Research Progress of the Attenuated Vaccine and the Integrated Prevention and Control Techniques of Mycoplasma hyopneumoniae
Mycoplasma hyopneumoniae is the major pathogen of mycoplasmal pneumonia of swine (MPS). The infection ratio of MPS is estimated to exceed 30-50%, which brought about a considerable economic loss of more than ten billion yuan annually. The lost of the more complex diseases, such as the porcine respiratory disease complex (PRDC) caused by the swine mycoplasma pneumonia and mycoplasma hyorhinis has been increasing. With the rapid development of the industrial and intensive pig production mode, the economic loss caused by PRDC remains in a steady increase each year, and it has become a bottle-neck restriction to swine industry. Swine mycoplasma pneumonia is the most important primary disease of PRDC, meanwhile, PRDC induces some other immunosuppressive diseases, particularly PRRS, which is still lack of effective measures currently. In the United States, PIC swine farms have got a good control by the immune swine mycoplasma pneumonia vaccine. It has become the consensus to control swine mycoplasma pneumonia so as to ensure the control of swine respiratory disease both at home and abroad.
Animal Diseases Control Laboratory, the Institute of Veterinary Medicine of JAAS, is one of the first institutions to carry out the research of swine mycoplasma pneumonia and bacterial respiratory disease in China. It has systematically studied on the pathogen, pathogenesis, diagnostics, purification as well as the creation of vaccines. The novel vaccine of swine mycoplasma pneumonia (168) has a great impact on domestic and foreign veterinary industry.
Team Members of Animal Diseases Control Laboratory
Currently, the "swine mycoplasma pneumonia vaccine and its comprehensive prevention and control technology research and promotion” has been progressed as following:
The study of Mycoplasma hyopneumoniae in JAAS started as early as 1970s. Mycoplasma hyopneumoniae strain 168 was isolated from an Er-hua-lian pig with typical MPS. The field strain was attenuated by the continual passage in vitro. After 300 passages, a stable attenuated strain named 168 was obtained, which was authorized as national patent for invention (ZL 99114276.4). The attenuated vaccine of Mycoplasma hyopneumoniae based on strain 168 was approved as the National New Veterinary Medical Certificate (Second Category) in 2007. "A Live Attenuated Vaccine Swine Mycoplasma Pneumonia and Its Application PCT/CN2014/071506" was applied for international PCT in 2013. At present, 12 strains of Mycoplasma hyopneumoniae and 10 strains of Mycoplasma hyorhinis are preserved in our lab. The whole genome sequences of the attenuated strain of Mycoplasma hyopneumoniae have been completed. The genetic differences between Maternal virulent strain and the attenuated strains have been found and published in BMC Genomics 2013 and Journal of Bacteriology 2011.
On the basis of the current commercial vaccine, great efforts have been taken to improve its route of delivery. Different approaches including aerosol immunization and intramuscular immunization have been tested.
Aerosol immunization: The first attenuated Mycoplasma hyopneumoniae aerosol vaccine was prepared based on strain 168 and showed a significant protection against infection. Recently, the new formulation of dry powder vaccine has been tested successfully. The National patent for the utility models on the equipment for aerosol vaccine (ZL 2012 20357811.3) has been authorized. The other patent for invention has been applied on the preparation and detection method for aerosol vaccine (201210275639.1). One scientific paper has been published on Veterinary Microbiology in 2013.
Intramuscular immunization: A new adjuvant has been successfully developed for intramuscular administration of the attenuated vaccine strain 168. Three patents for invention have been applied (2 of them have been authorized) on adjuvant formulations and the vaccine (ZL201010288021.X, ZL201010288025.8, 201310017232.3). One scientific paper has been published on the Veterinary Journal in 2014.
We have gained 6 grants on Mycoplasma hyopneumoniae study supported by the National Natural Science Foundation of China (charged 5, participated 1). Among the 18 excellent scientific articles listed in the research report of “IRPCM Porcine Team Report 2012” by the International Organisation for Mycoplasmology, 4 of them have been published by our team, including the article with the highest impact factor. We have found in our study that the lipid-associated membrane protein (LAMP) derived from Mycoplasma hyopneumoniae has the effect of apoptosis on the host immune cells. (Vet Immunol Immunopathol, 2013). The inhibition of GR nuclear translocation and binding to the TLR2 promoter, which results in diminished TLR2 expression, is associated with the protective effect of vaccination on Mycoplasma hyopneumoniae-induced lung lesions in the pigs (Vet Microbiology, 2013).
