WO2015109578A1 - 一种猪支原体肺炎减毒活疫苗及其应用 - Google Patents

一种猪支原体肺炎减毒活疫苗及其应用 Download PDF

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WO2015109578A1
WO2015109578A1 PCT/CN2014/071506 CN2014071506W WO2015109578A1 WO 2015109578 A1 WO2015109578 A1 WO 2015109578A1 CN 2014071506 W CN2014071506 W CN 2014071506W WO 2015109578 A1 WO2015109578 A1 WO 2015109578A1
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vaccine
mycoplasma hyopneumoniae
attenuated
live
strain
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PCT/CN2014/071506
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English (en)
French (fr)
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邵国青
熊祺琰
刘茂军
冯志新
韦艳娜
王海燕
白方方
甘源
王丽
张道华
刘冬霞
华利忠
武昱孜
白昀
王占伟
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江苏省农业科学院
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Priority to US15/113,909 priority Critical patent/US9849168B2/en
Priority to PCT/CN2014/071506 priority patent/WO2015109578A1/zh
Priority to EP14879895.2A priority patent/EP3098301B1/en
Publication of WO2015109578A1 publication Critical patent/WO2015109578A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/35—Mycoplasma

Definitions

  • the present invention relates to an attenuated vaccine strain of Mycoplasma hyopneumoniae, a live vaccine containing the attenuated strain, and the use thereof for protecting a disease associated with Mycoplasma hyopneumoniae infection.
  • Mycoplasma hyopneumoniae commonly known as swine asthma, is a chronic respiratory tract infection caused by Mycoplasma hyopneumoniae (Mhp). The main symptoms are cough and asthma. The disease mainly causes the pig's feed rate to decrease and growth to be blocked, with high incidence and low mortality. Mycoplasma pneumoniae is widespread worldwide and causes significant economic losses to the modern pig industry.
  • Mycoplasma hyopneumoniae often forms a mixed infection with other pathogens.
  • Pasteurella Multocida Streptococcus Suis (SS), Haemophilus Parasuis of Swine (HPS) or Actinobacillus Pleuropneumonia of Porcine (APP) Such as mixed infection, causing local epidemic pneumonia in pigs.
  • RIRSV Porcine Reproductive and Respiratory Syndrome
  • PCV2 Porcine Circovirus Type 2
  • SIV swine influenza virus
  • PRDC Porcine Respiratory Disease Complex
  • Mycoplasma pneumoniae is an intractable chronic infectious disease.
  • the effect of drug treatment is not ideal. It is easy to relapse after stopping the drug. Once the disease is popular in the farm, it is difficult to eradicate. Therefore, the prevention, control and purification of Mycoplasma hominis pneumonia requires comprehensive measures such as early diagnosis, timely isolation, antibiotic treatment or elimination of sick pigs, vaccine prevention, total access, strict disinfection, etc., in which vaccine prevention starts in the control of Mycoplasma pneumoniae A key role.
  • Mycoplasma hyopneumoniae binds to bronchial ciliated epithelial cells via respiratory tract infection to form colonization, resulting in persistent infection.
  • Inactivated vaccines stimulate a systemic immune response, and the effect of inducing a local immune response is poor.
  • the circulating antibodies produced by the inactivated vaccine must pass through the epithelial barrier and secrete into the tracheobronchial lumen, which is extremely difficult and inefficient.
  • the vaccine In order to achieve effective immunity, the vaccine must be able to stimulate a high level of systemic immune response. Of course, this requires a relatively high antigen dose and a strong adjuvant, which makes these vaccine costs more expensive.
  • the immune response produced by the inactivated vaccine can only inhibit the proliferation of wild-type infection and reduce the degree of tissue damage, and can not completely prevent the infection of wild-type virus, so it is difficult to achieve ideal control of the disease. Summary of the invention
  • the object of the present invention is to provide a live attenuated vaccine against Mycoplasma hyopneumoniae, which can be used for the prevention and control of diseases caused by Mycoplasma hyopneumoniae infection, including diseases caused by co-infection of other pathogenic bacteria with Mycoplasma hyopneumoniae.
  • the live attenuated vaccine of Mycoplasma hyopneumoniae is different from the inactivated vaccine. After inoculation, it can proliferate in the body and effectively activate the body's immune system. At the same time, the live vaccine can colonize the bronchial ciliated cells after entering the respiratory system, resulting in a "occupation". The effect "can make the wild poison unable to colonize and block the infestation process of wild poison from the source, thus playing a good anti-infective effect. At the same time, because live vaccines can proliferate in vivo and effectively activate the immune system, the required dose is much lower than inactivated vaccines, so it has obvious advantages over inactivated vaccines in cost control, which is conducive to saving social resources.
  • the key to the development of live vaccines is the weakening of the strains that increase the virulence of the strains while maintaining the immunogenicity of the highly immunogenic strains.
  • the present invention uses the first generation of the rabbit lungs to subculture, and after 100 passages, the cell-free medium is subdivided.
  • the method has the advantages that the in vivo passage of the animal in the early passage process can avoid the defect that the strain is easily weakened in the early stage and is not conducive to the maintenance of immunogenicity when the medium is used in vitro.
  • Subsequent use of cell-free medium for subsequent generation is beneficial to reduce the weaker generation and shorten the weakening process to a certain extent.
  • the specific source of the strain is: Screening with typical Mycoplasma hyopneumoniae infection without obvious other pathogens
  • the infected pig lung disease material was subcultured to the 100th generation using the lung of the rabbit, and then the M. hyopneumoniae strain was isolated and serially passaged by the medium, and simultaneously obtained by screening of a plurality of strains.
  • the selected strain was named as Mycoplasma hyopneumoniae (Mhp) attenuated strain AN306. After being weakened for a long time, the attenuated strain loses its virulence, is safe to animals, and retains good immunogenicity.
  • the attenuated strain AN306 can be used in the preparation of a Mycoplasma hyopneumoniae pneumonia vaccine or a multiple vaccine.
  • the attenuated strain of Mycoplasma hyopneumoniae was deposited on October 25, 2012 in the China Center for Type Culture Collection in Wushan, Wuchang, Wuhan, under the accession number CCTCC M 2012431.
  • Another object of the present invention is to disclose the live attenuated vaccine against Mycoplasma hyopneumoniae, which can be a Mycoplasma hyopneumoniae pneumonia vaccine or a multiple vaccine.
  • the above-mentioned Acoplasma hyopneumoniae (Mhp) attenuated strain AN306, and a pharmaceutically acceptable carrier or adjuvant are included.
  • Carriers or excipients mainly include three types of substances, which vary depending on the specific use state of the vaccine and the route of immunization.
  • One type is solvent or buffer system, including sterile physiological saline, sterile phosphate buffer; one type is lyoprotectant, including skim milk powder, gelatin, sucrose, when the vaccine is prepared in lyophilized form, it needs to be added to lyophilized
  • the system helps the live vaccine to maintain activity; the first type is a spray protectant, mainly glycerin.
  • a spray protectant mainly glycerin.
  • the live vaccine formulation of Mycoplasma hyopneumoniae of the present invention may further comprise an immunological adjuvant.
  • Adjuvants are a common ingredient in vaccine formulations and are used to enhance the immune stimulating ability of the vaccine itself.
  • the adjuvant used in the present invention is an adjuvant suitable for a live vaccine against Mycoplasma hyopneumoniae, i.e., does not cause significant damage to the viability of the live vaccine, and is suitable for the immune requirement of Mycoplasma pneumoniae.
  • the immunoadjuvant disclosed by the invention may be a single adjuvant component or a composite of a plurality of adjuvant components, and the adjuvant component may include a Chinese herbal polysaccharide, a carbomer, a chitosan, an immunostimulating complex matrix, One or more of levamisole, dextran, CpG, and capsular.
  • the various immunoadjuvant ingredients, ratios, and dosages used will vary depending on the particular dosage and route of administration of the live vaccine.
  • the live vaccine preparation of Mycoplasma hyopneumoniae pneumoniae of the present invention may further comprise at least one immunogen of another pathogen, and is formed into a joint vaccine for use.
  • Other pathogens include at least one of viruses, bacteria, fungi, and parasites, such as porcine circovirus, Haemophilus parasuis, porcine reproductive and respiratory syndrome virus, pig nose Mycoplasma, Mycoplasma hyopneumoniae, swine influenza virus, etc. These pathogens often form mixed infections or secondary infections in the respiratory tract with M. hyopneumoniae in the clinic, which promote each other, leading to aggravation of the disease and posing a major threat to the health of the pig population.
  • the combination of these pathogen vaccines with a live vaccine against Mycoplasma hyopneumoniae can be used to prevent mixed infections of Mycoplasma pneumoniae and other pathogens.
  • the immunogen of the other pathogens may be in the form of live bacteria or live poison, or may be in an inactivated form, a protein subunit form or the like. It is worth noting that the carriers, excipients and adjuvants used in other pathogen vaccines must be compatible with the system used in the live vaccine of Mycoplasma hyopneumoniae and must be tolerated for this live vaccine.
  • the live attenuated vaccine against Mycoplasma hyopneumoniae of the present invention can be immunized by various routes including intrapulmonary injection, intramuscular injection, aerosol immunization, oral immunization and intranasal immunization.
  • the preparation for the live attenuated vaccine may be an intrapulmonary injection, an intramuscular injection, an aerosol, an oral preparation or a nasal drop.
  • the choice of immunization route depends on the animal to be immunized, the immunization history, the farm environment, and the ease of operation of the vaccination personnel.
  • Mycoplasma hyopneumoniae infects the body and first binds to the bronchial epithelial ciliated cells of the lungs, causing the cilia to fall off, which in turn infects the tissue, induces inflammation, and opens the door for infection by other pathogens.
