Abstract
BackgroundImmunization 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.ResultsIn 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.ConclusionsThe 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.
Highlights
Immunization of pigs with an inactivated Mycoplasma hyopneumoniae vaccine generates hyperimmune serum that contains high concentrations of anti-M. hyopneumoniae IgG
Based on the characteristics of the Mhp366 protein, we developed two Enzyme-linked immunosorbent assay (ELISA), one for screening serological immunodominant protein antigens [12] and another for further screening the discriminative immunodominant proteins that can distinguish between antiM. hyopneumoniae IgG in inactivated bacterin-induced hyperimmune sera and convalescent sera resulting from natural M. hyopneumoniae infection [13]
One hundred and eighty serum samples collected from unvaccinated pigs at Farm A were negative for M. hyopneumoniae IgG by commercial IgG-ELISA and the P36 gene was not detected in the corresponding laryngeal swabs by nested PCR
Summary
Immunization of pigs with an inactivated Mycoplasma hyopneumoniae vaccine (bacterin) generates hyperimmune serum that contains high concentrations of anti-M. hyopneumoniae IgG. Available IgGELISA kits cannot distinguish between anti-M. hyopneumoniae IgG in inactivated bacterin-induced hyperimmune sera and convalescent sera resulting from natural M. hyopneumoniae infection. Diagnosis of M. hyopneumoniae infection may be achieved by isolation of the bacterium, molecular identification, and serological detection. Each of these methods is associated with several limitations. Real-time polymerase chain reaction has been successfully applied to identify and differentiate M. hyopneumoniae from M. hyorhinis and M. flocclare in PEP-like lesions, but this method is too expensive for routine use in testing laboratories, and sample collection can be challenging [6, 7]. There remains an unmet need for a more sensitive and convenient method to diagnose natural M. hyopneumoniae infection
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