gradual attenuation, which is disclosed in the following literature: Liu et al., 2013, Comparative genomic analyses of Mycoplasma hyopneumoniae pathogenic 168 strain and its high passaged attenuated strain.

[0024] Mycoplasma hyopneumoniae strain J: a medium-weak virulence strain, ATCC 25934.

[0025] Example 1 Effects of serum from different pig breeds and their content on the growth of Mycoplasma hyopneumoniae

[0026] This example investigates the effects of different pig breed sera and their contents on the growth of Mycoplasma hyopneumoniae strains 168, LH, 168L, and J.

[0027] Non-anticoagulated blood was collected from Landrace pigs, Duroc pigs and Bama pigs using sterile syringes. After overnight storage at 4°C, the blood was centrifuged at 3000 rpm and 4°C for 20 min, and the supernatant was obtained to obtain the serum of each breed of pigs. It is important not to cause hemolysis when collecting non-anticoagulated blood. The serum of each breed of pigs obtained should be clear brown-red in color.

[0028] Prepare the basic culture medium (i.e., serum-free KM2 culture medium): 1000 mL of Eagles solution, 10.0 g of hydrolyzed milk protein, 20 mL of fresh yeast extract, 600 mL of phosphate buffer (pH 7.4-7.6, concentration 0.01 mol / L), 4 million units of penicillin, 3.5 mL of 0.4% phenol red aqueous solution, and adjust the pH value to 7.4-7.6 with 10 g / L NaOH aqueous solution. The Eagles solution is prepared as follows: 48.47 g of MEM culture medium (Gibco) and 1.5 g of L-glutamine are dissolved in ultrapure water, and then the volume is adjusted to 5000 mL with ultrapure water to obtain Eagles solution.

[0029] Take 800 mL of the basal medium and 200 mL of Landrace pig, Duroc or Bama miniature pig serum, respectively, and mix them evenly to obtain KM2 medium containing different breeds of pig serum, at which time the serum content in the medium is 20%. Use the same method to prepare KM2 medium containing 10% Landrace pig, Duroc or Bama miniature pig serum. Use the same method to prepare KM2 medium containing 5% Landrace pig, Duroc or Bama miniature pig serum.

[0030] Take the seed batches (10 8 CCU 50First, add 1 mL of serum-free KM2 medium to each of them for reconstitution, and then inoculate them into KM2 medium containing 20% ​​Landrace pig serum at a volume ratio of 1:10. Incubate at 37°C with 5% CO 2 The cells were cultured in an incubator for 54 h (Xiong et al., 2014, Protective efficacy of a live attenuated Mycoplasma hyopneumoniae vaccine with an ISCOM-matrix adjuvant in pigs. Vet. J. 199, 268–274.) to recover, and then the same method was used to pass the cells for one generation to obtain the seed solution. The seed solution was inoculated into KM2 medium containing 20%, 10% and 5% serum of each breed of pigs at a volume ratio of 1:10, and each culture medium was repeated 3 times. The cells were cultured in a green bottle (the volume of the liquid in the 5 mL small green bottle was 2 mL) at 37°C and 5% CO 2 The color change unit (CCU) was used to determine the color change of the sample. 50 ) was used to quantify the viable titer of the Mhp strain (Leigh et al., 2008, The effects of increasing sodium chloride concentration on Mycoplasma gallisepticum vaccine survival in solution. Avian Dis. 52, 136-138.). In addition, the color change time of the Mycoplasma hyopneumoniae cultured in the culture medium containing 20%, 10% and 5% of different pig serum was observed, and the pH of the culture was detected.

[0031] Based on the test results of pH (Table 2 and Table 3), color change time (Table 4) and titer (Table 1) during the comprehensive culture process, it can be seen that when the titer reaches the highest, the corresponding pH is 6.5-6.8, and the culture medium changes from bright red to orange-yellow or close to bright yellow. Therefore, the speed of the increase in the bacterial count titer of the strain can be judged by first observing the time when the culture medium turns orange-yellow during the culture of Mycoplasma hyopneumoniae combined with the determination of pH value.

