Immunogenicity of Staphylococcus aureus LukM/F0-PV recombinant subunits: Validation of diagnostic potential and evaluation of protective efficacy in vitro

Leukotoxin M/F0-Panton Valentine (LukM/F0-PV), a beta pore-forming toxin secreted by
Staphylococcus aureus, is a major virulence factor involved in the pathogenesis of bovine
mastitis. The present study was aimed to determine immunogenicity of two recombinant
subunits of LukM/F0-PV, rLukM (MW 38 kDa) and rLukF (MW 39 kDa), develop and
validate an indirect enzyme linked immunosorbent assay (ELISA) using polyclonal
antibodies raised in rabbits, and evaluate applicability of the assay to diagnose clinical and
subclinical bovine mastitis. Additionally, in vitro assays were conducted to determine
abilities of antibodies to neutralize cytotoxicity of the native leukotoxin. A total of 87
bovine milk samples (healthy, subclinical and clinical mastitis) were evaluated for the
presence of toxin determinants. Receiver–operator characteristic curve for the experi-
mental ELISA values statistically interpreted a cut-off score of >0.109 OD405, with an assay
specificity of 100% and sensitivity in the range of 80–87.5%. In addition, area under curve of
0.93–0.98 revealed the test was accurate in categorizing samples from infected and noninfected
bovine. The rLukF IgG-ELISA was more sensitive than rLukM IgG-ELISA.
Furthermore, it was evident from MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium) dye reduction, indirect immunofluorescence and lactate dehydrogenase
assays that anti-rLukM/rLukF antibodies, with high neutralizing titers, inhibited in vitro
leukotoxic activity and protected bovine neutrophil membrane integrity from cytotoxicity
of native leukotoxin. The findings demonstrated that antibodies produced from
recombinant subunits contribute to specific and sensitive immunodiagnosis and may
also have the potential to provide passive therapeutic benefit in the management of bovine
mastitis.