  • the live vaccine of Mycoplasma hyopneumoniae When the live vaccine of Mycoplasma hyopneumoniae is immunized by intrapulmonary injection, the live vaccine can be directly injected into the target tissue, and bind to the ciliated cells, thereby occupying the binding site and preventing the infection of wild poison; when the live vaccine of Mycoplasma hyopneumoniae is immunized by aerosol By controlling the particle size of the droplets, the live vaccine can be allowed to reach the lower respiratory tract of the animal, effectively binding to the bronchial ciliated cells, and the use of an aerosol vaccine in a closed barn or space can reduce the number of personnel required for immunization; Intramuscular injection is the most commonly used method of inoculation for animal immunization.
  • the live vaccine of Mycoplasma hyopneumoniae When the live vaccine of Mycoplasma hyopneumoniae is immunized by intramuscular injection, it can induce systemic cellular and humoral immune responses in animals. Partial target tissue can inhibit the infection of Mycoplasma hyopneumoniae wild venom; When the live vaccine of Mycoplasma hyopneumoniae is inoculated by oral or intranasal, it can induce intestinal mucosal immune response in the intestine or nasal cavity, through the common mucosal immune system, lymphocytes Can homing to different mucosal sites, thus achieving The immune response of the bronchopulmonary tissues of the respiratory tract, to protect against the infection of wild mycoplasma pneumoniae.
  • the selected immunological adjuvant will also vary depending on the route of inoculation used. Therefore, the live attenuated vaccine of Mycoplasma hyopneumoniae of the present invention can be applied to the prevention and treatment of Mycoplasma hyopneumoniae infection.
  • the live attenuated vaccine against Mycoplasma hyopneumoniae of the present invention is prepared by using Mycoplasma hyopneumoniae (Mhp) attenuated strain AN306 with a pharmaceutically acceptable carrier or adjuvant, and at least one immunological adjuvant, at least one other pathogen. The immunogens are combined.
  • Mhp Mycoplasma hyopneumoniae
  • the live vaccine of Mycoplasma hyopneumoniae of the present invention may be administered in a single immunization, or two or more repeated inoculations may be used to enhance the immune response. It is specifically selected depending on the route of immunization and the condition of the animal.
  • the single immunization time is usually 3-10 days old, preferably 5-7 days old.
  • the first immunization time for two or more repeated inoculations is usually 3-10 days old, preferably 5-7 days old.
  • the time interval between two immunizations is typically 2-3 weeks.
  • Biological sample preservation information is typically 3-10 days old, preferably 5-7 days old.
  • Mycoplasma hyopneumonia AN306 Mycoplasma hyopneumonia AN306 (Mycoplasma hyopneumoniae AN306) was deposited on October 25, 2012, at the China Center for Type Culture Collection in Wushan, Wuchang, Wuhan, with the accession number CCTCC. M 2012431. Specific examples:
  • the present invention also verifies the live vaccine formulation of Mycoplasma hyopneumoniae pneumoniae, including a plurality of different immunization routes, A variety of different adjuvants, adding other different pathogen immunogens, can effectively prevent infection of Mycoplasma hyopneumoniae, control the occurrence of Mycoplasma pneumoniae, special experiments and preparations.
  • Example 1 Acquisition of Mycoplasma hyopneumoniae AN306
  • the selected 10 virulent tissue poisons were inoculated into local breeds of healthy susceptible pigs by intranasal drip, after challenge
  • the clinical symptoms of Mycoplasma hyopneumoniae such as cough, asthma, weight loss, and canine sitting were observed in the test animals.
  • typical shrimp-like changes were observed in the sharp leaves and heart leaves of the lungs.
  • clinical symptoms, incidence ratio, lung lesion degree and serum agglutination antibody titer of animal challenge the three most virulent and immunogenicity were selected from 10 virulent tissue poisons for passage. Passage weakened:
  • the virulent tissue was injected into the lungs of healthy rabbits, and the lungs of the rabbits were collected. After homogenization, the healthy rabbits were inoculated and subcultured. Only one of the above three virulent strains was successfully passaged in rabbits. The strains were divided into 20 generations, 40 generations, 60 generations, and 80 generations, and each of them was divided into successive passages and successive passages to 100 generations.
  • Mycoplasma hyopneumoniae was isolated from the lungs of 100 generations of lactating rabbits using the "sick lung immersion method".
  • the specific method is as follows: animal bloodletting death, aseptic operation of collecting lung tissue, cutting 1/2 sesame-sized pieces, washing with Hank's solution in KM2 medium, adding antibiotics, culturing at 37 ° C, daily observation and culture pH change and turbidity of the liquid.
  • the pH showed a significant change (down to 7.0-6.8) and there was uniform turbidity (about 48 h)
  • the above cultures were serially passaged for 1:5 to 5 inoculation with 1:5 inoculum and then subcultured with 1:10 inoculum. Press
  • the isolated microbial cells are polymorphic, with typical morphological characteristics of Mycoplasma hyopneumoniae.
  • a microagglutination reaction and a metabolic inhibition test were carried out using Mycoplasma hyopneumoniae standard positive serum, and identified as Mycoplasma hyopneumoniae.
  • the isolated M. hyopneumoniae was purified by three solid clones and identified again and stored lyophilized.
  • the freeze-preserved F100-derived Mycoplasma hyopneumoniae strain was resuscitated, and serial passage was weakened by using KM2 medium at 1:10. Each five generations were identified by PCR and stored frozen every 20 generations. From F100,
  • AN301 F240 grows well, 10 9 CCU/mL no apparent lesions, no pathological changes
  • AN302 F110 grows slowly, 10 2 CCU/mL - media passage lost
  • AN303 F280 grows well, 10 8 CCU/mL no apparent lesions, no pathological changes
  • AN304 F240 grows well, 10 7 CCU/mL no apparent lesions, no pathological changes AN305 F32 suckling rabbits are lost
  • AN306 F280 grows well, 10 y CCU/mL no apparent lesions, no pathological changes
  • AN307 F140 grows well, 10 8 CCU/mL no apparent lesions, mild inflammatory pathology
  • AN308 F300 grows well, 10 9 CCU/mL no apparent lesions, no pathological changes
  • AN309 F360 grows well, 10 8 CCU/mL no apparent lesions, no pathological changes
  • AN311 F260 grows slowly, 10 4 CCU/mL has no apparent lesions, no pathological changes
  • the strains screened above were subjected to immunogenicity assay and challenge immunoprotective assay. Lyophilized bacteria of different strains were prepared, and the titer of each lyophilized strain was determined. The freeze-dried vaccine was dissolved in a sterile phosphate buffer, and each strain was adjusted to the same concentration with a sterile phosphate buffer.
  • the immunoprotective power of the 5-7 day old healthy negative Meishan pig was determined. Animals were randomized into groups of 5 each. In the immunized group, 0.5 mL (10 5 CCU/head) of attenuated attenuated strain was injected into the lung. After 8 weeks of immunization, intratracheal injection of Mycoplasma hyopneumoniae was highly virulent, and 28 days later, the necropsy was performed. A healthy control group and a challenge control group were also set up.
  • Serum antibodies were measured by indirect hemagglutination method and were judged to be positive by 1:10"++" or more.
  • Anatomy score Animals were sacrificed 28 days after challenge, and lung injury in the experimental pigs was scored according to the method reported by MADEC and KOBISCH (1982). The entire lung is divided into left lobe (LCL), left lobe (LAL), left temporal lobe (LDL), right heart (RCL), right lobes (RAL), right temporal lobe (RDL) and accessory cotyledons (IL). ), a total of 7 lung lobe. The damage score for each lung was the sum of the dorsal and ventral lesion scores of the above seven lung lobe, with a total score of 28 points.
  • the AN306 attenuated strain has better immunogenicity, and the attacking test proves that it has the highest protective power. And the above test proves that the growth in vitro is fast, the titer is high, and the safety is good. Therefore, the AN306 strain is selected as the live vaccine strain, and the initial generation is F280.
  • Example 2 Culture of attenuated strain of Mycoplasma hyopneumoniae AN306 strain
  • KM2 liquid medium preparation (according to 2050mL): Eagles solution 1000 mL, hydrolyzed milk protein 10 g, pig serum 400 mL, fresh yeast leach juice 20 mL, Dulbecco phosphate buffer 600 mL, penicillin 4 million units, 0.4% Phenol red 3.5 mL, adjusted to pH 7.4 ⁇ 7.6 with 10 g/L NaOH.
  • lyoprotectant formulated as gelatin 1.5 g, sucrose 12.5 g, double distilled water 100 mL, autoclaved at 115 °C for 15 min, adjusted to pH 7.0 with sterile 1% NaOH. After harvesting the culture, the CCU content was measured, and the lyoprotectant was added in a ratio of 3:2 by volume of the bacteria and the sterilized lyoprotectant. Add the protective agent, mix well, and dispense into an ampoule, 0.5 mL/tube. The ampoules of the prepared strains are placed in a lyophilizer and lyophilized. After lyophilization, the mixture was sealed with nitrogen and stored at -40 °C. One mL of culture was extracted and DNA was extracted for PCR identification.
  • the attenuated strain AN306 culture obtained by this method has a titer of up to 10 9 CCU/mL, and the yield is upgraded.
  • Example 3 Evaluation of immune protection of intrapulmonary injection of live attenuated vaccine against Mycoplasma hyopneumoniae AN306 strain and comparison with other commercial live vaccines and inactivated vaccines
  • Attenuated live vaccine of Mycoplasma hyopneumoniae pneumonia live vaccine AN306 strain dissolve the freeze-dried seedlings with sterile phosphate buffer solution, and prepare a vaccine solution to make the live vaccine titer 2x10 5 CCU/mL. Each animal was intrapulmonarily injected with 0.5 mL, or 10 5 CCU/head.
