Porcine antibody profiles of 33 Mycoplasma hyopneumoniae fusion proteins from M. hyopneumoniae natural infection but not vaccination.

BACKGROUND: Mycoplasma hyopneumoniae, the primary pathogen responsible for porcine enzootic pneumonia, reduces average daily weight gain and causes substantial economic losses to the pig industry worldwide. Vaccination is the most common strategy to control this disease but offers partial protection. Therefore, developing next-generation vaccines by screening protective antigens is crucial. OBJECTIVES: The aim of this study was to evaluate the antibody response to 33 recombinant proteins in pigs naturally infected with M. hyopneumoniae. METHODS: The genes encoding 33 (hypothetical) membrane proteins or secretory proteins were ligated into pGEX-6P-1, pGEX-6P-2, pGEX-5X-3 or pGEX-4T-3 vectors and transformed into Escherichia coli BL21(DE3) or E. coli XL-1 Blue to construct recombinant bacteria and to express the recombinant proteins. The recombinant bacteria expressing the target proteins reacted with porcine convalescent sera and negative sera to screen immunodominant proteins by ELISA. Then, recombinant bacteria expressing immunodominant proteins were used to identify the discriminating immunodominant proteins that were recognised by convalescent sera nut not hyperimmune sera. RESULTS: All recombinant bacteria could express the target recombinant proteins in soluble form. Twenty-one proteins were shown to present immunodominant antigens, and four proteins were not recognised by convalescent sera. Moreover, six proteins were considered discriminating and reacted with convalescent sera but not with hyperimmune sera. CONCLUSIONS: The identified immunodominant proteins were antigenic and expressed during bacterial infection, suggesting that these proteins, especially those capable of discriminating between sera, can be used to identify protective antigens with the view to develop more effective vaccines against M. hyopneumoniae infection.

Elevated Mhp462 antibody induced by natural infection but not in vitro culture of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the respiratory pathogen of porcine enzootic pneumonia, a chronic respiratory infectious disease that causes substantial pecuniary losses to pig husbandry worldwide. Commercial bacterins only provide incomplete protection and do not prevent the colonization and transmission of M. hyopneumoniae. Identification of new protective antigens is a key imperative for the development of more effective novel vaccine. The objective of this study was to evaluate antibody responses of 27 recombinant proteins in convalescent sera obtained from pigs that were naturally infected with M. hyopneumoniae. Fifteen proteins were identified as serological immunodominant antigens, while 3 proteins were not recognized by any convalescent serum. Moreover, Mhp462, a leucine aminopeptidase, was found to be a discriminative serological immunodominant antigen which reacted with convalescent sera but not with hyperimmune sera. The serological immunodominant proteins were antigenic and were expressed during infection; this suggests that these proteins (especially the discriminative one) are potential candidate antigens for the development of next generation vaccines against M. hyopneumoniae.

Development of an indirect ELISA for detecting humoral immunodominant proteins of Mycoplasma hyopneumoniae which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera.

BACKGROUND: Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary pathogen of porcine enzootic pneumonia, which has been associated with economic losses due to reduced daily weight gain and feed efficiency. Although it has a small genome and no more than 1000 genes, M. hyopneumoniae can be cultured in cell free media. However, some proteins were not expressed or were only expressed in negligible amounts under culture conditions. Nevertheless, some of these proteins can be expressed at a high level and induce a strong and rapid immune response after M. hyopneumoniae infection. The unexpressed or less expressed proteins may play critical roles in pathogenesis and/or immune response. In order to find the differentially expressed proteins of M. hyopneumoniae between culture condition and infected animals, we established an indirect ELISA for the detection of humoral immunodominant proteins which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera by using Mhp366 protein which did not react with sera from bacterin-immunized pigs, but revealed a strong immunoreaction with porcine convalescent sera. RESULTS: The checkerboard titration method was done by using porcine convalescent sera as positive sera and inactivated bacterin-induced hyperimmune sera as negative sera. The bacterial lysates of fusion proteins and free GST protein without dilution were the optimal coating antigens. The optimal blocking buffer was PBS with 10% FBS and 2.5% skimmed milk. In the checkboard ELISAs, when the sera were diluted at 1:500 and the HRP-labeled rabbit anti-pig IgG were diluted at 1:20000, most positive result was obtained for the assay. CONCLUSIONS: This established indirect ELISA can be used as a tool for the detection of humoral immunodominant proteins of M. hyopneumoniae which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera.

[Development of an ELISA for identification of immunodominant protein antigens of Mycoplasma hyopneumoniae].

We developed a method to identify serological humoral immunodominant proteinic antigen of Mycoplasma hyopneumoniae (Mhp). After constructing the recombinant plasmid pGEX-6P-1-mhp366 and transforming it into Escherichia coli BL21(DE3), the recombinant GST-Mhp366 protein was expressed successfully. The lysates of the recombinant GST-Mhp366 and genetic engineering GST were added into glutathione coated plates and reacted with 17 positive sera or 13 negative sera. Meanwhile, the optimization of experimental conditions, including coated antigen, blocking buffer, dilutions of sera and second antibody were determined. The optimal concentration of the coated antigen was the original bacteria lysates without dilution, and the optimal blocking buffer contained 10% FBS and 2.5% skim milk in PBS. Besides, the working concentration of serum samples and the HRP-tagged rabbit anti-pig IgG secondary antibody were 1:500 and 1:40 000, respectively. Thus, an indirect ELISA was established for identification of immunodominant protein antigens of Mhp. Meanwhile, this method was confirmed by the identified serological humoral immunodominant proteinic antigen Mhp156 and Mhp364. This method can be used for identification of the candidate vaccine antigens on a genome-wide scale. Furthermore, it can lay the foundation for identifying the candidate vaccine antigens through colostra and the nasal mucosal secretions.