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.
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
.
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