  • the live vaccine of Mycoplasma hyopneumoniae produced by Jilin Zhengye Biological Products Co., Ltd. is a chicken embryo yolk sac tissue containing attenuated rabbit strain of Mycoplasma hyopneumoniae, hereinafter referred to as commercial vaccine 1, according to the vaccine instructions;
  • the live vaccine of Mycoplasma hyopneumoniae produced by Jilin Zhengye Biological Products Co., Ltd. is a chicken embryo yolk sac tissue containing attenuated rabbit strain of Mycoplasma hyopneumoniae, hereinafter referred to as commercial vaccine 1, according to the vaccine instructions;
  • Commercial vaccine 2 Zhibining, in vitro culture of attenuated strain of Mycoplasma hyopneumoniae 168 strain The following, referred to as commercial vaccine 2, is operated according to the vaccine instructions.
  • G1 group was a healthy control group, no immunization was not challenged; G7 group was a challenge control group, and no immunity was only used for challenge.
  • the G2-G6 group was the vaccine immunization group.
  • the specific grouping and immunization methods are shown in the following table. Animals in the G2-G7 group were intratracheally injected with M. hyopneumoniae for 8 weeks after immunization, and were necropsy after 28 days.
  • the Mycoplasma hyopneumoniae pneumonia lesions in the lungs of the animals were scored using the method described in Example 1.
  • the results showed that the lung lesions of the live attenuated M. pneumoniae attenuated vaccine AN306 strain were significantly reduced, compared with the control group (statistical method One-way ANOVA), the difference was extremely significant (P ⁇ 0.01), and the mean lesion index was obvious. It is lower than the commercial live vaccine and inactivated vaccine widely used in the market (commodity vaccine 1, P ⁇ 0.01; commercial vaccine 2, P ⁇ 0.05; commercial vaccine 3, P ⁇ 0.01; commercial vaccine 4, P ⁇ 0.01).
  • Description Live attenuated vaccine AN306 strain can help immunized animals to effectively resist Mycoplasma hyopneumoniae infection, and the protection is better than the vaccine widely used on the market.
  • Table 4 Evaluation of immunoprotection of intrapulmonary injection of live attenuated vaccine against Mycoplasma hyopneumoniae AN306 strain and comparison with other commercial live vaccines and inactivated vaccines
  • Attenuated live vaccine of Mycoplasma hyopneumoniae pneumonia attenuated vaccine AN306 strain dissolve the freeze-dried seedlings with vaccine dilution solution, and prepare a vaccine solution to make the live vaccine titer of 10 6 CCU/mL.
  • G1 group was healthy control group, no immunization was not challenged
  • G2 group was live vaccine aerosol immunization group, aerosol immunization AN306 live vaccine 0.5 mL (5xl0 5 CCU/head), re-immunized 2 weeks after the first immunization, challenged after immunization
  • G3 group is a commercial vaccine group, immunizing the commercially available Mycoplasma hyopneumoniae pneumonia inactivated vaccine RespiSure, according to the vaccine instructions Operation, challenge after immunization
  • G4 group is the control group of the challenge, not immune only to attack. Animals in G2, G3, and G4 were intratracheally injected with M. hyopneumoniae for 8 weeks after the first immunization, and were examined by necropsy 28 days later. result:
  • the Mycoplasma hyopneumoniae pneumonia lesions present in the lungs of the animals were scored using the method described in Example 1, as shown in the table below.
  • the results showed that the lung disease of the animal model of attenuated live attenuated Mycoplasma pneumoniae pneumoniae AN306 strain was significantly reduced, which was significantly different from the challenge control group (P ⁇ 0.01), and was significantly lower than the commercial inactivated vaccine (P). ⁇ 0.01). This indicates that the live attenuated vaccine can help immunized animals against M. hyopneumoniae infection by aerosol immunization, and the protection is better than the inactivated vaccine widely used on the market.
  • Table 5 Evaluation of immunoprotective capacity of AN306 strain of aerosol-activated live attenuated Mycoplasma pneumoniae pneumonia
  • Pulmonary lesion score 1.08 ⁇ 0.27 3.83 ⁇ 2.48 8.25 ⁇ 4.14 20.34 ⁇ 3.45
  • Vaccine preparation Attenuated live vaccine of Mycoplasma hyopneumoniae pneumoniae AN306 strain.
  • the adjuvant used was a mixture adjuvant containing 5 mg/mL carbomer and 30 mg/mL xanthine polysaccharide formulated in sterile phosphate buffer. Lyophilized vaccine adjuvant solution, the solution is formulated such that the vaccine wherein the live vaccine titer 2.5xl0 5 CCU / mL.
  • G1 group was healthy control group, no immunization was not challenged
  • G2 group was live vaccine intramuscular injection immunization group, intramuscular injection of AN306 live vaccine 2 mL ( 5xl0 5 CCU/head), re-immunized 2 weeks after the first immunization, challenged after immunization
  • G3 group is a commercial vaccine group, immunization is currently used on the market, a wide range of commercially available Mycoplasma hyopneumoniae inactivated vaccine RespiSure, according to the vaccine instructions
  • the G4 group was the challenge control group, and the non-immunization only attacked the poison.
  • Animals in G2, G3, and G4 were intratracheally injected with M. hyopneumoniae for 8 weeks after the first immunization, and were examined by necropsy 28 days later. result:
  • the Mycoplasma hyopneumoniae pneumonia lesions present in the lungs of the animals were scored using the method described in Example 1, as shown in the table below.
  • the results showed that the lung lesions of the muscle-injected live attenuated M. pneumoniae attenuated vaccine AN306 strain were significantly reduced, which was significantly different from the challenge control group (P ⁇ 0.01), and significantly lower than the commercial inactivated vaccine (P). ⁇ 0.01).
  • P live attenuated vaccine can help immunized animals to resist Mycoplasma hyopneumoniae infection by intramuscular injection, and the protection is better than the inactivated vaccine widely used on the market.
  • Table 6 Intramuscular Injection of Liver Attenuated Mycoplasma Pneumoniae Attenuated Live Vaccine Evaluation of Immunoprotection of AN306 Strain Gl G2 G3 G4
  • a chitosan-containing vaccine was prepared as follows: chitosan was dissolved in a 1% acetic acid solution to prepare a mother liquor containing 1% chitosan, and the pH was adjusted to 6.5 with a 0.1 M NaOH solution; using 0.1 M NaAc-HAc (pH 6) The solution of .5) was diluted with the above chitosan mother liquor to a final chitosan concentration of 0.2%.
  • the fresh culture of Mycoplasma hyopneumoniae AN306 strain was centrifuged at 12,000 rpm for 30 min to collect the cells, and resuspended in 1 mL of 50 mM Na 2 S0 4 to give a titer of 6.66 x 10 6 CCU/mL.
  • G1 group was healthy control group, no immunization was not challenged
  • G2 group was live vaccine intranasal immunization group, nasal immunization AN306 live vaccine 0.5 mL (5xl0 5 CCU/head), re-immunized 2 weeks after the first immunization, challenged after immunization
  • G3 group is a commercial vaccine group, immunization is currently used on the market, a wide range of commercially available Mycoplasma hyopneumoniae inactivated vaccine RespiSure, according to the vaccine instructions Operation, challenge after immunization
  • G4 group is the control group of the challenge, not immune only to attack. Animals in G2, G3, and G4 were intratracheally injected with M. hyopneumoniae for 8 weeks after the first immunization, and were examined by necropsy 28 days later. result:
  • the Mycoplasma hyopneumoniae pneumonia lesions present in the lungs of the animals were scored using the method described in Example 1, as shown in the table below.
  • the results showed that the lung lesions of the live attenuated M. pneumoniae attenuated vaccine AN306 strain were significantly reduced, compared with the control group (P ⁇ 0.01), and significantly lower than the commercial inactivated vaccine (P ⁇ 0.01). ).
  • the live attenuated vaccine can help immunized animals against Mycoplasma pneumoniae infection by intranasal immunization, and the protection is better than the inactivated vaccine widely used on the market.
  • Example 7 Evaluation of the immunoprotective effect of the live vaccine of Mycoplasma hyopneumoniae AN306 strain and the inactivated vaccine against Haemophilus parasuis
  • Serum type 5 H. parasuis XX0306 strain (this strain was isolated by the Institute of Veterinary Medicine of Jiangsu Academy of Agricultural Sciences, and has good immunogenicity. The test proved to be suitable for the preparation of inactivated vaccine against Haemophilus parasuis, and the protective effect is good)
  • the adjuvant was formulated with a 5 mg/mL carbomer and immunostimulating complex matrix in a sterile phosphate buffer (component: Quil A 50 ( ⁇ g/mL, cholesterol 10 (Vg/mL, phospholipid 10 (Vg/mL) a mixture of adjuvants.
  • the 0.5 mL M. hyopneumoniae fresh culture was mixed with 0.5 mL of serum type 5 H. parasuis inactivated antigen and adjuvant at a volume ratio of 1:1:2 to immunize the animals.
  • G1 group was healthy control group, 10 heads, no immunization and no attack, 5 of them were simultaneously necropsy with G2 and G3 animals, and 5 other heads. Simultaneous necropsy with G4 and G5 animals; G2 group was combined with M.
  • hyopneumoniae challenge group 5 heads, intramuscular injection of 2 mL of the above-mentioned prepared vaccine (AN306 live vaccine 2.5xl0 5 CCU/mL, serum type 5) Haemophilus parasuis XX0306 strain inactivated antigen 2.5xl0 9 CFU/mL), re-immunized 3 weeks after the first immunization, intratracheal injection of Mycoplasma hyopneumoniae virulent challenge 8 weeks after the first immunization; G3 group was Mycoplasma hyopneumoniae attack Toxicity control group, 5 heads, not immunized, same as G2 group animals; G4 group was combined with M.
  • the above-mentioned prepared vaccine AN306 live vaccine 2.5xl0 5 CCU/mL, serum type 5
  • Haemophilus parasuis XX0306 strain inactivated antigen 2.5xl0 9 CFU/mL
  • parahaemolyticus challenge group 5 heads, intramuscular injection of the above prepared 2 ml vaccine (AN306 live vaccine 2.5 Xl0 5 CCU/mL, serum type 5 Haemophilus parasuis XX0306 strain inactivated antigen 2.5xl0 9 CFU/mL), re-immunized 3 weeks after the first immunization, intraperitoneal injection of Haemophilus parasuis 6 weeks after the first immunization Toxic attack; G5 group was Haemophilus parasuis challenge control group, 5 heads, not immune, with Animals in group G4 also attacked.
