Development of an ELISA for distinguishing convalescent sera with Mycoplasma hyopneumoniae infection from hyperimmune sera responses to bacterin vaccination in pigs.

Vaccination with inactivated bacterin is the most popular and practical measure to control enzootic pneumonia. After immunisation with inactivated bacterin, Mycoplasma hyopneumoniae colonised on the respiratory tract and lung stimulates the humoural immune responses and produces IgG and IgA antibodies. ELISA is a widely used serological method to detect M. hyopneumoniae antibodies. However, commercial IgG-ELISA kit cannot distinguish between inactivated bacterin-induced hyperimmune sera and convalescent sera stimulated by natural infection. SIgA-ELISA method needs to collect nasal swabs, but collecting nasal swabs is not easy to operate. Establishment of a discriminative ELISA detecting humoural IgG from convalescent sera but not hyperimmune sera facilitates to evaluate the natural infection of M. hyopneumoniae after inactivated bacterin vaccination. We expressed and purified a recombinant protein named Mhp366-N which contains an epitope recognised by the convalescent sera but not hyperimmune sera. The developed discriminative IgG-ELISA could discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera and was reproducible, sensitive and specific to M. hyopneumoniae antibody produced by natural infection. Compared to SIgA-ELISA method, discriminative IgG-ELISA was more convenient to detect IgG antibody from sera than IgA from nasal swabs, although it has limited sensitivity in the early stages of infection. Additionally, to some extent, it has a potential to avoid the interference of maternally derived IgG antibodies. The established discriminative IgG-ELISA was efficient to judge the serological IgG antibodies induced from natural infection or inactivated vaccine stimulation and provided a useful method to investigate and evaluate the live organism infection after the application of inactivated bacterin.

Development of an indirect ELISA for detection of anti-Mycoplasma hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera.

BACKGROUND: Immunization of pigs with an inactivated Mycoplasma hyopneumoniae vaccine (bacterin) generates hyperimmune serum that contains high concentrations of anti-M. hyopneumoniae IgG. Commercially available IgG-ELISA kits cannot distinguish between anti-M. hyopneumoniae IgG in inactivated bacterin-induced hyperimmune sera and convalescent sera resulting from natural M. hyopneumoniae infection. Establishment of an ELISA to detect anti-M. hyopneumoniae IgG in convalescent sera will facilitate the evaluation of the M. hyopneumoniae status of pig farms. RESULTS: In this study, we expressed and purified recombinant Mhp366-N protein, which contains an epitope recognized by M. hyopneumoniae convalescent sera but not hyperimmune sera, for use as a coating antigen. For the M. hyopneumoniae convalescent serum IgG-ELISA, the optimal antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time and colorimetric reaction time were 0.25 µg/mL, 2.5 % skim milk, 1 h, 1:500, 0.5 h, 1:10,000, 1 h and 15 min, respectively. Validation of the M. hyopneumoniae convalescent serum IgG-ELISA showed a cut-off value of 0.323, the intra-assay CV ranged from 3.27 to 7.26 %, the inter-assay CV ranged from 3.46 to 5.93 %, and the assay was able to differentiate convalescent sera from antibodies to 7 other porcine respiratory pathogens. The convalescent serum IgG-ELISA detected no anti-M. hyopneumoniae IgG in hyperimmune serum samples while a commercial IgG-ELISA identified 95/145 of these sera as positive. The accuracy of the M. hyopneumoniae convalescent serum IgG-ELISA was comparable to the sIgA-ELISA but better than the commercial IgG-ELISA. CONCLUSIONS: The convalescent serum IgG-ELISA is a reproducible, sensitive, and specific indirect ELISA to detect anti-M. hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera. This ELISA could be used to carry out large scale surveillance of M. hyopneumoniae infection in pig farms regardless of vaccination status.

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.