[0032] As shown in Table 1, after different toxicity Mycoplasma hyopneumoniae (Mhp) strains were cultured in vitro for 48 hours with KM2 medium containing different breeds of pig serum, the strains of Mhp cultured with Bama Xiang pig serum grew faster and the titer increased by at least 0.50 titer compared with Landrace and Duroc serum; when the serum content of each breed of pig was 20% and 10%, the titer of each strain remained unchanged, but when the serum content was reduced to 5%, the serum titer decreased significantly. As shown in Table 4, when the serum concentration was 10%, the color change time of the culture medium was close to the culture condition with a serum concentration of 20%; when the Mycoplasma hyopneumoniae strains were cultured with culture medium containing different breeds of pig serum, the color change time was significantly different. Whether it was a strong or weak toxic strain, the color change time of Mycoplasma hyopneumoniae cultured with Bama Xiang pig serum was at least 12 hours earlier than that with Landrace and Duroc serum, indicating that adding 10% Bama Xiang pig serum to the culture medium will significantly increase the growth rate of the strain. Therefore, the serum in the culture medium of Mycoplasma hyopneumoniae is selected from Bama Xiang pig serum, and the amount of serum used is 10%.

[0033] Table 1 Effect of porcine serum species and concentration on the titer of each Mhp strain after 48 hours of culture (CCU 50 / 0.2mL)

[0034]

[0035] Table 2 pH changes of Mhp strains cultured in medium containing 20% ​​serum from different pig breeds

[0036]

[0037] Table 3 pH changes of Mhp strains cultured in medium containing 10% serum from different pig breeds

[0038]

[0039]

[0040] Table 4 Effect of pig serum type and concentration on color change time of Mhp strain under laboratory blue bottle static culture conditions

[0041]

[0042] Example 2 Effects of different factors on the growth of Mycoplasma hyopneumoniae

[0043] In order to obtain a low-serum culture medium that can rapidly proliferate Mycoplasma hyopneumoniae at high titers, a large number of substances were screened, and it was found that L-asparagine, myo-inositol, arachidonic acid, urea, histamine, sodium pyruvate, valproic acid, glyceric acid (2,3-dihydroxypropionic acid), lysophosphatidic acid, L-citrulline, L-glutamine and L-leucine had a significant effect on the titer.

[0044] 1. Investigate the effects of L-asparagine, myo-inositol, arachidonic acid, urea, histamine and sodium pyruvate on the titer of virulent strains of Mycoplasma hyopneumoniae

[0045] Medium A0: 3.82 mmol valproic acid, 2.55 mmol glyceric acid (2,3-dihydroxypropionic acid), 4 mmol lysophosphatidic acid, 3.71 mmol L-citrulline, 4 mmol L-glutamine and 3.66 mmol L-leucine were added to 900 mL of basal medium (same as Example 1).

[0046] Culture medium A1: Mix 900 mL of culture medium A0 with 100 mL of Bama Xiang pig serum.

[0047] Medium A2: Medium A1 was supplemented with L-asparagine at a final concentration of 200 μM.

[0048] Medium A3: Medium A1 was supplemented with myo-inositol to a final concentration of 100 mM.

[0049] Medium A4: Medium A1 was supplemented with arachidonic acid to a final concentration of 400 mM.

[0050] Medium A5: Medium A1 was supplemented with urea at a final concentration of 400 μM.

[0051] Medium A6: Medium A1 was supplemented with sodium pyruvate to a final concentration of 100 mM.

[0052] Medium A7: Medium A1 was supplemented with histamine at a final concentration of 500 nM.

[0053] Medium A8: Medium A1 was supplemented with L-asparagine and myo-inositol at a final concentration of 200 μM.

[0054] Medium A9: Medium A1 was supplemented with arachidonic acid at a final concentration of 400 mM and 400 μM urea.

[0055] Medium A10: Medium A1 was supplemented with sodium pyruvate and histamine at a final concentration of 100 mM and 500 nM.