  • the lung mycoplasma pneumoniae lesions were scored according to the method described in Example 1, and recorded in the table below. The results showed that the combined vaccine could significantly reduce the incidence of lung lesions ( ⁇ 0 ⁇ 01) ).
  • the clinical symptoms of Haemophilus parasuis are characterized by decreased appetite, rough hair, redness of the ears and body skin, increased nasal fluid, difficulty in breathing later, swelling of the joints, and lying down.
  • the pleural examination showed chest and peritoneal effusion, varying degrees of pleural and peritoneal adhesions, joint swelling, increased mucus in the joint cavity, lung bleeding, and swollen lymph nodes.
  • the incidence of Haemophilus parasuis in each group of animals was judged by the above indicators, and the summary is recorded in the following table. The results showed that the immunization of the vaccine could significantly reduce the incidence of Haemophilus parasuis in animals.
  • Table 8 Evaluation of immunoprotection of a combination of live vaccine of Mycoplasma hyopneumoniae AN306 and inactivated vaccine of Haemophilus parasuis
  • Healthy control group combined vaccine, pig, Mycoplasma pneumoniae, combined vaccine, pig swine, blood stasis, Mycoplasma pneumoniae, challenge control, paraglottic blood test, challenge group, challenge group, bacillus attack group
  • Immunization non-immunization, vaccine immunization, non-immunization, immunization with immunization, non-immunization, Mycoplasma hyopneumoniae, no challenge, attack, attack, no attack, no attack, no attack, attack, attack
  • Example 8 Evaluation of immunoprotection of live vaccine of Mycoplasma hyopneumoniae AN306 strain and live vaccine of porcine reproductive and respiratory syndrome
  • a mixture adjuvant containing 10 mg/mL chitosan and 10 mg/mL levamisole was prepared in sterile phosphate buffer.
  • the freeze-dried seedlings of Mycoplasma hyopneumoniae live vaccine AN306 were used to dissolve the freeze-dried M.
  • the two vaccine solutions were mixed at a ratio of 3:1 (volume ratio), and the animals were immunized, and each animal was inoculated with 2 mL, that is, one live vaccine of Mycoplasma hyopneumoniae and one live vaccine of Porcine Reproductive and Respiratory Syndrome.
  • Animal immunity and attack :
  • G1 group was healthy control group, 10 heads, no immunization and no attack, 5 of them were simultaneously necropsy with G2 and G3 animals, and 5 other heads. Simultaneous necropsy with G4 and G5 animals; G2 group was combined with M.
  • hyopneumoniae challenge group 5 heads, intramuscularly immunized 2 mL of the above-mentioned prepared vaccine, re-immunized 2 weeks after the first immunization, 8 weeks after the first immunization
  • G3 group was a Mycoplasma hyopneumoniae challenge control group, 5 heads, not immunized, same as G2 group of animals
  • G4 group was combined with vaccine against pig reproductive and respiratory syndrome virus attack Toxic group, 5 heads, intramuscularly immunized 2mL of the above-mentioned prepared vaccine, re-immunized 2 weeks after the first immunization, 6 months after the first immunization, intramuscular injection of porcine reproductive and respiratory syndrome virus virulent challenge
  • G5 group Pig Breeding and Respiratory Syndrome virus challenge control group, 5, not immune, and the same as G4 animals.
  • the lung mycoplasma pneumoniae lesions were scored according to the method described in Example 1, and recorded in the table below. The results showed that the combined vaccine could significantly reduce the incidence of lung lesions ( ⁇ 0 ⁇ 01) ).
  • the criteria for the diseased pigs were: high fever above 41 °C for more than 3 days, decreased mental and appetite, respiratory conjunctivitis and respiratory symptoms such as cough and gasp, and death.
  • the necropsy showed a flaky consolidation.
  • the results showed that all the animals in the control group of the porcine reproductive and respiratory syndrome virus challenged, 2 of which died, and the animals in the combined vaccine group had no obvious symptoms and all survived.
  • Table 9 Evaluation of the immunoprotective effect of the live vaccine of Mycoplasma hyopneumoniae AN306 strain and live vaccine of porcine reproductive and respiratory syndrome
