Mycoplasma hyopneumoniae culture medium capable of significantly improving strain titer and application thereof

Abstract

The application provides a mycoplasma hyopneumoniae culture medium capable of significantly improving the strain titer and an application thereof, 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 pig serum. The culture medium can significantly improve the growth rate and viable bacterial titer of the mycoplasma hyopneumoniae under a low serum concentration, and reduces the production cost.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products, specifically relating to a culture medium for Mycoplasma hyopneumoniae that can significantly improve strain titer and shorten discoloration time, and its preparation method. Background Technology

[0002] Swine mycoplasmal pneumonia, commonly known as swine enzootic pneumonia, is a chronic respiratory infectious disease caused by Mycoplasma hyopneumoniae (Mhp). It is one of the most common and difficult-to-eradicate diseases in swine herds. Currently, the main measures for controlling Mycoplasma hyopneumoniae infection are the use of antibiotics and vaccines. However, because Mycoplasma hyopneumoniae lacks a cell wall, antibiotics that work by destroying the cell wall are ineffective against it. Furthermore, due to the increasing prevalence of drug resistance, vaccines play a particularly crucial role in the prevention and control of swine mycoplasmal pneumonia. my country has successfully developed several swine mycoplasmal pneumonia vaccines, which have played an important role in prevention and control. However, the high nutritional requirements, slow growth, and low bacterial density of Mycoplasma hyopneumoniae increase the difficulty of studying its biology and pathogenic mechanisms, and also seriously affect the production efficiency and distribution of vaccines.

[0003] Mycoplasma hyopneumoniae has limited biosynthetic and metabolic capabilities and stringent nutrient requirements, necessitating the addition of porcine serum for in vitro culture. Porcine serum plays a crucial role in the growth and reproduction of Mycoplasma hyopneumoniae. Current culture methods still rely on the addition of high concentrations of porcine serum. However, the use of porcine serum presents several problems: First, variations in source and batch can significantly affect the growth rate and titer of Mycoplasma hyopneumoniae. Second, pathogen-negative porcine serum, especially negative for porcine respiratory pathogens, is expensive and difficult to obtain, leading to high vaccine production costs and hindering the widespread application of Mycoplasma hyopneumoniae monotherapy and combination vaccines. How to efficiently culture Mycoplasma hyopneumoniae at low serum concentrations remains an urgent international challenge. Summary of the Invention

[0004] The purpose of this invention is to provide a culture medium for Mycoplasma hyopneumoniae that can significantly improve the titer of strains. It can significantly improve the growth rate and viable bacterial titer of Mycoplasma hyopneumoniae at low serum concentrations, thereby reducing production costs.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A culture medium for Mycoplasma hyopneumoniae that can significantly increase the titer of strains is obtained by adding L-asparagine, inositol, arachidonic acid, urea, histamine, sodium pyruvate, valproic acid, glyceric acid, lysophosphatidic acid, L-citrulline, L-glutamine and L-leucine to KM2 medium containing serum from Bama miniature pigs.

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

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

[0009] In this 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 this invention, the culture medium contains 18-220 μM L-asparagine, 9-110 mM muscle inositol, 90-440 mM arachidonic acid, 180-440 μM urea, 18-110 mM sodium pyruvate, and 90-550 nM histamine.

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

[0012] In this invention, the culture medium contains 180-220 μM L-asparagine, 90-110 mM muscle 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 the application of the culture medium in the culture of Mycoplasma pneumoniae in pigs.

[0014] This invention provides a culture medium with clearly defined components. The optimal pig breed for promoting the growth of *Mycoplasma hyopneumoniae* strains, Bama fragrant pig serum, was screened. Through screening of other components, a high-titer culture medium for *Mycoplasma hyopneumoniae* was finally obtained, exhibiting a promoting effect on both virulent and weakly virulent strains, with clearly defined components and significantly reduced serum concentration. This provides high-titer mycoplasma for the production of live and inactivated subunit vaccines against *Mycoplasma hyopneumoniae*, promoting the healthy development of the pig industry and possessing significant theoretical and practical value. Attached Figure Description

[0015] Figure 1 The titers of highly virulent strain LH of Mycoplasma hyopneumoniae were determined by static incubation of each culture medium in blue bottles for 48 hours. * indicates significant difference (p<0.05); *** indicates extremely significant difference (p<0.01).

[0016] Figure 2The titers of highly virulent strain 168 of Mycoplasma hyopneumoniae were determined by static incubation in blue bottles of various culture media for 48 hours. * indicates significant difference (p<0.05); *** indicates extremely significant difference (p<0.01).