[0056] Medium A11: Medium A1 was supplemented with L-asparagine at a final concentration of 200 μM, myo-inositol at 100 mM, arachidonic acid at 400 μM, urea at 400 mM, sodium pyruvate at 100 mM, and histamine at 500 nM.

[0057] Take the virulent strains of Mycoplasma hyopneumoniae LH and 168 seed batches (10 8 CCU 50 The bacteria were freeze-dried and stored), and 1 mL of serum-free KM2 medium (Example 1) was added to each of them for reconstitution, and then inoculated into KM2 medium containing 20% ​​Landrace pig serum at a volume ratio of 1:10, and incubated at 37°C with 5% CO 2 The seed solution was inoculated into culture medium A1-A11 at a volume ratio of 1:10, and each culture medium was repeated 3 times. The seed solution was inoculated into culture medium A1-A11 at a volume ratio of 1:10, and the ... 2 The color change unit (CCU) was used to determine the color change of the sample. 50 ) The viable bacterial titer of the Mhp strain was quantitatively tested. In addition, the color change time during the culture of Mycoplasma hyopneumoniae in the above different culture media was observed.

[0058] Table 5 Color change time

[0059] Culture medium name Mycoplasma hyopneumoniae strain LH Mycoplasma hyopneumoniae strain 168 A1 26h 25.3h A2 26.67h 23.33h A3 20.67h 24.33h A4 22h 24.67h A5 21h 25.33h A6 21.33h 25.33h A7 21h 25h A8 17.33h 21.33h A9 17.33h 21h A10 16h 22h A11 12.67h 18h

[0060] The results of culturing the virulent strains LH and 168 were shown in the following table: Figure 1 and Figure 2 Compared with medium A1 supplemented with valproic acid, glyceric acid, lysophosphatidic acid, L-citrulline, L-glutamine, L-leucine and Bama Xiang pig serum, the growth and color change time of the strain can be advanced by 7-13 hours (Table 5). Under the static culture conditions of laboratory small green bottles, the viable titer of the highly virulent strain LH increased by 1.4 CCU. 50 The viable titer of the highly virulent strain 168 increased by 1.25 CCU 50 Under fermentation tank culture conditions, the viable bacterial titer can be increased by 1.75-2.5 CCU 50 Compared with medium A2, medium A11 increased the titer of LH strain by 0.5 and that of 168 strain by 0.5.

[0061] 2. Investigate the effects of L-asparagine, myo-inositol, arachidonic acid, urea, histamine and sodium pyruvate on the growth titer of weakly virulent strains of Mycoplasma hyopneumoniae

[0062] Medium A0: 3.82 mmol valproic acid, 2.55 mmol glyceric acid (2,3-dihydroxypropionic acid), 4 mmol lysophosphatidic acid, 3.71 mmol L-citrulline, 4 mmol L-glutamine and 3.66 mmol L-leucine were added to 900 mL of basal medium (same as Example 1).

[0063] Culture medium B1: Mix 900 mL of culture medium A0 and 100 mL of Bama Xiang pig serum.

[0064] Medium B2: Medium B1 was supplemented with L-asparagine at a final concentration of 20 μM.

[0065] Medium B3: Medium B1 was supplemented with myo-inositol to a final concentration of 10 mM.

[0066] Medium B4: Medium B1 was supplemented with arachidonic acid at a final concentration of 100 mM.

[0067] Medium B5: Medium B1 was supplemented with urea at a final concentration of 200 μM.

[0068] Medium B6: Medium B1 was supplemented with sodium pyruvate to a final concentration of 20 mM.

[0069] Medium B7: Medium B1 was supplemented with histamine at a final concentration of 100 nM.

[0070] Medium B8: Medium B1 was supplemented with L-asparagine and myo-inositol at a final concentration of 20 μM.

[0071] Medium B9: Medium B1 was supplemented with arachidonic acid at a final concentration of 100 mM and urea at 200 μM.

[0072] Medium B10: Medium B1 was supplemented with sodium pyruvate and histamine at a final concentration of 20 mM and 100 nM.