  • Healthy control group combined vaccine, pig, Mycoplasma pneumoniae, vaccine vaccine, pig breeding and respiratory, Mycoplasma pneumoniae, challenge control, pig breeding and sucking syndrome, attack group, respiratory syndrome, poison challenge, virus attack, virus group
  • Immunization, non-immunization, immunization, immunization, immunization, immunization, immunization, immunization Mycoplasma pneumoniae does not attack and attack, does not attack, does not attack, breeds pigs, breeds and breaths, does not attack, does not attack, does not attack, does not attack

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Abstract

本发明公开了一种猪支原体肺炎减毒活疫苗及其应用。本发明筛选具有典型猪肺炎支原体感染并且无明显其他病原感染的猪肺脏病料，利用乳兔肺传代至100代，然后分离猪肺炎支原体菌株，利用培养基进行连续传代，通过多个菌株的筛选获得猪肺炎支原体AN306，其保藏号为CCTCC M 2012431。本发明还公开了基于该弱毒株制备的猪支原体肺炎活疫苗制剂，包含活的弱毒株、药学上可接受的载体或辅料，并可以包含佐剂和其他病原的免疫原。

Description

一种猪支原体肺炎减毒活疫亩及其应用 技术领域
本发明涉及一种猪支原体肺炎减毒疫苗株， 含有该减毒株的活疫苗， 及其在 保护猪肺炎支原体感染相关疾病中的应用。 说
背景技术
猪支原体肺炎， 俗称猪气喘病， 是由猪肺炎支原体（Mycoplasma hyopneumoniae, Mhp)引起的一种猪的慢性呼书吸道传染性疾病， 主要症状为咳嗽 和气喘。 该病主要引起猪的伺料转化率下降和生长受阻， 发病率高、 死亡率低。 猪支原体肺炎在世界范围内广泛流行， 给现代养猪业造成重大经济损失。
猪肺炎支原体常常与其他病原形成混合感染。 例如与多杀性巴氏杆菌 ( Pasteurella Multocida ) 猪链球菌 （Streptococcus Suis , SS )、 副猪嗜血杆菌 (Haemophilus Parasuis of Swine, HPS ) 或猪胸膜肺炎放线杆菌 （ Actinobacillus Pleuropneumonia of Porcine, APP)等混合感染， 引发猪地方流行性肺炎。 当猪肺 炎支原体与猪繁殖呼吸综合征病毒（Porcine Reproductive and Respiratory Syndrome \irus, PRRSV)、 猪圆环病毒 2型 （Porcine Circovirus Type 2， PCV2) 和 /或猪流感病毒 (Swine Influenza \irus, SIV)混合感染时， 就会发生常见的猪呼 吸道疾病综合征 (Porcine Respiratory Disease Complex, PRDC), 不仅可以引起猪 的呼吸***疾病， 同时也会导致猪的繁殖能力下降。
猪支原体肺炎是一种顽固性慢性传染病， 药物治疗效果不够理想， 停药后容 易复发， 该病一旦在猪场流行， 就很难根除。 因此， 预防、 控制和净化猪支原体 肺炎须采取早期诊断、及时隔离、抗生素治疗或淘汰病猪、疫苗预防、全进全出、 严格消毒等综合性措施， 其中疫苗预防在控制猪支原体肺炎中起着关键性的作 用。
尽管目前猪支原体肺炎灭活疫苗已经在临床上进行了大规模的应用， 但是对 该病的控制效果却并不尽如人意， 且通常情况下， 灭活疫苗可以一定程度上降低 患病猪肺脏的病变指数，但并不能有效降低患病猪的比例。猪支原体肺炎因此被 认为是流行范围最广、最难净化的猪传染病之一。这些疫苗效果不佳的原因可能 与其灭活疫苗的本质以及猪肺炎支原体感染的特性有关。
猪肺炎支原体通过呼吸道感染结合于支气管纤毛上皮细胞上形成定殖， 从而 造成持续感染。灭活疫苗免疫后激发全身性免疫应答， 而诱发局部免疫应答的效 力不佳， 其产生的循环抗体必须通过上皮屏障， 分泌到气管支气管管腔， 而该过 程异常困难， 而且效率低下。 为了能够达到有效免疫， 疫苗必须能够刺激高水平 的***免疫应答。 当然， 这需要相对较高的抗原剂量和较强的佐剂， 这样就使这 些疫苗成本增加价格昂贵。 同时， 灭活疫苗产生的免疫应答只能抑制野毒感染后 的增殖和减轻组织破坏程度， 并不能完全阻止野毒的感染， 因而难以达到对 该病的理想控制。 发明内容
本发明的目的在于提供一种猪支原体肺炎减毒活疫苗， 该猪支原体肺炎减 毒活疫苗可用于预防和控制猪肺炎支原体感染导致的疾病， 包括其他病原 菌与猪肺炎支原体共同感染导致的疾病。
猪支原体肺炎减毒活疫苗的作用机制不同于灭活疫苗， 其接种后可在 体内增殖， 有效激活机体免疫***， 同时活疫苗进入呼吸***后可定殖于 支气管纤毛细胞上， 产生"占位效应"， 使得野毒无法定殖， 从源头上阻断 野毒的侵染过程， 从而起到很好的抗感染作用。 同时， 由于活疫苗可以在 体内增殖， 有效激活免疫***， 所需要的剂量远远低于灭活疫苗， 因此在 成本控制上较灭活疫苗有明显优势， 有利于节约社会资源。
活疫苗研制的关键在于高免疫原性菌株的获得和致弱过程中使菌株毒 力下降同时保持免疫原性的致弱技术。
本发明根据毒株的特点， 使用前 100代乳兔肺继代， 100代以后无细胞 培养基继代的致弱方法。 该方法的优点在于在前期传代过程中采用动物体 内继代， 可以避免使用培养基体外继代时容易导致菌株在早期过快致弱、 不利于免疫原性维持的缺陷。 后期采用无细胞培养基继代， 有利于一定程 度上减少致弱代次， 縮短致弱过程。
该毒株具体来源为： 筛选具有典型猪肺炎支原体感染同时无明显其他病原感 染的猪肺脏病料， 利用乳兔肺继代传至 100代， 然后分离猪肺炎支原体菌株， 利 用培养基进行连续传代， 同时通过多个菌株的筛选获得。筛选得到的毒株命名为 猪肺炎支原体 (Mycoplasma hyopneumoniae, Mhp)弱毒株 AN306。该弱毒株经过 长期多次传代致弱后， 丧失致病力， 对动物安全， 并保留了良好的免疫原性， 能 为免疫动物提供免疫保护， 防止猪肺炎支原体感染疾病的发生。经过五代毒力返 强试验证实弱毒株 AN306不会回复突变成为有致病力的强毒株。 因此， 该弱毒 株 AN306可在制备猪支原体肺炎疫苗或多联疫苗中应用。
该猪肺炎支原体 (Mycoplasma hyopneumoniae, Mhp)弱毒株已于 2012年 10 月 25 日， 保藏于位于武汉市武昌珞珈山的中国典型培养物保藏中心， 保藏号为 CCTCC M 2012431 ο
本发明的另一个目的在于公开该猪支原体肺炎减毒活疫苗， 该减毒活疫苗可 为猪支原体肺炎疫苗或多联疫苗。 包括上述猪肺炎支原体（Mycoplasma hyopneumoniae, Mhp) 弱毒株 AN306, 以及药学上可接受的载体或辅料。 载体 或辅料主要包括三类物质，根据疫苗的具体使用状态和免疫途径而不同。一类为 溶剂或缓冲体系， 包括无菌生理盐水、 无菌磷酸盐缓冲液； 一类为冻干保护剂， 包括脱脂奶粉、 明胶、蔗糖， 当疫苗制备为冻干形式时需添加入冻干体系中帮助 活疫苗维持活性； 一类为喷雾保护剂， 主要为甘油， 当活疫苗采用气雾免疫时需 加入帮助活疫苗在喷雾过程中维持活性。
同时本发明的猪支原体肺炎活疫苗制剂可进一步含有一种免疫佐剂。
佐剂是疫苗制剂中的常见成分， 用于增强疫苗本身的免疫刺激能力。 本发明 中使用的佐剂是适合于猪支原体肺炎活疫苗的佐剂，即对该活疫苗的活力不造成 明显损伤， 同时适合猪支原体肺炎的免疫需求。
本发明所公开的免疫佐剂， 可为单一佐剂成份或由多种佐剂成分复合而成， 所含佐剂成份可包括中草药多糖、 卡波姆、 壳聚糖、 免疫刺激复合物基质、 左旋 咪唑、 葡聚糖、 CpG、 囊素中的一种或多种。 各种免疫佐剂成分、 配比和使用剂 量因活疫苗具体使用的剂量及给药途径的不同而不同。
本发明的猪支原体肺炎活疫苗制剂中可进一步含有至少一种其他病原的免 疫原， 形成联苗进行使用。 其他的病原包括病毒、 细菌、 真菌、 寄生虫中的 至少一种， 例如猪圆环病毒、 副猪嗜血杆菌、 猪繁殖与呼吸综合征病毒、 猪鼻 支原体、猪滑液支原体、猪流感病毒等。 这些病原在临床上常与猪肺炎支原体在 呼吸道中形成混合感染或继发感染， 相互促进， 导致病情加重， 给猪群健康带来 重大威胁。因此将这些病原的疫苗与猪支原体肺炎活疫苗联用， 可用于预防猪肺 炎支原体及其他病原的混合感染。所述其他病原的免疫原既可以为活菌或活毒形 式， 也可以为灭活形式、 蛋白亚单位形式等。 值得注意的是， 其他病原疫苗所使 用载体、辅料和佐剂必须与本猪支原体肺炎活疫苗使用的体系兼容， 同时必须要 能够为本活疫苗所耐受。
