Mycoplasma hyopneumoniae attenuated strain and application thereof in preparation of attenuated vaccines

[0023] Example 1

[0024] The Mycoplasma hyopneumoniae ES-2L strain of the present invention is based on the local clinically isolated virulent strain ES-2 strain (deposit number CCTCC NO: M 2018570) which is continuously propagated to 200 generations in vitro. The specific passaging process is as follows: the cryopreserved ES-2 strain is revitalized and activated, and the revitalized bacterial solution is used as P1 generation; After culturing in the environment for 2 to 3 days, harvest as P2 generation; in this way, continuous subculture is carried out. When it is passed to the 197th generation, the harvested bacterial liquid is coated on the modified Friis solid medium and placed in a 37°C, 5% carbon dioxide environment. After 7 to 10 days of medium culture, pick a single colony under a low magnification microscope and insert it into Friis liquid medium for cultivation; in this way, the clone purification and identification were carried out for three consecutive times, and the

[0035]Example 2

[0036] The virulence of Mycoplasma hyopneumoniae ES-2 strain P1 and ES-2L strains were evaluated. Twelve 25-day-old landrace boars and large white sows binary crossbred pigs were divided into three groups, A, B, and C, with 4 pigs in each group. Among them, group A was injected with Mycoplasma hyopneumoniae of P1 generation by intratracheal injection. Intratracheal injection of challenge Mycoplasma hyopneumoniae ES-2L strain in group B, and intratracheal injection of normal saline in group C (blank control group). The challenge doses of both groups A and B were 7×10 8 CCU. After the challenge, the patients were dissected after 42 days of observation, and the body temperature and body weight were measured and clinical symptoms were observed during the period.

[0037] The virulence evaluation experiment showed that there was no increase in body temperature in each group after the challenge; the body weight was measured and the average daily weight gain was cal

[0042] Example 3

[0043] Mycoplasma hyopneumoniae ES-2L strain was cultured in a modified Friis liquid medium at 37°C in a 5% carbon dioxide environment to a stable stage, then the bacterial liquid was harvested, and the harvested bacterial liquid was diluted with the modified Friis liquid medium to adjust the titer to 1 ×10 7 CCU / mL, prepared into attenuated vaccine. Fifteen 14-day-old landrace boars and large white sows binary crossbred pigs were divided into groups A, B and C, with 5 pigs in each group. Group A was the intramuscular injection immunization group, 14-day-old neck intramuscular injection was immunized once, and 14 days after the first immunization, booster immunization was performed once with the same dose and procedure, 2 mL / head each time; group B was the normal saline immunization group (challenge drug) control group), and group C was the blank control group. The immunization dose is 2 × 10 7 CCU, except the blank control group, the other two groups r