[0017] Figure 3 The titers of attenuated strain J of Mycoplasma hyopneumoniae were determined by static incubation in blue bottles of various culture media for 48 hours. * indicates significant difference (p<0.05); *** indicates extremely significant difference (p<0.01).

[0018] Figure 4 The titers of attenuated strain 168L of Mycoplasma hyopneumoniae in porcine culture were determined by static incubation in blue bottles of various culture media for 48 hours. * indicates significant difference (p<0.05); *** indicates extremely significant difference (p<0.01). Detailed Implementation

[0019] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention.

[0020] The source of the porcine Mycoplasma pneumoniae strain used in this invention:

[0021] Mycoplasma hyopneumoniae strain 168: Strain 168 is a highly virulent strain isolated and cultured from pigs, exhibiting typical symptoms of porcine mycoplasma pneumonia. It is published 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 is a highly virulent strain, published in GenBank, NCBI GenBank accession number CP079799.

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

[0024] Mycoplasma hyopneumoniae strain J: is a moderately virulent strain, ATCC 25934.

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

[0026] This embodiment investigates the effects of serum and its content from different pig breeds on the growth of Mycoplasma hyopneumoniae strains 168, LH, 168L, and J.

[0027] Non-anticoagulated blood was collected from Landrace, Duroc, and Bama miniature pigs using sterile syringes. After incubation at 4°C overnight, the blood was centrifuged at 3000 rpm for 20 minutes at 4°C, and the supernatant was collected to obtain the serum of each breed. Care should be taken to avoid hemolysis during the collection of non-anticoagulated blood; the final serum of each breed should be a clear brownish-red color.

[0028] Prepare the basal culture medium (i.e., serum-free KM2 medium): 1000 mL Eagles' solution, 10.0 g hydrolyzed milk protein, 20 mL fresh yeast extract, 600 mL 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 to 7.4-7.6 with 10 g / L NaOH aqueous solution. Eagles' solution is prepared as follows: Dissolve 48.47 g of MEM medium (Gibco) and 1.5 g of L-glutamine in ultrapure water, then bring the volume to 5000 mL with ultrapure water to obtain Eagles' solution.

[0029] Take 800 mL of basal culture medium and mix it thoroughly with 200 mL of serum from Landrace, Duroc, or Bama miniature pigs to obtain KM2 culture media containing serum from different pig breeds. At this point, the serum content in the culture medium is 20%. Using the same method, prepare KM2 culture media containing 10% Landrace, Duroc, or Bama miniature pig serum. Using the same method, prepare KM2 culture media containing 5% Landrace, Duroc, or Bama miniature pig serum.

[0030] Take seed batches (10) of each strain 8 CCU 50The bacteria were lyophilized and preserved. First, 1 mL of serum-free KM2 medium was added to each culture to reconstitute them. Then, they were inoculated at a volume ratio of 1:10 into KM2 medium containing 20% ​​Landrace porcine serum and incubated statically at 37°C with 5% CO2 for 54 h (Xiong et al., 2014, Protective efficacy of a live attenuated Mycoplasma hyopneumoniae vaccine with an ISCOM-matrix adjuvant inpigs. Vet. J. 199, 268–274.). After resuscitation, the culture was passaged using the same method to obtain the seed culture. The seed culture was then inoculated at a volume ratio of 1:10 into KM2 medium containing 20%, 10%, and 5% serum of each porcine breed, with three replicates for each medium. The culture was incubated statically at 37°C with 5% CO2 in small 5 mL vials (containing 2 mL of the culture) for 48 h. The culture was then analyzed using 50% color change units (CCU). 50 The viable titer of *Mycoplasma hyopneumoniae* strains was quantitatively tested (Leigh et al., 2008, The effects of increasing sodium chloride concentration on *Mycoplasma gallisepticum* vaccine survival in solution. Avian Dis. 52, 136-138.). Additionally, the color change time of *Mycoplasma hyopneumoniae* cultured in mediums containing 20%, 10%, and 5% serum from different breeds of pigs was observed, and the pH of the cultures was measured.

[0031] Based on the results of pH (Tables 2 and 3), color change time (Table 4), and titer (Table 1) measurements during the culture process, it can be seen that when the titer reaches its highest value, the corresponding pH is 6.5-6.8. At this time, the culture medium changes from bright red to orange-yellow or close to bright yellow. Therefore, the rate of increase in bacterial count titer can be determined by observing the time it takes for the culture medium to turn orange-yellow during the culture of Mycoplasma hyopneumoniae and measuring the pH value.