本发明的猪支原体肺炎减毒活疫苗可通过多种途径进行免疫， 包括肺内注 射、 肌肉注射、 气雾免疫、 口服免疫和滴鼻免疫。 所述减毒活疫苗采用的制剂可 为肺内注射剂、 肌肉注射剂、 气雾剂、 口服剂或滴鼻剂。 免疫途经的选择取 决于将进行免疫接种的动物、 免疫接种史、猪场环境， 以及给动物进行疫苗接种 人员的操作方便性。猪肺炎支原体感染机体后首先结合在肺部支气管上皮纤毛细 胞上， 导致纤毛脱落， 继而进一步侵染组织、 诱发炎症， 同时为其他病原菌的感 染打开门户。猪支原体肺炎活疫苗采用肺内注射途径免疫时活疫苗能够直接注射 入靶组织， 结合到纤毛细胞上， 从而占据结合位点， 阻止野毒的感染； 当猪支原 体肺炎活疫苗采用气雾途径免疫时通过控制雾滴粒径能够使得活疫苗到达动物 的下呼吸道内，有效结合在支气管纤毛细胞上， 同时在封闭的畜棚或空间内使用 气雾剂疫苗可以减少进行免疫接种所需的人员；肌肉注射为动物免疫最常用的接 种方式， 操作简便， 可接受度高， 当猪支原体肺炎活疫苗采用肌肉注射途径免疫 时， 能够诱发动物产生全身性的细胞及体液免疫应答， 该应答反应到肺部靶组织 局部， 可抑制猪肺炎支原体野毒的侵染； 当猪支原体肺炎活疫苗采用口服或滴鼻 方式接种时， 可诱导肠道或鼻腔局部黏膜免疫应答， 通过共同黏膜免疫***， 淋 巴细胞可归巢到不同的黏膜部位， 从而实现下呼吸道支气管肺部组织的免疫应 答， 实现对猪肺炎支原体野毒感染的保护。根据所使用的接种途径， 其所选用的 免疫佐剂亦随之发生变化。 因此， 本发明所述猪支原体肺炎减毒活疫苗可在防 治猪肺炎支原体感染疾病中应用。 本发明猪支原体肺炎减毒活疫苗的制备方 法是将猪肺炎支原体 (Mycoplasma hyopneumoniae， Mhp)弱毒株 AN306与药学 上可接受的载体或辅料， 以及至少一种免疫佐剂， 至少一种其他病原的免疫 原组合在一起。 本发明的猪支原体肺炎活疫苗可以采用单次免疫， 亦可采用 2次或多次重复 接种来增强免疫应答。根据免疫接种的途径及动物状况不同而具体选择。单次免 疫接种时间通常为 3-10日龄， 优选为 5-7日龄。 2次或多次重复接种的首次免疫 接种时间通常为 3-10 日龄， 优选为 5-7 日龄。 两次免疫接种之间的时间间隔一 般为 2-3周。 生物样品保藏信息：
上述猪肺炎支原体弱毒株 AN306 , 分类命名为： 猪肺炎支原体 AN306 (Mycoplasma hyopneumoniae AN306) 已于 2012年 10月 25日， 保藏位于武汉市 武昌珞珈山的中国典型培养物保藏中心， 保藏号为 CCTCC M 2012431。 具体实 式：
结合具体实施例对本发明进行进一步描述和论证，但是本发明所包括的内容 并不局限于此。
为了说明本发明的猪支原体肺炎活疫苗菌株的筛选、传代、鉴定过程以及活 疫苗菌株的免疫原性和安全性， 同时验证本发明的猪支原体肺炎活疫苗制剂，包 括采用多种不同免疫途径、 多种不同佐剂、添加其他不同病原免疫原的情况， 能 够有效防止猪肺炎支原体的感染，控制猪支原体肺炎的发生，特做以下实验和制 备。 实施例 1 : 猪肺炎支原体 AN306的获得
强毒株的获得：
1975年江苏省农业科学院根据农业部要求集中了全国 20多个省的农业大学 和国家、省级农业科学院的猪支原体肺炎典型病料进行猪肺炎支原体强毒致病株 的筛选工作。共搜集全国各地的疑似猪支原体肺炎肺脏病料二百余份， 从中筛选 出具有典型猪肺炎支原体感染并无明显其他病原感染的病料 10份， 作为猪支原 体肺炎的强毒组织毒冻干保存。同时进行本动物攻毒发病试验和连续传代致弱试 验。
将所选的 10个强毒组织毒以滴鼻方式接种地方品种健康易感猪， 攻毒后均 可观察到试验动物出现咳嗽、 气喘、 消瘦、 犬坐等猪支原体肺炎临床症状， 剖检 时其肺部尖叶、心叶可见典型虾肉样实变。根据动物攻毒发病潜伏期、临床症状、 发病比例、 肺脏病变程度、 血清凝集抗体效价， 从 10个强毒组织毒中挑选毒力 最强、 免疫原性最好的 3个进行传代。 传代致弱：
将强毒组织毒肺内注射接种健康乳兔， 采集攻毒兔肺脏， 匀浆后再接种健康 乳兔， 如此继代。上述 3个强毒仅 1株成功用兔体进行传代。 该毒株在传代时分 别于 20代、 40代、 60代、 80代时各多分设一平行传代组， 连续传代至 100代。
采用"病肺块浸泡法 "从 100代乳兔肺脏中分离猪肺炎支原体。 具体方法为： 动物放血致死， 无菌操作采集肺组织， 剪下 1/2芝麻大小的小块， 用 Hank's液 洗涤浸泡于 KM2 培养液中， 加入抗生素， 置于 37°C培养， 每天观察培养液的 pH变化和混浊度。 当 pH呈现明显变化 （降至 7.0-6.8) 且有均匀混浊 (约 48h)产 生时， 上述培养物以 1:5接种量连续传代 4-5代后， 再以 1:10接种量继代。 按
Gurr 改良的姬姆萨染色法染色， 油镜镜检， 所分离的微生物菌体呈多形态， 具 有猪肺炎支原体的典型形态特征。利用猪肺炎支原体标准阳性血清进行微粒凝集 反应和代谢抑制试验， 鉴定为猪肺炎支原体。将所分离获得的猪肺炎支原体通过 三次固体克隆纯化并再次鉴定后冻干保存。
将冻干保存的 F100代猪肺炎支原体菌株复苏， 利用 KM2培养基按 1 : 10继 续进行连续传代致弱。 每五代采用 PCR鉴定， 每 20代冻干保存。 分别从 F100、
F140、 F180、 F220 F240 F280 、 F320代中另分一株同样进行传代， 传至不同 代次。 表 1 : 致弱菌株的初步筛选
菌株 代次 体外培养生长情况 攻毒发病情况 备注
AN301 F240 生长良好， 109CCU/mL 无表观病变， 无病理改变
AN302 F110 生长缓慢， 102CCU/mL - 培养基传代丢失
AN303 F280 生长良好， 108CCU/mL 无表观病变， 无病理改变
AN304 F240 生长良好， 107CCU/mL 无表观病变， 无病理改变 AN305 F32 乳兔继代丢失
AN306 F280 生长良好， 10yCCU/mL 无表观病变， 无病理改变
AN307 F140 生长良好， 108CCU/mL 无表观病变， 轻度炎性病理
改变
AN308 F300 生长良好， 109CCU/mL 无表观病变， 无病理改变
AN309 F360 生长良好， 108CCU/mL 无表观病变， 无病理改变
AN310 F12 乳兔继代丢失
AN311 F260 生长缓慢， 104CCU/mL 无表观病变， 无病理改变
AN312 F64 乳兔继代丢失 通过传代情况、 生长性能、 毒力几个方面对不同传代菌株进行综合分析， 选 择生长迅速、滴度较高、毒力致弱的 5个菌株 AN301、 AN303、 AN306、 AN308、 AN309进行后续毒力返强试验和免疫保护能力测定。 致弱菌株的毒力返强试验：
将上述筛选的菌株进行连续 5代毒力返强试验。 将 108CCU/mL猪肺炎支原 体培养物， 向 3头健康猪气管内注射 3mL， 隔离伺养 28d， 剖检观察实验猪肺脏 是否出现病变， 同时进行病理切片， 观察是否产生炎性改变。 无菌取肺脏心叶、 尖叶和膈叶前缘组织 5-7g剪碎， 碾磨成糊状， 加入含有 1000 IU/mL青霉素的 Hank's溶液 lOOmL, 配制成 50g/L的肺悬液， 吸取上清液于当日再次接种健康 猪 3头， 每头 3mL， 如此反复传代 5次， 观察其是否出现毒力返强的现象。 表 2: 致弱菌株的毒力返强试验筛选
菌株 AN301 AN303 AN306 AN308 AN309
代次 F240 F280 F280 F300 F360
第一次 0/3 0/3 0/3 0/3 0/3
第二次 0/3 0/3 0/3 0/3 0/3
第三次 0/3 0/3 0/3 0/3 0/3
第四次 1/3 0/3 0/3 0/3 0/3 第五次 2/3 0/3 0/3 0/3 0/3
结果显示， AN301 菌株在第四及第五代时出现有动物肺脏发生炎性病理改 变，其余 4株 5次接种的动物均未出现明显的猪支原体肺炎临床症状， 剖检未发 现肺脏实变，病理切片检查未发现淋巴组织增生、淋巴细胞与巨噬细胞浸润等炎 性病理改变。 由此证明这 4株传代菌株为弱毒株且不具备毒力返强能力。 致弱菌株的免疫试验：
将上述筛选的菌株进行免疫原性测定和攻毒免疫保护力测定。制备不同菌株 的冻干菌， 测定各冻干菌株滴度。用无菌磷酸盐缓冲液溶解冻干苗， 并用无菌磷 酸盐缓冲液将各菌株调整至同样浓度进行试验。
用 5-7日龄健康阴性梅山猪进行攻毒免疫保护力测定。 动物随机分组， 每组 5头。免疫组动物肺内注射免疫弱毒株 0.5mL(105CCU/头)， 免疫后 8周气管内注 射猪肺炎支原体强毒攻毒， 28天后剖检。 另设健康对照组及攻毒对照组。
用间接血凝法测定血清抗体， 1:10"++"以上判定为阳性。
剖检评分： 攻毒后 28天处死动物， 参照 MADEC和 KOBISCH ( 1982) 报 道的方法对实验猪肺部的损伤进行评分。 整个肺脏分左心叶 （LCL)、 左尖叶 (LAL)、 左膈叶 （LDL)、 右心叶 （RCL)、 右尖叶 （RAL)、 右膈叶 （RDL) 禾口 副叶 （IL)， 共 7个肺叶。 每个肺脏的损伤分数为上述 7个肺叶的背侧面和腹侧 面损伤得分之和， 总分为 28分。 每个肺叶按照损伤的面积分别打 0-4分， 其中 无损伤为 0分， 1-25%面积损伤为 1分， 26-50%面积损伤为 2分， 51-75%面积损 伤为 3分， 76-100%面积损伤为 4分。 表 3: 致弱菌株的免疫试验筛选
免疫菌株 健康对照 AN303 AN306 AN308 AN309 攻毒对照 代次 F280 F280 F300 F360
抗体 - - + + - - 猪肺脏病变评 0.24土 0.15 6.23+3.39 3.01+1.45 7.73+4.38 11.10+6.77 20.29土 5.42 分
由结果分析， AN306弱毒株的免疫原性较好， 攻毒试验证明其保护力最高， 且前述试验证明其体外培养生长快、 滴度高， 安全性好， 故最终选择 AN306株 为活疫苗菌株使用， 初始代次为 F280。
实施例 2: 猪肺炎支原体弱毒株 AN306株的培养
