A virulent strain of Mycoplasma hyorhinis and its application in establishing pathogenic model and preparing inactivated vaccine

Abstract

The present invention discloses a strong strain of Mycoplasma hyorhinalis and its application in establishing a disease model and preparing an inactivated vaccine, and belongs to the field of microbial technology. The strong strain of Mycoplasma hyorhinalis of the present invention is Mycoplasma hyorhinalis ZZ-2021 with a deposit number of CCTCC NO: M 20232234. The strain of the present invention grows fast and has a high titer. It can induce a variety of typical lesions of Mycoplasma hyorhinalis infection including multiple serositis (pleurisy, pericarditis, peritonitis, etc.), arthritis and pneumonia by attacking piglets of different ages. It has strong virulence and rapid onset. Typical clinical manifestations can appear within 3 days at the fastest. The onset is serious, and it can be used to establish a Mycoplasma hyorhinalis disease model with typical symptoms. The Mycoplasma hyorhinalis strong strain of the present invention is prepared into an inactivated vaccine, which can effectively prevent and treat multiple pathological changes such as polyserositis, pneumonia, and arthritis caused by Mycoplasma hyorhinalis, and has a good attack and protection effect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strong strain of Mycoplasma hyorhinis and application thereof in establishing a disease model and preparing an inactivated vaccine. Background Art

[0002] Mycoplasmajn hyorhinis (Mhr) was first discovered and named in 1955. It is common in pigs, with a high incidence and low mortality. It mainly infects the upper respiratory tract of pigs, and can cause systemic inflammation with typical characteristics of arthritis and polyserositis. It has also been reported to be co-infected with a variety of other pathogens. Clinically, Mhr is often mixed with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2), causing severe porcine respiratory syndrome, resulting in a significant increase in the mortality rate of weaned piglets, causing huge economic losses to the pig industry, and endangering the healthy development of the pig industry.

[0003] At present, the domestic pig farming industry has little knowledge of Mycoplasma hyorhinis, because its clinical symptoms are extremely similar to those caused by porcine contagious pleuropneumonia, Haemobacterium parasuis infection, etc., and misdiagnosis often leads to persistent infection of Mycoplasma hyorhinis in pig farms, resulting in a decline in the production performance of the pig herd. Due to the prevalence of Mycoplasma hyorhinis infection, common antibiotics in clinical practice have developed a certain degree of drug resistance. At present, the pathogenic mechanism of Mycoplasma hyorhinis is still unclear, there is a lack of specific serum diagnostic kits, and there is no commercially available vaccine in China. It is urgent to establish a disease model with obvious symptoms, typical and stable. However, most of the Mycoplasma hyorhinis disease models that have been made public have mild onset or single lesions, which are different from the typical clinical symptoms of pig farms, have weak virulence, mild onset, and lack certain representativeness.

[0004] In addition, the infection of Mycoplasma hyorhinis is becoming more and more serious. There is an urgent need to isolate a Mycoplasma hyorhinis strain with good immunogenicity and stable titer as a vaccine candidate strain, and further develop vaccines to prevent various diseases caused by Mycoplasma hyorhinis. Summary of the invention

[0005] The purpose of the present invention is to provide a strong strain of Mycoplasma hyorhinis with fast growth, high titer and good immune protection effect. The strain grows fast and has high titer. The titer can reach 10 10.0～11.0CCU / mL. In addition to inducing multiple serositis (pleurisy, pericarditis, peritonitis, etc.), arthritis and other typical lesions of swine mycoplasma infection, the strain can also induce less common pneumonia symptoms. It has strong virulence and rapid onset. Typical clinical symptoms can appear within 3 days at the fastest. The incidence is serious and can occur in pigs of different ages. The symptoms are the same as those of swine mycoplasma infection in pig farms, which is representative. The strain is prepared into an inactivated vaccine, which can provide good immune stimulation ability and anti-toxic protection efficacy, and can be used to prevent or treat various diseases such as swine polyserositis (pleurisy, pericarditis, peritonitis, etc.), arthritis, pneumonia, etc. caused by swine mycoplasma. Based on the above performance, the purpose of the present invention is also to provide the use of the strong strain of swine mycoplasma in establishing a disease model and preparing an inactivated vaccine.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The virulent strain of Mycoplasma hyorhinis provided by the present invention is named ZZ-2021, and its preservation information is as follows: preservation unit: China Center for Type Culture Collection (address: Wuhan University, Wuhan, China); classification name: Mycoplasma hyorhinis ZZ-2021; preservation number: CCTCC NO: M 20232234; preservation date: November 15, 2023.