[0032] As shown in Table 1, after culturing Mycoplasma hyopneumoniae (Mhp) strains of different virulences in KM2 medium containing serum from different breeds of pigs for 48 hours, the Mhp strains cultured with Bama miniature pig serum showed faster growth rates and at least a 0.50 titer increase compared to Landrace and Duroc serum. The titers of each strain remained unchanged at serum concentrations of 20% and 10%, but decreased significantly when the serum concentration was reduced to 5%. Table 4 shows that at a serum concentration of 10%, the discoloration time of the medium was close to that at a serum concentration of 20%. Significant differences in discoloration time were observed when Mycoplasma hyopneumoniae strains were cultured with medium containing serum from different breeds of pigs. For both highly virulent and weakly virulent strains, the discoloration time was at least 12 hours earlier when cultured with Bama miniature pig serum compared to Landrace and Duroc serum, indicating that adding 10% Bama miniature pig serum to the medium significantly improved the growth rate of the strains. Therefore, Bama fragrant pig serum was selected as the serum used in the culture medium for Mycoplasma hyopneumoniae, and the amount of serum used was 10%.

[0033] Table 1. Titers (CCU) of porcine serum strains after 48 hours of culture for each Mhp strain, based on serum type and concentration. 50 Effect of ( / 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. Effects of porcine serum type and concentration on the discoloration time of *Mhp* strain under laboratory static culture conditions in blue flasks.

[0041]

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

[0043] To obtain a low-serum culture medium that can rapidly proliferate porcine mycoplasma pneumoniae at high titers, a large number of substances were screened. It was found that L-asparagine, 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 titers.

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

[0045] Culture medium A0: Add 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 to 900 mL of basal culture medium (same as in Example 1).

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

[0047] Culture medium A2: Add L-asparagine to culture medium A1 to a final concentration of 200 μM.

[0048] Culture medium A3: Add muscle inositol to culture medium A1 to a final concentration of 100 mM.

[0049] Culture medium A4: Arachidonic acid was added to culture medium A1 to a final concentration of 400 mM.

[0050] Culture medium A5: Add urea to culture medium A1 to a final concentration of 400 μM.

[0051] Culture medium A6: Add sodium pyruvate to culture medium A1 to a final concentration of 100 mM.

[0052] Culture medium A7: Add histamine to culture medium A1 to a final concentration of 500 nM.

[0053] Culture medium A8: Add L-asparagine to culture medium A1 at a final concentration of 200 μM and muscle inositol at 100 mM.

[0054] Culture medium A9: Arachidonic acid and urea at a final concentration of 400 mM were added to culture medium A1.

[0055] Culture medium A10: Add sodium pyruvate to culture medium A1 to a final concentration of 100 mM and histamine to a final concentration of 500 nM.

[0056] Culture medium A11: Add L-asparagine to culture medium A1 at a final concentration of 200 μM, 100 mM muscle inositol, 400 mM arachidonic acid, 400 μM urea, 100 mM sodium pyruvate and 500 nM histamine.

[0057] Take the highly virulent strain LH of Mycoplasma hyopneumoniae and 168 seed batches (10 8 CCU 50 The strains were first reconstituted by adding 1 mL of serum-free KM2 medium (Example 1) to each culture medium. Then, they were inoculated at a volume ratio of 1:10 into KM2 medium containing 20% ​​Landrace porcine serum and incubated statically at 37°C with 5% CO2 for 54 hours for recovery. After one generation using the same method, seed cultures were obtained. The seed cultures of each strain were inoculated at a volume ratio of 1:10 into media A1-A11, with three replicates for each medium. The cultures were incubated statically in 5 mL vials (containing 2 mL of liquid in each vial) at 37°C with 5% CO2 for 48 hours. The culture was then analyzed using 50% color change units (CCU). 50 The viable bacterial titer of the Mhp strain was quantitatively tested. Additionally, the discoloration time during the culture of *Mycoplasma hyopneumoniae* in different culture media was observed.

[0058] Table 5 Color Change Time

[0059] 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] Valproic acid, glyceric acid, lysophosphatidic acid, L-citrulline, L-glutamine, and L-leucine, along with Bama miniature pig serum, L-asparagine, muscle inositol, arachidonic acid, urea, sodium pyruvate, and histamine (medium A11), were added to the basal medium. The results of culturing highly virulent strains LH and 168 are as follows: Figure 1 and Figure 2 As shown in Table 5, compared with culture medium A1 containing valproic acid, glyceric acid, lysophosphatidic acid, L-citrulline, L-glutamine, L-leucine, and Bama miniature pig serum, the growth and color change time of the strain was advanced by 7-13 hours. Under the laboratory static culture conditions in small blue vials, 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 CCUs. 50 Under fermenter culture conditions, the viable cell titer can be increased by 1.75-2.5 CCUs. 50 Compared to culture medium A2, culture medium A11 increased the titer of LH strain by 0.5 titers and increased the titer of 168 strains by 0.5 titers.

