title: Immunogenicity of Staphylococcus aureus LukM/F'-PV recombinant subunits: validation of diagnostic potential and evaluation of protective efficacy in vitro.
abstract: Leukotoxin M/F'-Panton Valentine (LukM/F'-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/F'-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 experimental 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 non-infected 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.
authors.affiliation: Department of Food Microbiology, CSIR-Central Food Technological Research Institute, Mysore 570 020, India.
authors.forename: R J
authors.initials: RJ
authors.lastname: Padmaja
authors.affiliation: Department of Food Microbiology, CSIR-Central Food Technological Research Institute, Mysore 570 020, India. Electronic address: prakashalami@cftri.res.in.
authors.forename: P M
authors.initials: PM
authors.lastname: Halami
mesh_terms: D000818:Animals; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards / Q000662:veterinary*; D005098:Exotoxins / Q000235:genetics* / Q000276:immunology*; D005260:Female; D008414:Mastitis, Bovine / Q000175:diagnosis* / Q000382:microbiology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000175:diagnosis* / Q000662:veterinary*; D013211:Staphylococcus aureus / Q000276:immunology*; D037521:Virulence Factors
chemical_list: D005098:Exotoxins; D011994:Recombinant Proteins; D037521:Virulence Factors; C018579:leukotoxin
keywords: Bovine mastitis; IgG-ELISA; Immunofluorescence; Neutralization; Recombinant leukotoxin subunits; Staphylococcus aureus