In the meantime, inactivated vaccine against Mycoplasma hyopneumoniae(NJ strain) is also under the development. The clinical test has already been completed, and the vaccine is to be applied for drug registration at present. Two patents for invention have been applied (201310608564.9 and 201310605986.0).
2. R&D of Novel Vaccine
The attenuated vaccine of Mycoplasma hyopneumoniae (strain 168) was approved as the National New Veterinary Medical Certificate (Second Category) in 2007. More than 70,000,000 doses of attenuated vaccines have been applied in pig farms, which showed a significant protection to MPS. Based on this project, the team gained the first prize for China Agricultural Science and Technology Award (2010-2011), the first prize for the 5th Golden Bridge Awards, the second prize for Scientific and Technological Progress of Jiangsu Province and the support by Agricultural Science and Technology Achievement Transformation Fund of China. The patent of the attenuated vaccine gained the prize for Excellent Patent Award of Jiangsu and the prize for Excellent Patent Award of Nanjing.
Up to now, the development of new administration route of the attenuated vaccine has been primarily completed, including the aerosol and intramuscular immunization method. 4 patents for invention have been applied (2 have been authorized), and 3 patents for utility models have been applied (3 have been authorized).
The preclinical and clinical study of the inactivated vaccine against Mycoplasma hyopneumoniae (stain NJ) has been finished. The product has been transferred to 4 biological products enterprises, including Nanjing Tech-bank Bio-Industry Co., Ltd, Chengdu Tecbond Biological Products Co., Ltd, PULIKE BIOLOGICAL ENGINEERING, INC. and Shanghai Hile Bio-pharmaceutical Co., Ltd. The product is currently under the application for veterinary medicine registration.
Stable diagnostic methods have been established on the antigen and antibody detecting for Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. 7 patents for invention have been applied (4 have been authorized). Two scientific articles have been published on SCI journals. 4 provincial industrial standards have been authorized and 1 national standard has been applied.
Nested PCR diagnostic kit for Mycoplasma hyopneumoniae, anti-Mycoplasma hyopneumoniae IgG ELISA kit and anti-Mycoplasma hyopneumoniae SIgA ELISA kit have been developed. One scientific article has been published on Clinical Laboratory Publications GmbH in 2013. Two patents for invention on LAMP diagnostic kit for Mycoplasma hyopneumoniae and Mycoplasma hyorhinis have been authorized (ZL 201210170783.9 and ZL 201110033271.3).
Based on the study of successful international eradication methods, the appropriate eradication ways for China swine farming industry have been developed and successfully applied in the pig farms. The assessment system for eradication efficacy has also been designed. One national standard has been applied on the PCR detection method of Mycoplasma hyopneumoniae (20130479-T-326). Three patents for utility models have been authorized (ZL 2012 20435911.3, ZL 201220357811.3 and ZL 201320497672.9)
Papers: More than 100 articles about the study of MPS and some other respiratory diseases of swine have been published. Among these, 11 papers were published on SCI journals and the other 11 papers were published in the international conference symposium.
Books: Pig Production and Health Management Operations, 3rd edition;
Mycoplasmal pneumonia of swine, 1st edition (500,000 words ) is to be finished within 2014.
Websites: www.piginfo.com, over 32759 page view.
Conferences: Over 100 technical sessions have been co-organized with biological products enterprises in the prevention and control techniques of MPS and some other respiratory diseases of swine during 2010-2013. More than 20,000 managers and technicians of pig farms have been trained. The 4th national MPS congress 2014 will be held in Nanjing. The experts of MPS from all over the world will be invited to attend the meeting.
In conclusion, via the medians of paper, book, website and conference, we have made great efforts to establish a good cooperation and promotion system for the integrated prevention and control techniques against MPS and other respiratory diseases of swine with large biological products enterprises.
Certificate of China National Patent for Invention
Attenuated Mycoplasma hyopneumoniae vaccine (strain 168)
New Veterinary Medical Registration Certificate for Attenuated Mycoplasma hyopneumoniae vaccine (strain 168), 2007
First prize for China Agricultural Science and Technology Award (2010-2011), “The Creation of Mycoplasma hyopneumoniae Vaccine and Its Integrated Application for the Prevention and Control Techniques” awarded by Ministry of Agriculture
- Feng ZX, Bai Y, Yao JT, Pharr GT, Wan XF, Xiao SB, Chi LZ, Gan Y, Wang HY, Wei YN, Liu MJ, Xiong QY, Bai FF, Li B, Wu XS, Shao GQ. Use of serological and mucosal immune responses to Mycoplasma hyopneumoniae antigens P97R1, P46 and P36 in the diagnosis of infection. Vet J. 2014 Jul 1. pii: S1090-0233(14)00276-7. doi: 10.1016/j.tvjl.2014.06.019.