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

[0062] Culture medium A0: Add 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 to 900 mL of basal culture medium (same as in Example 1).

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

[0064] Culture medium B2: Add L-asparagine to culture medium B1 to a final concentration of 20 μM.

[0065] Culture medium B3: Add muscle inositol to culture medium B1 to a final concentration of 10 mM.

[0066] Culture medium B4: Arachidonic acid was added to culture medium B1 to a final concentration of 100 mM.

[0067] Culture medium B5: Add urea to culture medium B1 to a final concentration of 200 μM.

[0068] Culture medium B6: Add sodium pyruvate to culture medium B1 to a final concentration of 20 mM.

[0069] Culture medium B7: Add histamine to culture medium B1 to a final concentration of 100 nM.

[0070] Culture medium B8: Add L-asparagine to culture medium B1 at a final concentration of 20 μM and muscle inositol at 10 mM.

[0071] Culture medium B9: Arachidonic acid and urea at a final concentration of 100 mM and 200 μM were added to culture medium B1.

[0072] Culture medium B10: Add sodium pyruvate to culture medium B1 to a final concentration of 20 mM and histamine to a final concentration of 100 nM.

[0073] Culture medium B11: Add L-asparagine to culture medium B1 at a final concentration of 20 μM, 10 mM muscle inositol, 100 mM arachidonic acid, 200 μM urea, 20 mM sodium pyruvate and 100 nM histamine.

[0074] Take attenuated strain J of Mycoplasma hyopneumoniae and seed batch 168L (10 8 CCU 50The strains were first reconstituted by adding 1 mL of serum-free KM2 medium (Example 1) to each culture medium. Then, they were inoculated at a volume ratio of 1:10 into KM2 medium containing 20% ​​Landrace porcine serum and incubated statically at 37°C with 5% CO2 for 54 hours for recovery. After one generation using the same method, seed cultures were obtained. The seed cultures of each strain were inoculated at a volume ratio of 1:10 into media B1-B11, with three replicates for each medium. The cultures were incubated statically at 37°C with 5% CO2 in small 5 mL vials (containing 2 mL of the culture medium) for 48 hours. The culture was then analyzed using 50% color change units (CCU). 50 The viable bacterial titer of the Mhp strain was quantitatively tested. Additionally, the discoloration time during the culture of *Mycoplasma hyopneumoniae* in different culture media was observed.

[0075] Table 6 Color Change Time

[0076] 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 culture medium B1, the growth and color change time of attenuated strain J and attenuated strain 168L in culture medium B11 can be advanced by 10-14 hours. Under the static culture conditions of laboratory small blue bottles, the viable titer of attenuated strain J can be increased by up to 1.25 CCU. 50 For the attenuated strain 168L, the viable titer increased by up to 1.2 CCU50. Under fermenter culture conditions, the viable titer could be increased by 1.75-2.5 CCU. 50 .

Claims

1. A culture medium for Mycoplasma hyopneumoniae that can significantly increase the titer of bacterial strains, characterized in that: The culture medium is obtained by adding L-asparagine, inositol, arachidonic acid, urea, histamine, sodium pyruvate, valproic acid, glyceric acid, lysophosphatidylcholine, L-citrulline, L-glutamine, and L-leucine to KM2 medium containing Bama miniature pig serum. The volume percentage concentration of Bama miniature pig serum in the culture medium is 8%-12%. The concentrations of valproic acid, glyceric acid, lysophosphatidylcholine, L-citrulline, L-glutamine, and L-leucine in the culture medium are as follows: valproic acid 3.4-4.2 mM, glyceric acid 2.2-2.8 mM, lysophosphatidylcholine 3.6-4.4 mM, L-citrulline 3.3-4.1 mM, L-glutamine 3.6-4.4 mM, and L-leucine 3.2-4.0 mM. The concentrations of L-asparagine, inositol, arachidonic acid, urea, and sodium pyruvate are as follows: 18-220 μM, 9-110 mM, 90-440 mM, 180-440 μM. mM, histamine 90-550 nM.

2. The culture medium according to claim 1, characterized in that... The culture medium contained 18-22 μM L-asparagine, 9-11 mM muscle inositol, 90-110 mM arachidonic acid, 180-220 μM urea, 18-22 mM sodium pyruvate, and 90-110 nM histamine.

3. The culture medium according to claim 1, characterized in that... The culture medium contains 180-220 μM L-asparagine, 90-110 mM muscle inositol, 360-440 mM arachidonic acid, 360-440 μM urea, 90-110 mM sodium pyruvate, and 450-550 nM histamine.

4. The use of the culture medium described in claim 1 in the culture of Mycoplasma pneumoniae in pigs.

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