Isolation And Characterization Of Mycoplasma Hyopneumoniae And Development Of Its Genetic Engineering Vaccine

Mycoplasma hopneumoniae (MHP) is an etiologic agent of enzootic pneumonia (EP). MHP is also a primary contributory agent in porcine respiratory disease complex. Although the primary disease is an important, more significant is the fact that this bacterial predisposes to colonization and disease with other viral or bacterial pathogens, resulting in higher morbidity and more mortality. This study reported here focused on the isolated pathogen, pathogen diagnostic methods, new recombinant Salmonella vaccine and new recombinant Actinobacillus pleuropneumoniae vaccine, which provided very important materials for basic research and contributed for preventing and control of MHP in China. What’s more, it is provided the basis for further studies on the pathogenic mechanisms of MHP. The results are as follows.1. Isolation and identification of MHP from pigs and the MHP genes sequence analysis.One MHP strain, HNYY, was isolated from lesion in lungs of pigs with MHP positive. On the basis of typical clone shape, electron microscopy, sequence analysis of genes test, one of MHP strain was isolated and purified. During the course of cultivation, the conglobate colonies like "fried egg" on solid medium could be observed under a microscope. By negative staining and electron microscope the morphological characters of the bacteria were observed to be circular. To evaluate sequence variations in MHP isolate from China, the complete P36 and P46 genes and a part of 16sRNA gene were sequenced. The alignment results revealed that the P36, P46 and 16sRNA were highly identical as much as 100%,93% and 96%, respectively, with other MHP. To establish the relationship between MHP isolate and other MHP strains, the obtained P36 and P46 DNA sequences were aligned with the previously published sequences of MHP. The phylogenetic trees were constructed using the neighbor-joining, minimum evolution and maximum parsimony programs available in MEGA 4.0. The phylogeny showed that the MHP isolate was most closely related to MHP 232 strain.2. Studies on pathogenesis of MHP isolate.Twelve piglets, (21-day old) from a farm in China, found to be serologically negative for MHP and PRRSV were divided into two groups; the first one is the challenge group (seven piglets) and the other group represented the negative control group (five piglets). Groups were individually housed in separate rooms. Five piglets of challenge group were challenged with 5 ml MHP HNYY culture containing 107 color change units (CCU) via trachea intubation for two days (one dose/day), the other two piglets employed as sentinel piglets. For 28 days following challenge, pigs were under continuous observation to evaluate body condition, appetite, presence of dyspnea, coughing or behavioural changes. The average rectal temperatures, physically and mentally, clinical symptoms were not significantly different within inoculation group members. The lung lesion of piglets inoculated with MHP isolate did not develop significant pathological changes compared to the negative control group. The average weighs challenge group and negative group at the beginning of the study varied between 8.14±1.18 and 9.35±1.03 kg, respectively, at 0 DPI and between 18.00±2.51 and 22.07±1.36 kg, respectively, at 28 DPI. The ADG per group varied between 0.35±0.63 and 0.45±0.54 kg for the challenge group and negative group, respectively. Not all animals had sero-converted in MHP inoculation group at 28 DPI. Macroscopic lesions associated to MHP isolate infection were found to be bilateral in the apical, cardiac and intermediate lobes. Histopathological study showed interstitial pneumonia, perivascular and peribronchial infiltration in the lungs, whereas, trachea showed exfoliated bronchial cells cilia.3. Construction of Real-time PCR for the detection of MHPDevelopment of real-time fluorescent quantitative PCR assay-based on TaqMan probe was designed for the detection of MHP using the lactate dehydrogenase (P36) element as a target. The standardized intra-assay pMD18-P36 DNA at initial concentrations of 1.0×106,1.0×107 and 1.0×108 copies/μl showed 0.6%,0.5% and 0.5% coefficients of variation of Ct values, respectively. While in inter-assay pMD18-P36 