[0008] The virulent strain of Mycoplasma hyorhinis was isolated from the lung tissue of piglets with suspected Mycoplasma hyorhinis infection symptoms such as joint swelling, lameness, asthma, depression, and emaciation. It was cultured in a modified Friis medium for 12 to 16 hours and the titer could reach 10 10.0～11.0 CCU / mL.

[0009] The advantages and characteristics of the virulent strain of Mycoplasma hyorhinis ZZ-2021 include: first, it can induce a variety of typical clinical symptoms of Mycoplasma hyorhinis, including polyserositis and arthritis, and can also induce pneumonia and other symptoms that are less common in other strains. The established disease model is representative and comprehensive; second, it has strong virulence and rapid onset. At the same time, the titer required for the virus to cause pigs to become ill is low; third, the Mycoplasma hyorhinis has the advantages of simple growth conditions, fast growth, and high proliferation titer. The titer can reach 10 after 12 to 16 hours of culture on a cell-free culture medium. 10.0～11.0 CCU / mL, these characteristics are conducive to the establishment and wide application of artificial disease models.

[0010] The virulent strain of Mycoplasma hyorhinis is directly inoculated with the strain culture for attack, and is injected through multiple routes of the nasal cavity, lung, and abdominal cavity. Symptoms such as rough fur, fever, depression, and loss of appetite may appear 2 to 3 days after inoculation. Characteristic clinical symptoms such as joint swelling and lameness may appear as early as the 3rd day. Autopsy on the 28th day after attack shows multiple serositis (pleurisy, pericarditis, peritonitis, etc.), pneumonia, arthritis, etc. Based on this, the present invention also provides the use of the virulent strain of Mycoplasma hyorhinis in establishing a Mycoplasma hyorhinis pathogenesis model. The Mycoplasma hyorhinis pathogenesis model can be used for vaccine efficacy testing, Mycoplasma hyorhinis pathogenic mechanism research, anti-Mycoplasma hyorhinis infection drug screening, etc. Preferably, the Mycoplasma hyorhinis pathogenesis model is established by attacking with the pure culture of the virulent Mycoplasma hyorhinis strain.

[0011] The present invention also provides the use of the virulent strain of Mycoplasma hyorhinis in the preparation of drugs for preventing or treating swine diseases caused by Mycoplasma hyorhinis infection, wherein the swine diseases include: multiple serositis (pleurisy, pericarditis, peritonitis, etc.), pneumonia, arthritis, etc.

[0012] The present invention also provides the use of the virulent strain of Mycoplasma hyorhinis in the preparation of an inactivated Mycoplasma hyorhinis vaccine.

[0013] An inactivated vaccine of Mycoplasma hyorhinis, which uses the inactivated virulent strain of Mycoplasma hyorhinis as an active ingredient. The inactivated vaccine also contains an adjuvant, which is an oily adjuvant or an aqueous adjuvant, preferably mineral oil.

[0014] The method for preparing the inactivated vaccine of Mycoplasma hyorhinis comprises the following steps: (1) culturing the virulent strain of Mycoplasma hyorhinis to obtain a bacterial solution; (2) adding an inactivator to the bacterial solution to inactivate the bacterial solution; (3) adding an adjuvant to the inactivated bacterial solution, mixing evenly, and emulsifying to obtain an inactivated vaccine of Mycoplasma hyorhinis. Preferably, the bacterial solution is inactivated in the following manner: adding formaldehyde to the bacterial solution to a final concentration of 0.4% (v / v), and inactivating by shaking at 37°C for 72 to 96 hours before the inactivation is terminated. The adjuvant is preferably the commercialized MONTANIDETM ISA15AVG adjuvant of Air Liquide Healthcare; the ratio of the adjuvant to the inactivated bacterial solution is preferably 15:85 by volume.

