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We developed a new recombinant enzyme-linked immunosorbent assay (rELISA) for serodiagnosis of contagious agalactia (CA), a disease caused by Mycoplasma agalactiae in sheep and goats. The assay is based on two M. agalactiae surface proteins, namely, P80 and P55. Identification of these immunodominant and common antigens was accomplished by examining the antibody response elicited in sheep during experimental infection and comparing it to the protein expression profiles of 75 M. agalactiae field strains. Our rELISA was tested with 343 sera, collected from sheep with a laboratory-confirmed diagnosis of CA (n ‫؍‬ 223) and from healthy animals (n ‫؍‬ 120). All sera had previously been tested by Western blotting (WB) for reactivity against M. agalactiae. In addition, our rELISA was compared with a commercial routine ELISA based on inactivated antigens (CHEKiT). Among the 223 samples that were WB positive for M. agalactiae, 209 (93.7%) tested positive for rP80-P55 with our ELISA, whereas only 164 (73.8%) tested positive with the CHEKiT ELISA. Among the 120 samples tested that were WB negative for M. agalactiae, 96.7% were confirmed as negative with our rELISA, while only 75.8% were confirmed as negative with the CHEKiT ELISA. A comparison of the results with receiver operating characteristic curves indicated that the differences observed between our rELISA and the CHEKiT ELISA are statistically significant. The use of recombinant peptides instead of inactivated antigens could significantly improve the discrimination of positive and negative animals, bringing significant advantages in controlling the import/export of live animals and helping in eradication of this economically detrimental disease.

In this study, an immunoproteomic approach was used to identify immunodominant proteins from Mycoplasma mycoides subsp. capri isolates. Membrane proteins, extracted through TX-114 phase partitioning, were separated using mono- and two-dimensional electrophoresis and detected by Western blotting with pooled sera from naturally infected goats. A total of 27 immunoreactive spots, corresponding to 13 different proteins, were identified using nanoLC-ESI-MSMS. Function annotation revealed that most of these proteins were metabolic enzymes involved in carbohydrate and energy metabolism. The immunogenic proteins identified in this study: pyruvate dehydrogenase, dihydrolipoamide acetyltransferase, dihydrolipoyl dehydrogenase, phosphate acetyltransferase, phosphopyruvate hydratase, adenine phopshoribosyltransferase, transketolase, translation elongation factor G, translation elongation factor Ts, FMN-dependent NADH-azoreductase, peptide methionine sulfoxide reductase, inorganic diphosphatase and trigger factor may be used as biomarkers for the serological diagnosis of contagious agalactia caused by M. mycoides subsp. capri.

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