DNA the same previous initial concentrations revealed 7.6%,7.9% and 3.4% as coefficients of variation of Ct values, respectively. The sensitivity of Real-time PCR was 100 times as conventional PCR. Real-time PCR method for the MHP detection has been established to be specific, sensitive as well as reproducible.4. Expression and reactivity analysis of MHP antigens in E. coliIt was based on the recent related researches on antigenic and immunogenic analysis of MHP. We chose and analyzed the antigenicity of the P46, P65, P36 and P97R1NrdF genes of MHP. In this research, three TGA codons in the cloned P46 gene were found, at position 208,301 and 760. One TGA codon in the cloned P65 gene was found, at position 631. A repeat region of P97R1 has been shown to include both cilium- and antibody-binding sites and is reported to function independently from other P97 regions. The R2 subunit of prokaryotic class I ribonucleotide reductases (NrdF) was an 11-KDa protein. P46, P65, P36 and P97RlNrdF genes were cloned into prokaryotic expression pGEX-KG vector, resulting in the recombinant plasmids pGEX-KGP46, pGEX-KGP65, pGEX-KGP36 and pGEX-KGP97R1N, respectively. Then pGEX-KGP46, pGEX-KGP65, pGEX-KGP36 or pGEX-KGP97R1N was transformed into E. coli JM105. Expression and purification were assayed as well. The molecular weight of P46, P65, P36 and P97RlNrdF protein were about 72 KDa,91 KDa,62 KDa and 50 KDa, respectively, all of them including the 26 KDa GST tag from the vector.5. Construction of Samonella choleraesuis C501 expressing the recombinant immunodominant genes of MHP.The P46, P65, P36 and P97RlNrdF DNA fragments specifying the important immunodominant proteins of MHP were cloned into the pYA3493 vector, resulting in the recombinant plasmids pYAP46, pYAP65, pYAP36 and pYAP97R1N, respectively. Then pYAP46, pYAP65, pYAP36 and pYAP97R1N were electrotransformed to C501 strain, resulting in the recombinant Samonella strain C501(pYAP46), C501(pYAP65), C501(pYAP36) and C501(pYAP97R1NrdF), respectively. In this study, the recombinant strains maintained the serotype and energy source utilization characteristics of parental C501 strain, and the recombinant heterologous antigens were highly expressed.6. Vaccination efficacy of the recombinant Salmonella vaccine strains in BALB/c mice.To evaluate the vaccination efficacy of the recombinant Samonella strains C501(pYAP46), C501(pYAP65), C501(pYAP36) and C501(pYAP97R1NrdF), two sets of mice were vaccinated with 4×108 CFU of C501(pYAP46), C501(pYAP65), C501(pYAP36) or C501(pYAP97R1NrdF) via oral route or injected intramuscularly (IM) on days 0 and 14. The results showed that only P46-specific IgG antibodies were detected in serum and lung samples. However, in lung sample only IM immunized group P46-specific IgG response was induced. P46-specific IgA antibodies were not detected in serum and lung in all immunized groups. Similarly, the results illustrated that only P65-specific IgG antibodies were detected in serum and lung samples, P65-specific IgA antibodies were not detected in serum and lung in all immunized groups. Nevertheless, the P65-specific IgG antibodies titers in serum were higher than P46-specific IgG, and significantly higher than 168 vaccine groups (P<0.05). The P65-specific IgG antibodies titers in lung were lower than P46-specific IgG. The C501(pYAP36) and C501(pYAP97R1NrdF) were induced immune response better than C501(pYAP46) and C501(pYAP65). The present study findings demonstrated that P36- and P97RlN-specific serum IgG and IgA antibodies were detected in serum and lung samples. Animals vaccinated by IM had higher levels of P36-specific IgA and lower levels of P36-specific IgG than those vaccinated by oral route. Mice immunized with the 168 vaccine by IM elicited significantly higher levels of P36-specific IgA in lungs (P<0.05). Mice immunized with the C501(pYAP36) vaccine by orally elicited significantly higher levels of P36-specific IgG in lungs (P<0.05). P97R1-specific serum IgG and IgA antibodies were also detected in serum, but in lung sample only P97R1-specific IgG response was induced. On the other hand, animals vaccinated with C501(pYAP97RlNrdF) by IM had higher levels of P97R1N-specific IgG and IgA than those vaccinated by oral route. Furthermore, mice immunized with the C501 vaccine elicited