[0015] The immunization method of the inactivated vaccine of Mycoplasma swine rhinis comprises the following steps: immunizing twice through the neck muscle, with an immunization dose of 2×10 7.0 ~2×10 9.0 CCU / head dose, the interval between two immunizations is 2 weeks.

[0016] Advantages and beneficial effects of the present invention:

[0017] (1) The virulent strain of Mycoplasma hyorhinis of the present invention grows fast and has a high titer. The titer can reach 10 after culturing in the modified Friis medium for 12 to 16 hours. 10.0～11.0 CCU / mL.

[0018] (2) The virulent strain of Mycoplasma hyorhinis of the present invention can induce a variety of typical Mycoplasma hyorhinis symptoms including polyserositis and arthritis when infecting piglets of different age groups. It can also induce pneumonia and other symptoms that are less common in infection with other strains. The strain is highly virulent and develops quickly, with characteristic clinical manifestations appearing within 3 days at the fastest. It can be used to establish a Mycoplasma hyorhinis disease model with typical symptoms, which is consistent with the clinical disease characteristics of pig farms and is representative.

[0019] (3) The inactivated vaccine prepared from the virulent strain of Mycoplasma hyorhinis of the present invention can effectively prevent and treat various pathological changes such as polyserositis (pericarditis, pleurisy), pneumonia, arthritis, etc. caused by Mycoplasma hyorhinis, and has a good anti-virus protection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the colony morphology of Mycoplasma hyorhinis ZZ-2021 strain on a solid plate.

[0021] Figure 2 This is the Giemsa staining image of Mycoplasma hyorhinis strain ZZ-2021.

[0022] Figure 3 This is a scanning electron micrograph of Mycoplasma hyorhinis strain ZZ-2021.

[0023] Figure 4 The results of PCR amplification of 16sRNA and specific gene P37 of Mycoplasma hyorhinis ZZ-2021 strain. 1: 16sRNA; 2: P37.

[0024] Figure 5 This is the MLST typing of Mycoplasma hyorhinis ZZ-2021 strain.

[0025] Figure 6 The clinical symptoms of pigs infected with Mycoplasma hyorhinis ZZ-2021 strain include: emaciation and lameness of piglets, and swollen joints of piglets.

[0026] Figure 7 The symptoms of autopsy after infection with Mycoplasma hyorhinis strain ZZ-2021 are: pleurisy; pericarditis; pneumonia; peritonitis; arthritis. DETAILED DESCRIPTION

[0027] The embodiments of the present invention will be described in more detail by the following examples, but it should be understood that the examples are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0028] The modified Friis medium used in the following examples was prepared as follows: 4.0 g of PPLO broth powder, 3.0 g of brain heart infusion, 30 mL of 10× Hank's solution, 30 mL of yeast infusion, 200 U / mL of penicillin, 250 mL of porcine serum, and 10 mL of 0.25% phenol red. The pH was adjusted to 7.6 with 1 M NaOH, and the volume was made up to 1 L with pure water. After sterile filtration through a 0.22 μm filter membrane, the medium was divided into portions and placed at 4° C. for use.

[0029] Example 1 Isolation, purification and identification of Mycoplasma hyorhinis strain ZZ-2021