- Feng Z, Baroch JA, Long LP, Xu Y, Cunningham FL, Pedersen K, Lutman MW, Schmit BS, Bowman AS, Deliberto TJ, Wan XF. Influenza a subtype h3 viruses in feral Swine, United States, 2011-2012. Emerging Infectious Diseases. 2014. 20(5):843-6. [PDF]
- Hua LZ, Wu YZ, Bai FF, William KK, Feng ZX, Liu MJ, Yao JT, Zhang X, Shao GQ. Comparative analysis of mucosal immunity to Mycoplasma hyopneumoniae in Jiangquhai porcine lean strain and DLY piglets. Genet Mol Res. 2014.13(3):5199-206.
- Xiong Q, Wei Y, Xie H, Feng Z, Gan Y, Wang C, Liu M, Bai F, Xie F, Shao G. Effect of different adjuvant formulations on the immunogenicity and protective effect of a liveMycoplasma hyopneumoniae vaccine after intramuscular inoculation. Vaccine. 2014.32(27):3445-51.
- Feng Z, Gomez J, Bowman AS, Ye J, Long LP, Nelson SW, Yang J, Martin B, Jia K, Nolting JM, Cunningham F, Cardona C, Zhang J, Yoon KJ, Slemons RD, Wan XF. Antigenic characterization of H3N2 influenza A viruses from Ohio agricultural fairs. J Virol. 2013. 87(13):7655-67. [PDF]
- Du GM, Liu MJ, Wu YZ, Xiong QY, Bai FF, Feng ZX, Shao GQ. Development of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasmahyorhinis. Clin Lab. 2013;59(11-12):1363-71.
- Xiong Q, Wei Y, Feng Z, Gan Y, Liu Z, Liu M, Bai F, Shao G. Protective efficacy of a live attenuated Mycoplasma hyopneumoniae vaccine with an ISCOM-matrix adjuvant in pigs. Vet J. 2014.99(2):268-74.
- Sun Z, Liu M, Zou H, Li X, Shao G, Zhao R. Vaccination inhibits TLR2 transcription via suppression of GR nuclear translocation and binding to TLR2 promoter in porcine lung infected with Mycoplasma hyopneumoniae. Vet Microbiol. 2013. 67(3-4):425-33.
- Feng ZX, Wei YN, Li GL, Lu XM, Wan XF, Pharr GT, Wang ZW, Kong M, Gan Y, Bai FF, Liu MJ, Xiong QY, Wu XS, Shao GQ. Development and validation of an attenuated Mycoplasma hyopneumoniae aerosol vaccine. Vet Microbiol. 2013. 167(3-4):417-24.
- Bai F, Ni B, Liu M, Feng Z, Xiong Q, Xiao S, Shao G. Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce apoptosis in porcine alveolar macrophage via increasing nitric oxide production, oxidative stress, and caspase-3 activation. Vet Immunol Immunopathol. 2013.155(3):155-61.
- Liu W, Xiao S, Li M, Guo S, Li S, Luo R, Feng Z, Li B, Zhou Z, Shao G, Chen H, Fang L. Comparative genomic analyses of Mycoplasma hyopneumoniae pathogenic 168 strain and its high-passaged attenuated strain. BMC Genomics. 2013.14:80.
- Liu W, Fang L, Li M, Li S, Guo S, Luo R, Feng Z, Li B, Zhou Z, Shao G, Chen H, Xiao S. Comparative genomics of Mycoplasma: analysis of conserved essential genes and diversity of the pan-genome. PLoS One. 2012;7(4):e35698.
- Liu W, Feng Z, Fang L, Zhou Z, Li Q, Li S, Luo R, Wang L, Chen H, Shao G, Xiao S. Complete genome sequence of Mycoplasma hyopneumoniae strain 168. J Bacteriol. 2011.193(4):1016-7.
- Liu W, Fang L, Li S, Li Q, Zhou Z, Feng Z, Luo R, Shao G, Wang L, Chen H, Xiao S. Complete genome sequence of Mycoplasma hyorhinis strain HUB-1. J Bacteriol. 2010 .192(21):5844-5.
- Feng ZX, Shao GQ, Liu MJ, Wang HY, Gan Y, Wu XS. Development and validation of a SIgA-ELISA for the detection of Mycoplasma hyopneumoniae infection. Vet Microbiol. 2010 .143(2-4):410-6.