KM2液体培养基制备 （按 2050mL计）： Eagles氏液 1000 mL, 水解乳蛋白 10 g, 猪血清 400 mL， 鲜酵母浸出汁 20 mL， Dulbecco磷酸盐缓冲液 600 mL， 青霉素 400万单位， 0.4%酚红 3.5 mL， 用 10 g/L NaOH 调 pH值为 7.4〜7.6。
取猪肺炎支原体弱毒株 AN306冻干菌种 1支，用 0.5 mL培养基溶解后取 0.5 mL接种于 4.5 mL培养基中， 于封闭青霉素瓶中 37°C培养， 当培养基颜色由红 色变成黄色时及时收获培养物， 按体积比 1:10 的比例接种入新鲜培养基中扩大 培养， 当培养基颜色由红色变成黄色及时收获培养物， 依此培养扩大至 5000 mL 并收获菌种。
配制冻干保护剂， 配方为明胶 1.5 g、 蔗糖 12.5 g、 双蒸水 lOO mL, 115°C高 压灭菌 15 min, 使用灭菌的 1% NaOH调节 pH至 7.0。 收获培养物后先测定其 CCU含量，按菌种与灭菌冻干保护剂体积比为 3:2的比例添加冻干保护剂。添加 保护剂后混匀， 分装至安瓿瓶， 0.5 mL/管。 将分装好菌种的安瓿瓶放至冻干机 中， 进行冻干。 冻干结束用氮气封口， -40°C保存。 抽取 1 mL培养物提取 DNA 进行 PCR鉴定。
通过此方法获得的弱毒株 AN306培养物， 滴度可达 109CCU/mL， 产量为升 级。
实施例 3: 肺内注射免疫猪支原体肺炎减毒活疫苗 AN306株的免疫保护力评价 及与其他商品化活疫苗及灭活疫苗的比较
疫苗制备：
猪支原体肺炎减毒活疫苗 AN306株冻干苗， 用无菌磷酸盐缓冲液溶解冻干 苗， 配制疫苗溶液使得其中活疫苗滴度为 2xl05CCU/mL。 每头动物肺内注射 0.5mL， 即 105CCU/头。
商品化活疫苗： 本试验使用目前市场仅有的两种猪支原体肺炎活疫苗作为对 照活疫苗。 （1 )吉林正业生物制品股份有限公司生产的猪支原体肺炎活疫苗， 为 含猪肺炎支原体兔化弱毒株的鸡胚卵黄囊组织， 以下简称为商品疫苗 1， 按照疫 苗说明书操作； （2)商品名： 支必宁， 为猪肺炎支原体弱毒株 168株的体外培养 物， 以下简称为商品疫苗 2， 按照疫苗说明书操作。
商品化灭活疫苗： 本试验使用目前市场上使用较为广泛的两种猪支原体肺炎 灭活疫苗作为对照灭活疫苗。 （1 ) 商品名： Res Sure, 以下简称为商品疫苗 3， 按照疫苗说明书操作。； （2)商品名： MycoFLEX, 以下简称为商品疫苗 4， 按照 疫苗说明书操作。
动物免疫及攻毒：
健康仔猪 70头， 随机分成 7组： G1组为健康对照组， 不免疫不攻毒； G7 组为攻毒对照组， 不免疫只攻毒。 G2-G6组为疫苗免疫组， 具体分组及免疫方法 见下表， 免疫后攻毒。 G2-G7组动物于免疫后 8周气管内注射猪肺炎支原体强毒 攻毒， 28天后剖检。
试验重复两次。 分别使用苏钟猪和托佩克猪两个品种进行。
结果：
采用实施例 1中描述方法对动物肺脏的猪支原体肺炎病变进行评分。 结果显 示免疫猪支原体肺炎减毒活疫苗 AN306株的动物肺脏病变明显减轻， 与攻毒对 照组相比统计学（统计方法 One-way ANOVA) 差异极显著（P<0.01 )， 其病变指 数均值明显低于目前市场上广泛使用的商品化活疫苗及灭活疫苗 （商品疫苗 1， P < 0.01 ; 商品疫苗 2， P < 0.05; 商品疫苗 3， P < 0.01 ; 商品疫苗 4， P < 0.01 )。 说明减毒活疫苗 AN306株能够帮助免疫动物有效抵抗猪肺炎支原体感染， 保护 力好于目前市场上广泛使用的疫苗。
表 4: 肺内注射免疫猪支原体肺炎减毒活疫苗 AN306株的免疫保护力评价及与 其他商品化活疫苗及灭活疫苗的比较
组号 G1 G2 G3 G4 G5 G6 G7 组别 健康对照 活疫苗组 商品疫苗 商品疫苗 商品疫苗 商品疫苗 攻毒对照 组 1 2 3 4 组 免疫途径 - 肺内注射 胸腔注射 肺内注射 肌肉注射 肌肉注射 - 肺脏病变评 0.50 2.60 5.00 3.80 10.60 11.90 21.90 分 （试验 1 ) 土 0.71 土 0.84 土 1.15 土 1.14 土 2.37 土 3.41 土 2.02 肺脏病变评 0.40 2.80 5.10 4.00 8.30 6.00 17.90 分 （试验 2) ±0.70 土 1.40 土 2.18 土 1.25 土 2.41 土 2.17 土 3.21 实施例 4: 气雾免疫猪支原体肺炎减毒活疫苗 AN306株的免疫保护力评价 疫苗稀释液配制：
含 5%-10%甘油的无菌去离子水溶液， pH值 6.8-7.5。
疫苗配制：
猪支原体肺炎减毒活疫苗 AN306株冻干苗， 用疫苗稀释液溶解冻干苗， 配 制疫苗溶液使得其中活疫苗滴度为 106CCU/mL。
动物免疫及攻毒：
5-7 日龄健康阴性托佩克猪 40头， 随机分成 4组： G1组为健康对照组， 不 免疫不攻毒； G2 组为活疫苗气雾免疫组， 气雾免疫 AN306 活疫苗 0.5 mL (5xl05CCU/头）， 于首次免疫后 2周再次免疫， 免疫后攻毒； G3组为商品化疫 苗组， 免疫目前市场上广泛使用的商品化猪支原体肺炎灭活疫苗 RespiSure, 按照 疫苗说明书操作， 免疫后攻毒； G4组为攻毒对照组， 不免疫只攻毒。 G2、 G3、 G4组动物于首次免疫后 8周气管内注射猪肺炎支原体强毒攻毒， 28天后剖检。 结果：
采用实施例 1中描述方法对动物肺脏中出现的猪支原体肺炎病变进行评分， 具体见下表。 结果显示气雾免疫猪支原体肺炎减毒活疫苗 AN306株的动物肺脏 病变明显减轻， 与攻毒对照组相比统计学差异极显著 （P<0.01 )， 并显著低于商 品化灭活疫苗 （P<0.01 )。 说明该减毒活疫苗通过气雾免疫能够帮助免疫动物抵 抗猪肺炎支原体感染， 保护力好于目前市场上广泛使用的灭活疫苗。
表 5: 气雾免疫猪支原体肺炎减毒活疫苗 AN306株的免疫保护力评价
组别 Gl G2 G3 G4
健康对照组 活疫苗组 商品化灭活 攻毒对照组
疫苗组
肺脏病变评分 1.08 ± 0.27 3.83 ± 2.48 8.25 ± 4.14 20.34 ± 3.45 实施例 5: 肌肉注射免疫猪支原体肺炎减毒活疫苗 AN306株的免疫保护力评价 疫苗制备： 猪支原体肺炎减毒活疫苗 AN306株冻干苗。 所使用佐剂为用无菌磷酸盐缓 冲液配制的含有 5 mg/mL卡波姆和 30 mg/mL黄芪多糖的混合物佐剂。用佐剂溶 液溶解冻干苗， 配制疫苗溶液使得其中活疫苗滴度为 2.5xl05CCU/mL。
动物免疫及攻毒：
5-7 日龄健康阴性苏钟猪 40头， 随机分成 4组： G1组为健康对照组， 不免 疫不攻毒； G2组为活疫苗肌肉注射免疫组， 肌肉注射免疫 AN306活疫苗 2 mL (5xl05CCU/头）， 于首次免疫后 2周再次免疫， 免疫后攻毒； G3组为商品化疫 苗组， 免疫目前市场上使用广泛的商品化猪支原体肺炎灭活疫苗 RespiSure, 按照 疫苗说明书操作， 免疫后攻毒； G4组为攻毒对照组， 不免疫只攻毒。 G2、 G3、 G4组动物于首次免疫后 8周气管内注射猪肺炎支原体强毒攻毒， 28天后剖检。 结果：
采用实施例 1中描述方法对动物肺脏中出现的猪支原体肺炎病变进行评分， 具体见下表。 结果显示肌肉注射免疫猪支原体肺炎减毒活疫苗 AN306株的动物 肺脏病变明显减轻， 与攻毒对照组相比统计学差异极显著 （P<0.01 )， 并显著低 于商品化灭活疫苗 （P<0.01 )。 说明该减毒活疫苗通过肌肉注射免疫能够帮助免 疫动物抵抗猪肺炎支原体感染， 保护力好于目前市场上广泛使用的灭活疫苗。 表 6: 肌肉注射免疫猪支原体肺炎减毒活疫苗 AN306株的免疫保护力评价 组别 Gl G2 G3 G4
健康对照组 活疫苗组 商品化灭活 攻毒对照组
疫苗组
月巿脏病变评分 0.98 ± 0.43 4.57 ± 2.32 7.88 ± 4.78 20.65 ± 7.51 实施例 6: 滴鼻免疫猪支原体肺炎减毒活疫苗 AN306株的免疫保护力评价 疫苗制备：
猪肺炎支原体 AN306株新鲜培养物， 滴度为 5xl06CCU/mL。 如下制备含壳 聚糖佐剂的疫苗：用 1%乙酸溶液溶解壳聚糖配制成含 1%壳聚糖的母液，用 0.1M NaOH溶液调节 pH值至 6.5; 用 0.1M NaAc-HAc(pH6.5)的溶液稀释上述壳聚糖 母液至壳聚糖终浓度为 0.2%。将猪肺炎支原体 AN306株新鲜培养物 12000rpm 30 min离心收集菌体， 用 1 mL 50mM Na2S04重悬， 使滴度为 6.66xl06CCU/mL。 取 5 mL 0.2%壳聚糖溶液，加入 1 mL含猪肺炎支原体的 Na2S04溶液，振荡混匀， 边振荡边缓慢滴加 660 三聚磷酸钠 （4 mg/mL) 溶液呈均一乳白色悬浊液， 即得。
动物免疫及攻毒：
5-7 日龄健康阴性托佩克猪 32头， 随机分成 4组： G1组为健康对照组， 不 免疫不攻毒； G2 组为活疫苗滴鼻免疫组， 滴鼻免疫 AN306 活疫苗 0.5 mL (5xl05CCU/头）， 于首次免疫后 2周再次免疫， 免疫后攻毒； G3组为商品化疫 苗组， 免疫目前市场上使用广泛的商品化猪支原体肺炎灭活疫苗 RespiSure, 按照 疫苗说明书操作， 免疫后攻毒； G4组为攻毒对照组， 不免疫只攻毒。 G2、 G3、 G4组动物于首次免疫后 8周气管内注射猪肺炎支原体强毒攻毒， 28天后剖检。 结果：
采用实施例 1中描述方法对动物肺脏中出现的猪支原体肺炎病变进行评分， 具体见下表。 结果显示免疫猪支原体肺炎减毒活疫苗 AN306株的动物肺脏病变 明显减轻， 与攻毒对照组相比统计学差异极显著 （P<0.01 )， 并显著低于商品化 灭活疫苗 （P<0.01 )。 说明该减毒活疫苗通过滴鼻免疫能够帮助免疫动物抵抗猪 肺炎支原体感染， 保护力好于目前市场上广泛使用的灭活疫苗。
