A Western blot immunoassay (WBI) was developed as a confirmatory test for 2 commercial Mycoplasma hyopneumoniae (Mhyo) ELISAs. The WBI detected at least 5 antigen bands (150, 130, 74, 70, and 30 kDa) from Mhyo whole membrane proteins that were not present in the antigens prepared from M. hyorhinis and M. hyosynoviae. Among discrepant sera from vaccinated pigs (n = 17) and field samples (n = 91) assayed by WBI: 1) 2 of the ELISA-positive samples reacted with all 5 antigens bands; 2) all blocking ELISA-positive samples (n = 53) bound 150-, 130-, 74-, and 70-kD antigen bands; and 3) all indirect ELISA-positive samples (n = 55) bound 150-, 130-, and 30-kD antigens. We conclude that the WBI targeting the top 4 antigen bands is a useful confirmatory test for samples initially screened using the commercial Mhyo ELISAs.
AbivenPBBlanchardCSaillardM: 1992, A specific DNA probe for detecting Mycoplasma hyopneumoniae in experimentally infected piglets. Mol Cell Probe6: 423–429.
2.
AhrensPFriisNF: 1991, Identification of Mycoplasma hyopneumoniae with DNA probe. Lett Appl Microbiol12: 249–253.
3.
AmanfuWWengCRossRFBarnesH: 1984, Diagnosis of mycoplasmal pneumonia of swine: Sequential study by direct immunofluorescence. Am J Vet Res45: 1349–1352.
4.
Ameri-MahabadiMZhouE-MHsuWH: 2005, Comparison of two swine Mycoplasma hyopneumoniae ELISAs for detection of antibodies from vaccinated pigs and field serum samples. J Vet Diagn Invest17: 61–64.
5.
ArmstrongCHFreemanMJSands-FreemanL: 1983, Comparison of the enzyme-linked immunosorbent assay and the indirect hemagglutination and complement fixation tests for detecting antibodies to Mycoplasma hyopneumoniae. Can J Comp Med47: 464–470.
6.
BereiterMYoungTFJooHSRossRF: 1990, Evaluation of the ELISA and comparison to the complement fixation test and radial immunodiffusion enzyme assay for detection of antibodies against Mycoplasma hyopneumoniae in swine serum. Vet Microbiol25: 177–192.
7.
FeldNCQvistPAhrensP: 1992, A monoclonal blocking ELISA detecting serum antibodies to Mycoplasma hyopneumoniae. Vet Microbiol30: 35–46.
8.
FriisNF: 1975, Some recommendations concerning primary isolation of Mycoplasma suipneumoniae and Mycoplasma flocculare. Nord Veterinaermed27: 337–339.
LaemmliUK: 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)227: 680–685.
11.
L'EcuyerCBoulangerP: 1970, Enzootic pneumonia of pigs: Identification of a causative mycoplasma in infected pigs and in cultures by immunofluorescent staining. Can J Comp Med34: 38–46.
RazinS: 1994, DNA probes and PCR in diagnosis of mycoplasma infection. Mol Cell Probes8: 497–511.
14.
RossRF: 1999, Mycoplasmal diseases. In: Diseases of swine, ed. StrawBED'AllaireSMengelingWLTaylorDJ, 8th ed., pp. 495–509. Iowa State University Press, Ames, IA.
15.
ScarmanALChinJCEamensGJ: 1997, Identification of novel species-specific antigens of Mycoplasma hyopneumoniae by preparative SDS-PAGE ELISA profiling. Microbiology143(Pt 2): 663–673.
16.
StärkKDCNicoletJFreyJ: 1998, Detection of Mycoplasma hyopneumoniae by air sampling with a nested PCR assay. Appl Environ Microbiol64: 543–548.
17.
ThackerELHalburPRossRF: 1999, Mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia. J Clin Microbiol37: 620–627.
18.
ThackerELThackerBJJankeBH: 2001, Interaction between Mycoplasma hyopneumoniae and swine influenza virus. J Clin Microbiol39: 2525–2530.
19.
WallgrenPArturssonKFossumCAlmGV: 1993, Incidence of infections in pigs bred for slaughter revealed by elevated serum levels of interferon and development of antibodies to Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae. Zentralbl Veterinarmed B40: 1–12.
20.
ZhouE-MChanMKH: 1996, Immunoblot assay as a confirmatory test of competitive ELISA for serodiagnosis of bluetongue. J Vet Diagn Invest8: 365–367.