[0073] Medium B11: Medium B1 was supplemented with L-asparagine at a final concentration of 20 μM, myo-inositol at 10 mM, arachidonic acid at 100 mM, urea at 200 μM, sodium pyruvate at 20 mM, and histamine at 100 nM.

[0074] Take the low-virulence strains of Mycoplasma hyopneumoniae J and 168L seed batches (10 8 CCU 50The bacteria were freeze-dried and stored), and 1 mL of serum-free KM2 medium (Example 1) was added to each of them for reconstitution, and then inoculated into KM2 medium containing 20% ​​Landrace pig serum at a volume ratio of 1:10, and incubated at 37°C with 5% CO 2 The seed solution was inoculated into medium B1-B11 at a volume ratio of 1:10, and each medium was repeated 3 times. The seed solution was inoculated into medium B1-B11 at a volume ratio of 1:10, and the ... 2 Incubate incubator for 48 h and use 50% color change unit (CCU 50 ) The viable bacterial titer of the Mhp strain was quantitatively tested. In addition, the color change time during the culture of Mycoplasma hyopneumoniae in the above different culture media was observed.

[0075] Table 6 Color change time

[0076] Culture medium name Mycoplasma hyopneumoniae strain J Mycoplasma hyopneumoniae strain 168L B1 24.33h 27h B2 21.33h 27.33h B3 28h 22h B4 28h 22h B5 28h 21.33h B6 28.33h 21.66h B7 28h 21.33h B8 21.33h 26.67h B9 29h 26.67h B10 28.67h 26.33h B11 14h 13.33h

[0077] As shown in Table 6, Figure 3 and Figure 4 As shown, compared with medium B1, the color change time of weak virulence strain J and weak virulence strain 168L in medium B11 can be advanced by 10-14 hours. Under the laboratory small green bottle static culture conditions, the viable bacterial titer of weak virulence strain J can be increased by up to 1.25 CCU 50 For the weakly virulent strain 168L, the live bacterial titer can be increased by up to 1.2 CCU50. Under fermentation tank culture conditions, the live bacterial titer can be increased by 1.75-2.5 CCU 50 .

Claims

1. A Mycoplasma hyopneumoniae culture medium capable of significantly improving strain titer, characterized in that: The culture medium is obtained by adding L-asparagine, myo-inositol, arachidonic acid, urea, histamine, sodium pyruvate, valproic acid, glyceric acid, lysophosphatidic acid, L-citrulline, L-glutamine and L-leucine to a KM2 culture medium containing Bama Xiang pig serum.

2. The culture medium according to claim 1 or 2, characterized in that: The volume percentage concentration of Bama Xiang pig serum in the culture medium is 8%-20%.

3. The culture medium according to claim 2, characterized in that The volume percentage concentration of Bama Xiang pig serum in the culture medium is 8%-12%.

4. The culture medium according to claim 3, characterized in that: The culture medium contains 3.4-4.2 mM valproic acid, 2.2-2.8 mM glyceric acid, 3.6-4.4 mM lysophosphatidic acid, 3.3-4.1 mM L-citrulline, 3.6-4.4 mM L-glutamine, and 3.2-4.0 mM L-leucine.

5. The culture medium according to claim 4, characterized in that The culture medium contains 18-220 μM L-asparagine, 9-110 mM myo-inositol, 90-440 mM arachidonic acid, 180-440 μM urea, 18-110 mM sodium pyruvate, and 90-550 nM histamine.

6. The culture medium according to claim 5, characterized in that The culture medium contains 18-22 μM L-asparagine, 9-11 mM myo-inositol, 90-110 mM arachidonic acid, 180-220 μM urea, 18-22 mM sodium pyruvate, and 90-110 nM histamine.

7. The culture medium according to claim 4, characterized in that The culture medium contains 180-220 μM L-asparagine, 90-110 mM myo-inositol, 360-440 mM arachidonic acid, 360-440 μM urea, 90-110 mM sodium pyruvate, and 450-550 nM histamine.

8. Use of the culture medium according to claim 1 in culturing Mycoplasma hyopneumoniae.

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