表 7: 滴鼻免疫猪支原体肺炎减毒活疫苗 AN306株的免疫保护力评价
组别 Gl G2 G3 G4
健康对照组 活疫苗组 商品化灭活 攻毒对照组
疫苗组
肺脏病变评分 0.21 ± 0.18 3.03 ± 2.68 8.72 ± 4.19 22.68 ± 4.67
实施例 7: 猪支原体肺炎活疫苗 AN306株与副猪嗜血杆菌病灭活疫苗二联苗的 免疫保护作用评价
疫苗制备：
猪肺炎支原体 AN306株新鲜培养物， 滴度为 lxl06CCU/mL。
血清 5型副猪嗜血杆菌 XX0306株（该菌株为江苏省农业科学院兽医研究所 自行分离得到， 免疫原性好， 试验证实适合用于制备副猪嗜血杆菌病灭活疫苗， 保护效果良好）新鲜培养物， 活菌计数后用终浓度 0.2%甲醛溶液灭活 12h， 加入 焦亚硫酸钠 0.2%终止灭活。 将菌数浓縮成 lxl01QCFU/mL。
佐剂采用以无菌磷酸盐缓冲液配制的含有 5mg/mL卡波姆和免疫刺激复合物 基质 （成份为 Quil A 50(^g/mL、 胆固醇 10(Vg/mL、 磷脂 10(Vg/mL) 的混合物 佐剂。
将 0.5 mL猪肺炎支原体新鲜培养物与 0.5 mL血清 5型副猪嗜血杆菌灭活抗 原及佐剂按照体积比 1:1:2混合均匀， 免疫动物。
动物免疫及攻毒：
10日龄左右的健康阴性苏钟猪 30头， 随机分成 4组： G1组为健康对照组， 10头， 不免疫不攻毒， 其中 5头与 G2、 G3组动物同时剖检， 另外 5头与 G4、 G5组动物同时剖检； G2组为联苗免疫猪肺炎支原体攻毒组， 5头， 肌肉注射免 疫上述制备的联苗 2 mL(AN306活疫苗 2.5xl05CCU/mL,血清 5型副猪嗜血杆菌 XX0306株灭活抗原 2.5xl09CFU/mL)， 于首次免疫后 3周再次免疫， 首次免疫 后 8周气管内注射猪肺炎支原体强毒攻毒； G3组为猪肺炎支原体攻毒对照组， 5 头， 不免疫， 与 G2组动物同样攻毒； G4组为联苗免疫副猪嗜血杆菌攻毒组， 5 头， 肌肉注射免疫上述制备的联苗 2mL(AN306活疫苗 2.5xl05CCU/mL,血清 5 型副猪嗜血杆菌 XX0306株灭活抗原 2.5xl09CFU/mL)， 于首次免疫后 3周再次 免疫， 于首次免疫后 6周腹腔注射副猪嗜血杆菌强毒攻毒； G5组为副猪嗜血杆 菌攻毒对照组， 5头， 不免疫， 与 G4组动物同样攻毒。
结果：
( 1 ) 对猪肺炎支原体感染的保护作用
攻毒后 28天剖检， 采用实施例 1中描述方法对肺部猪支原体肺炎病变进行 评分， 记录于下表， 结果可见联苗免疫后可显著减轻肺脏病变的发生程度 (Ρ<0·01 )。
(2) 对副猪嗜血杆菌感染的保护作用
副猪嗜血杆菌病临床症状表现为食欲降低，被毛粗乱，耳朵及全身皮肤发红， 鼻液增多， 后期出现呼吸困难， 关节肿胀， 卧地不起。剖检可见胸腔、腹腔积液， 有不同程度的胸膜、 腹膜粘连， 关节肿胀、 关节腔粘液增多， 肺脏出血， *** 肿胀等病变。 由上述指标判断各组动物副猪嗜血杆菌病的发病情况， 总结记录于 下表。 结果表明联苗免疫后可显著减少动物副猪嗜血杆菌病的发病率。 表 8: 猪支原体肺炎活疫苗 AN306株与副猪嗜血杆菌病灭活疫苗二联苗的免疫 保护作用评价
组别 G1 G2 G3 G4 G5
健康对照组 联苗免疫猪 猪肺炎支原体 联苗免疫 副猪嗜血杆 肺炎支原体 攻毒对照组 副猪嗜血 菌攻毒对照 攻毒组 杆菌攻毒 组
组
免疫 不免疫 联苗免疫 不免疫 联苗免疫 不免疫 猪肺炎支原体 不攻毒 攻毒 攻毒 不攻毒 不攻毒 攻毒
副猪嗜血杆菌 不攻毒 不攻毒 不攻毒 攻毒 攻毒 攻毒
猪支原体肺炎 0.54土 0.28 5.37土 4.36 19.81 ± 6.57 未评价 未评价 病变评分
副猪嗜血杆菌 0/5 未评价 未评价 1/5 5/5 病发病情况评
价
实施例 8: 猪支原体肺炎活疫苗 AN306株与猪繁殖与呼吸综合征活疫苗二联苗 的免疫保护作用评价
疫苗制备：
用无菌磷酸盐缓冲液配制含有 10 mg/mL壳聚糖和 10 mg/mL左旋咪唑的混 合物佐剂。 取猪支原体肺炎活疫苗 AN306株冻干苗， 用 1.5 mL佐剂溶解猪肺炎 支原体 AN306株冻干苗， 得到 1头份（5xl05CCU) /1.5mL的猪支原体肺炎活疫 苗溶液。
商品化猪繁殖与呼吸综合征活疫苗 （ATCC VR-2332株）。 用生理盐水溶解 活疫苗， 得到 2头份 /mL的猪繁殖与呼吸综合征活疫苗溶液。
将两种疫苗溶液按 3:1(体积比)混合， 免疫动物， 每头动物接种 2 mL， 即 1 头份猪支原体肺炎活疫苗和 1头份猪繁殖与呼吸综合征活疫苗。 动物免疫及攻毒：
10日龄左右的健康阴性苏钟猪 30头， 随机分成 4组： G1组为健康对照组， 10头， 不免疫不攻毒， 其中 5头与 G2、 G3组动物同时剖检， 另外 5头与 G4、 G5组动物同时剖检； G2组为联苗免疫猪肺炎支原体攻毒组， 5头， 肌肉注射免 疫上述制备的联苗 2mL， 于首次免疫后 2周再次免疫， 首次免疫后 8周气管内 注射猪肺炎支原体强毒攻毒； G3组为猪肺炎支原体攻毒对照组， 5头， 不免疫， 与 G2组动物同样攻毒； G4组为联苗免疫猪繁殖与呼吸综合征病毒攻毒组， 5头， 肌肉注射免疫上述制备的联苗 2mL， 于首次免疫后 2周再次免疫， 于首次免疫 后 6周颈部肌肉注射猪繁殖与呼吸综合征病毒强毒攻毒； G5组为猪繁殖与呼吸 综合征病毒攻毒对照组， 5头， 不免疫， 与 G4组动物同样攻毒。
结果：
( 1 ) 对猪肺炎支原体感染的保护作用
攻毒后 28天剖检， 采用实施例 1中描述方法对肺部猪支原体肺炎病变进行 评分， 记录于下表， 结果可见联苗免疫后可显著减轻肺脏病变的发生程度 (Ρ<0·01 )。
(2) 对猪繁殖与呼吸综合征病毒感染的保护作用
攻毒后每日对动物进行观察并测温， 发病猪的判定标准为： 持续 3天以上出 现 41 °C以上高热， 精神、 食欲下降， 出现眼结膜炎及咳嗽、 喘气等呼吸道症状， 死亡， 剖检肺出现片状实变。结果发现猪繁殖与呼吸综合征病毒攻毒对照组动物 全部发病， 其中 2头死亡， 联苗免疫组动物无明显发病症状， 全部存活。
表 9: 猪支原体肺炎活疫苗 AN306株与猪繁殖与呼吸综合征活疫苗二联苗的免 疫保护作用评价
组别 G1 G2 G3 G4 G5
健康对照组 联苗免疫猪 猪肺炎支原体 联苗免疫 猪繁殖与呼 肺炎支原体 攻毒对照组 猪繁殖与 吸综合征病 攻毒组 呼吸综合 毒攻毒对照 征病毒攻 组 毒组
免疫 不免疫 联苗免疫 不免疫 联苗免疫 不免疫 猪肺炎支原体 不攻毒 攻毒 不攻毒 不攻毒 猪繁殖与呼吸 不攻毒 不攻毒 不攻毒
综合征病毒攻 猪支原体肺炎 0.22 + 0.15 5.05 + 3.89 23.24 + 3.72 未评价 未评价 病变评分
猪繁殖与呼吸 0/5 未评价 未评价 0/5 5/5 综合征发病情
况

Claims

权 利 要 求 书
1. 一种猪肺炎支原体 (Mycoplasma hyopneumoniae, Mhp)弱毒株 AN306, 其保 藏于中国典型培养物保藏中心， 保藏号为 CCTCC M 2012431。
2. 权利要求 1 所述猪肺炎支原体 (Mycoplasma hyopneumoniae, Mhp)弱毒株 AN306在防治猪肺炎支原体感染疾病中的应用。
3. 权利要求 1 所述猪肺炎支原体 (Mycoplasma hyopneumoniae, Mhp)弱毒株 AN306在制备猪支原体肺炎疫苗或多联疫苗中的应用。
4. 一种猪支原体肺炎减毒活疫苗， 其特征在于所述疫苗包括权利要求 1所述猪 肺炎支原体 (Mycoplasma hyopneumoniae, Mhp)弱毒株 AN306。
5. 如权利要求 4所述猪支原体肺炎减毒活疫苗， 其特征在于所述疫苗含有药学 上可接受的载体或辅料。
6. 如权利要求 4或 5所述猪支原体肺炎减毒活疫苗， 其特征在于所述疫苗含有 至少一种免疫佐剂。
7. 如权利要求 4或 5所述猪支原体肺炎减毒活疫苗， 其特征在于所述疫苗含有 至少一种其他病原的免疫原。
8. 如权利要求 7所述猪支原体肺炎减毒活疫苗， 其特征在于所述其他病原包括 病毒、 细菌、 真菌、 寄生虫中的至少一种。
9. 如权利要求 8所述猪支原体肺炎减毒活疫苗， 其特征在于所述其他病原为猪 圆环病毒、 副猪嗜血杆菌、 猪繁殖与呼吸综合征病毒、 猪鼻支原体、 猪滑 液支原体、 猪流感病毒。
10. 如权利要求 4-9 中的任一权利要求所述猪支原体肺炎减毒活疫苗， 其特 征在于所述疫苗采用的制剂为肺内注射剂、 肌肉注射剂、 气雾剂、 口服剂或 滴鼻剂。
11. 一种制备猪支原体肺炎减毒活疫苗的方法， 其特征在于， 将权利要求 1 所述的猪肺炎支原体 (Mycoplasma hyopneumoniae， Mhp)弱毒株 AN306与药学 上可接受的载体或辅料， 以及至少一种免疫佐剂， 至少一种其他病原的免疫 原组合在一起。
12. 权利要求 4-9 中的任一权利要求所述疫苗在防治猪肺炎支原体感染疾 病的中的应用。
13. 如权利要求 12所述的应用， 所述猪肺炎支原体感染相关疾病包括猪肺 炎支原体单独感染及与其他病原混合感染所导致的猪支原体肺炎疾病。
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CN108251338B (zh) * 2018-03-12 2021-01-29 江苏省农业科学院 猪鼻支原体强毒株及其应用
CN112875277A (zh) * 2021-01-12 2021-06-01 高凤琴 一种注射科用西林瓶分离回收设备

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