Quadruple inactivated vaccine of mycoplasma pneumoniae and haemophilus parasuis, streptococcus suis, and actinobacillus pleuropneumoniae and application thereof
A technology of Mycoplasma hyopneumoniae and Haemophilus suis, applied in vaccines, multivalent vaccines, veterinary vaccines, etc., can solve the problems of lack of immune cross-protection, achieve good immunogenicity, reduce costs, and reduce stress effects
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Embodiment 1
[0051] Embodiment 1, the separation and identification of Mycoplasma hyopneumoniae
[0052] 1.1 Collection of disease data
[0053] The disease material came from the lungs of a 2-month-old nursery pig that had severe pneumonia and dyspnea in a large pig farm in Puyang City, Henan Province in April 2017.
[0054] 1.2 Preparation of medium
[0055] PPLO liquid medium: 10.5 g of PPLO broth powder, 2.5 g of glucose, and 2.5 g of yeast powder, dissolved in 440 mL of ultrapure water, sterilized at 115°C for 15 min, added with 5 mL of MEM medium, 50 mL of horse serum, penicillin 80,000 units, 10 mL of sterile 10% arginine and 500 μL of 1% (w / v) phenol red. After the culture was sterilized at 115°C for 15 minutes, it was stored at 4°C for later use;
[0056] PPLO solid medium: PPLO liquid medium with 1.5% (w / v) agar powder added. After the culture was sterilized at 115°C for 15 minutes, it was stored at 4°C for later use.
[0057] 1.3 Isolation and culture of mycoplasma
[0058...
Embodiment 2
[0070] Example 2: Isolation and identification of strain HNHPS1
[0071] 2.1 Collection of disease data
[0072] The disease material came from a 2-month-old nursery pig that died of severe pneumonia, dyspnea and neurological symptoms in a large pig farm in Ruyang County, Henan Province in November 2016.
[0073] 2.2 Preparation of medium
[0074] TSB liquid medium: Dissolve 30 g of TSB broth powder (Tryptic Soy broth, tryptone soybean broth) in 1000 mL of ultrapure water, sterilize at 115 °C for 15 min, add 50 mL of fetal bovine serum, sterile 1% NAD (Nicotinamide adeninedinucleotide, nicotinamide adenine dinucleotide, coenzyme Ⅰ) 1 mL;
[0075] TSA solid medium: Dissolve 40g of TSA agar powder (Tryptic Soy Agar, tryptone soybean agar) in 1000mL ultrapure water, sterilize at 115°C for 15min, add 50mL of fetal bovine serum, 1mL of sterile 1% NAD, in 4 Store at ℃ for later use.
[0076] 2.3 Isolation and culture of bacteria
[0077] Aseptically collect the brain tissue of ...
Embodiment 3
[0097] Example 3: Isolation and Identification of Bacterial Strain HN1570
[0098] 3.1 Culture medium preparation
[0099] TSB liquid medium and TSA solid medium were prepared according to the method in Example 2.
[0100] 3.2 Isolation and cultivation of strain HN1570
[0101] In 2017, the applicant collected the heart blood of nursery pigs with typical polyserositis under aseptic conditions, inoculated them on TSA solid medium containing NAD, cultured them in a constant temperature incubator at 37°C for 36 hours, and picked suspected colonies for passage Purify and culture for another 24-48 hours to observe the colony morphology of Haemophilus parasuis, and grow consistent needle-shaped, colorless, transparent, smooth, moist colonies with a diameter of 1-2 mm (see Figure 7 ); while purified and inoculated on NAD-free TSA solid medium, it cannot grow on NAD-free TSA medium;
[0102] Pick a single purified colony for Gram staining and microscopic examination, and observe t...
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