[0030] In a pig farm in Hunan, some piglets showed symptoms of suspected Mycoplasma hyorhinis infection, such as swollen joints, lameness, wheezing, depression, and weight loss. The lung tissues of the sick pigs were collected, ground and centrifuged at 3000r / min for 5 minutes. 0.5mL of the supernatant was inoculated into 2.0mL of modified Friis medium containing penicillin and cultured in an incubator at 37°C. When the bacterial solution turned orange-yellow, it was diluted 1:1000 and spread on a solid plate. After 4-6 days of culture, it was observed under a microscope. A single colony was picked up and expanded in a liquid culture medium to complete the first purification. The purification was repeated three times to obtain a purified strain. The strain obtained after purification was identified by morphological and biochemical characteristics, molecular biological characteristics, etc.: The strain was cultured in modified Friis medium for 12-16 hours, and the titer could reach 10 10.0～11.0 CCU / mL; after dilution, spread on the plate, and a round, centrally protruding "fried egg-like" colony can be observed under a microscope in 3 to 5 days ( Figure 1 ); Giemsa staining microscopy showed that the bacteria were mainly spherical ( Figure 2 ); Scanning electron microscopy results showed that the bacteria were small (0.2-0.4 μm) and mainly spherical ( Figure 3 ); extract the nucleic acid of the strain to amplify the 16sRNA (1450bp) and specific gene P37 (550bp) of Mycoplasma hyorhinis, and the corresponding target bands appeared ( Figure 4 ), sequencing showed that the nucleotide sequence of 16sRNA was as shown in SEQ ID NO.1; the six housekeeping genes (dnaA, rpoB, gyrB, gltx, adk, gmk) of the strain were sequenced and MLST typing was performed, and it was identified as a new ST type Mycoplasma hyorhinis ( Figure 5). Thus, the obtained strain was determined to be a strain of Mycoplasma hyorhinis, named as strain ZZ-2021.

[0031] The Mycoplasma hyorhinis ZZ-2021 strain was deposited, and the preservation information is as follows: Depository unit: China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China); Classification name: Mycoplasma hyorhinis ZZ-2021; Deposit number: CCTCC NO: M 20232234; Deposit date: November 15, 2023.

[0032] Example 2 Challenge and Pathogenesis Model 1 of Mycoplasma hyorhinis ZZ-2021 strain

[0033] Experimental materials and methods: Five healthy, 12- to 15-day-old, non-vaccinated, dual-hybrid piglets were selected and each pig was inoculated with a total of 10 mL (5 × 10 8.0 CCU / head), and 5 piglets with the same conditions were set as blank control group and raised under the same conditions.

[0034] Disease determination criteria: After 28 days of continuous observation after infection, the piglets were autopsied and scored according to the "Pathological Scoring System" of Mycoplasma hyorhinis (Table 1 below). A disease was determined if the lesion score was higher than 3 points.

[0035] Table 1

[0036]

[0037]

[0038] Test results: After the infection with ZZ-2021 strain of Mycoplasma suorhinis, 5 / 5 piglets became ill: 2-3 days after infection, the experimental pigs showed typical clinical symptoms of Mycoplasma suorhinis, such as rough fur, mild fever, depression, loss of appetite, difficulty walking and joint swelling, and the above clinical symptoms lasted for more than 2 days. 15 days after infection, the experimental pigs were obviously emaciated, and 4 piglets had joint swelling. The autopsy mainly showed joint swelling, increased joint fluid, and varying degrees of cheesy exudate; pericardial adhesions, varying degrees of effusion and cheesy exudate; thoracic adhesions, accompanied by fibrinous exudate; abdominal adhesions, varying degrees of cheesy exudate; milky white fibrinous exudate on the surface of the liver, spleen and intestines; 2 piglets had "shrimp-like" lesions in the lungs. All piglets also had multiple serositis and arthritis lesions, and a small number of piglets also had pneumonia symptoms. 5 / 5 piglets became ill.

[0039] The above results show that ZZ-2021 strain can cause obvious clinical and autopsy changes after infection. Pigs develop the disease quickly, and characteristic clinical manifestations of joint swelling can appear within 3 days ( Figure 6 ), the lesions were typical and comprehensive, 5 / 5 of the piglets were sick, and the disease was serious. The above experimental results show that the ZZ-2021 strain of Mycoplasma hyorhinis is a highly virulent strain.

[0040] Example 3 Challenge and Pathogenesis Model 2 of Mycoplasma hyorhinis ZZ-2021 Strain

[0041] Experimental materials and methods: Five healthy, grown-up, dual-hybrid piglets aged 40 to 42 days that had not been vaccinated with any mycoplasma-related vaccines were selected and each piglet was inoculated with a total of 10 mL (5 × 10 8.0 CCU / head), and 5 piglets with the same conditions were set as blank control group and raised under the same conditions.