significantly higher levels of IgA and IgG in lungs and serum (P<0.05).ELISA assays demonstrated that IFN-γand IL-4 production was induced in culture supernatants from splenocytes stimulated with specific antigens in vitro. MHP whole cell-specific antibody was induced in vaccinated mice and a good immunogenic effect by this vaccine was indicated. Taken together, these results suggested that IM vaccination with the C501 vaccine might provide an effective method of inducing an immune response to MHP.7. Vaccination and challenge efficacy of the recombinant Salmonella vaccine strains in piglets.To test the immune efficacy of the recombinant vaccine strain for piglets. Eighteen piglets (21-day old) from a farm serologically negative for MHP and PRRSV were divided into three vaccination groups of five piglets and one negative control group of three piglets. Groups were individually housed in separate rooms. The three groups were vaccinated twice via IM with 3×109CFU of C501(pYAP46/P65/P36/P97R1NrdF) C501(pYAP97R1NrdF) and 1 ml M+PACRat 2-week intervals, respectively. The antibody of each antigen was detected by ELISA. The results showed that P46-, P65-, P36- and P97R1NrdF-specific IgM antibodies were detected in serum samples. Moreover, piglets vaccinated with C501(pYAP97R1NrdF) had higher levels of specific IgM than other groups. Specific IgG antibodies were detected in serum, but no specific IgA antibodies, except for P97R1 and P97R1N. Piglets vaccinated with C501(pYAP97R1NrdF) had significantly higher specific IgG immune response than other groups(P<0.05). M.hyo whole cell-specific antibody was induced in vaccinated piglets, but the M+PACR vaccine group was induced higher M.hyo-specific antibodies than other groups. The recombinant vaccine induced local and systemic immune responses confirmed by IgGl and IgG2a EILSA.Piglets were challenged with MHP velogen strain, three weeks after the second vaccination dose, via trachea intubation. The protective efficacy of immunization was evaluated by inflammatory cytokines detection, lung lesion score and pathological section analysis. The results demonstrated that vaccination groups inflammatory cytokines level of IL-6 and TNF-a ELISA were significantly higher than control challenged group (P<0.05). However, the recombinant C501(pYAP97R1NrdF) vaccination induced TNF-αsignificantly lower than M+PACR vaccine groups did (P<0.05); lower than the recombinant C501(pYAP46/P65/P36/P97R1NrdF) group. Pathological section results showed that pathological changes were different not only between the groups but in the same group. But the lung lesion scores of piglets of control challenged group were significantly higher compared to the C501(pYAP97R1NrdF) (P<0.01) and other vaccine groups (P<0.05). Given that, the recombinant C501(pYAP97R1NrdF) vaccine IM can provide piglets with reduced lung lesions and protection against infections with MHP velogen strain. Consequently, recombinant C501(pYAP97R1NrdF) showed the potential as a new recombinant Salmonella vaccine against MHP.8. Vaccination efficacy of the recombinant Atinobacillus pleuropneumoniae vaccine strain in BALB/c miceIn this study, a mutant strain of A. pleuropneumoniae, SLW36, was constructed by replacing the urease structural gene of parental mutant strain SLW03 of A. pleuropneumoniae with the L-lactate dehydrogenase gene (p36) of M. hyopneumoniae by transconjugation and counter selection. The urease function and the growth kinetics of SLW36 were measured. Protein expression of P36 was analyzed by SDS-PAGE and Western blotting. The attenuated virulence and immunity of SLW36 were analyzed in a mouse model. The mutant strain SLW36 was urease negative and 4-fold less virulent than the parental strain SLW03. There were no differences in expression levels of p36 at different culture time-points and the foreign gene was stable after in-vitro passage. IgG response against p36 antigen and M. hyopneumoniae whole-cell antigens were detected. The mutant strain SLW36 can induce antibody against p36 and M. hyopneumoniae. Therefore, the mutant strain SLW36 has the potential to be used as a live vaccine for protection against A. pleuropneumoniae and M. hyopneumoniae. Studies on pigs are needed to confirm protective levels of antibodies and to check for rare side effects of the vaccine.