[0042] Disease determination criteria: same as above.

[0043] Test results: After infection with Mycoplasma hyorhinis ZZ-2021 strain, 4 / 5 piglets became ill: 7 days after infection, the test pigs showed typical clinical symptoms of Mycoplasma hyorhinis, such as rough fur, mild fever, depression, loss of appetite, difficulty walking and swollen joints, and the above clinical symptoms lasted for more than 2 days. 15 days after infection, the test pigs were significantly emaciated, and 3 piglets had swollen joints. The autopsy mainly showed swollen joints, increased joint fluid, and varying degrees of caseous exudates; pericardial adhesions with varying degrees of effusion; pleural adhesions with fibrinous exudates; mild caseous exudates in the abdominal cavity; milky white fibrinous exudates on the surface of the liver, spleen and intestines; and 1 piglet had "shrimp-like" lesions in the lungs. Most piglets had multiple serositis and arthritis lesions, and a small number of piglets had symptoms of pneumonia ( Figure 7 ), 4 / 5 piglets were sick.

[0044] From the above results, it can be seen that after the ZZ-2021 strain is challenged, obvious clinical and autopsy changes can occur. The pigs develop the disease quickly, and characteristic clinical manifestations of joint swelling can appear within 7 days. The autopsy lesions are typical and comprehensive. 4 / 5 piglets are sick, and the disease is serious. The above experimental results show that the ZZ-2021 strain of Mycoplasma suorhinis is a strong strain.

[0045] It can be seen from the challenge experiments of Examples 2 to 3 that after challenge with the ZZ-2021 strain, the disease can occur in piglets of different ages, and the lesions are typical and comprehensive, indicating that the ZZ-2021 strain of Mycoplasma hyorhinis is a highly virulent strain.

[0046] Example 4 Preparation of inactivated vaccine of Mycoplasma hyorhinis ZZ-2021 strain

[0047] Preparation of bacterial solution: In this embodiment, the ZZ-2021 strain of Mycoplasma hyorhinis was inoculated into a modified Friis medium at a dose of 1:10, cultured at a constant temperature of 37° C., and the harvest time was 12 to 16 hours.

[0048] Inactivation: Select a formaldehyde solution with a final concentration of 0.4% to inactivate the ZZ-2021 strain of Mycoplasma hyorhinis. After shaking inactivation at 37°C for 72 hours, the ZZ-2021 strain of Mycoplasma hyorhinis is completely inactivated. The inactivated antigen is inoculated into the modified Friis medium at a ratio of 1:10 and cultured for 7 days. Observe whether the culture medium changes color to ensure complete inactivation.

[0049] Emulsification: The inactivated antigen was centrifuged at 12000r / min for 15min, the supernatant was discarded, and then fully resuspended with PBS buffer. Then, the volume of the aqueous phase antigen and the oil phase adjuvant was 85:15, and the mixture was stirred and mixed with a homogenizer and a magnetic stirrer in a sterile room. The oil phase adjuvant used was the commercialized MONTANIDETM ISA15AVG adjuvant of Air Liquide Healthcare. The emulsification procedure was to first stir the aqueous phase in a magnetic stirrer, then slowly add the oil phase adjuvant, and stir continuously at 800r / min for 30 minutes to obtain an oil-in-water formulation of the vaccine. After standing for 30 minutes, it was packaged to stabilize the interface and eliminate foam to achieve the best emulsification effect.

[0050] Example 5 Immune protection test of inactivated vaccine of Mycoplasma hyorhinis ZZ-2021 strain

[0051] Vaccine preparation: The inactivated vaccine was prepared according to Example 4, and the antigen concentration was adjusted to 1×10 7.0 CCU / mL, 1×10 8.0 CCU / mL, 1×10 9.0 CCU / mL, and three doses of inactivated vaccine were obtained: low, medium and high.

[0052] Experimental materials and methods: 25 healthy and grown binary hybrid piglets aged 12 to 15 days without any mycoplasma-related vaccines were selected and randomly divided into 5 groups: A (low-dose immunization group), B (medium-dose immunization group), C (high-dose immunization group), D (blank challenge group), and E (blank control group). The piglets in groups A, B, and C were injected with different doses of 2 mL of inactivated vaccine of Mycoplasma suorhinis ZZ-2021 strain into the neck muscle, and then boosted with immunization once 2 weeks later. 28 days after the second immunization, all piglets except the blank control group were inoculated with 10 mL of fresh culture of Mycoplasma suorhinis ZZ-2021 strain (5×10 8.0 CCU / head portion). Necropsy was performed 28 days after challenge.

[0053] Evaluation of vaccine protection: Piglet autopsy scores were performed with reference to the above-mentioned disease standards, and the lesion reduction rate was calculated according to the following formula: lesion reduction rate = (average autopsy score of blank challenge group - average autopsy score of immunized group) / average score of blank challenge × 100%.

[0054] Test results: Except for one piglet in the low-dose immunization group that developed disease and joint swelling, the piglets in the low, medium, and high-dose immunization groups did not show clinical symptoms and autopsy symptoms related to Mycoplasma suis. All five piglets in the blank challenge group developed disease, and no abnormalities were found in the blank control group. The lesion reduction rates in each group were 86.11%, 97.22%, and 100%, respectively. The autopsy scores and lesion reduction rates in each group are shown in Table 2.

[0055] Table 2

[0056] Group Mean necropsy score Reduced lesions Low dose group 1 86.11％ Medium dose group 0.2 97.22％ High dose group 0 100％ Blank challenge group 7.2 / Blank control group 0 /

[0057] The above results show that 2×10 8.0 CCU / head, 2×10 9.0 The CCU / Toufen inactivated vaccine of Mycoplasma hyorhinis ZZ-2021 strain has a good protective effect, with the lesion reduction rates reaching 97.22% and 100% respectively.

Claims

1. A virulent strain of Mycoplasma hyorhinis, characterized in that: The virulent strain of Mycoplasma hyorhinis is Mycoplasma hyorhinis ZZ-2021 with a deposit number of CCTCC NO: M 20232234.

2. Use of the virulent strain of Mycoplasma hyorhinis according to claim 1 in establishing a Mycoplasma hyorhinis pathogenesis model.

3. The application according to claim 2, characterized in that: The Mycoplasma hyorhinis challenge and pathogenesis model is used for testing vaccine efficacy and screening drugs for preventing or treating Mycoplasma hyorhinis infection.

4. The use according to claim 2 or 3, characterized in that: The pure culture of the virulent strain of Mycoplasma hyorhinis described in claim 1 is used for challenge to establish a Mycoplasma hyorhinis pathogenesis model.

5. Use of the virulent strain of Mycoplasma hyorhinis according to claim 1 in the preparation of a drug for preventing or treating swine diseases caused by Mycoplasma hyorhinis infection.

6. The use according to claim 5, characterized in that: The swine diseases caused by Mycoplasma hyorhinis include: polyserositis, pneumonia, and arthritis.

7. Use of the virulent strain of Mycoplasma hyorhinis according to claim 1 in the preparation of an inactivated Mycoplasma hyorhinis vaccine.

8. An inactivated vaccine of Mycoplasma hyorhinis, characterized in that: The active ingredient is the inactivated virulent strain of Mycoplasma hyorhinis according to claim 1.

9. The inactivated Mycoplasma hyorhinis vaccine according to claim 8, characterized in that: An adjuvant is also included.

10. A method for preparing an inactivated vaccine of Mycoplasma hyorhinis, characterized in that: The following steps are involved: (1) Cultivating the virulent strain of Mycoplasma hyorhinis according to claim 1 to obtain a bacterial liquid; (2) adding an inactivating agent to the bacterial solution to inactivate the bacterial solution; (3) Add an adjuvant to the inactivated bacterial solution, mix evenly, and emulsify to obtain an inactivated Mycoplasma hyorhinis vaccine.

Images