Characterization of leukotoxin producing Staphylococcus aureus and development of antibody based method for leukotoxin detection in bovine milk samples.

Padmaja, R. J. (2015) Characterization of leukotoxin producing Staphylococcus aureus and development of antibody based method for leukotoxin detection in bovine milk samples. PhD thesis, University of Mysore.

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Abstract

Bovine mastitis, an acute bacterial disease caused by Staphylococcus aureus affects dairy farming regions where cattle are professionally exploited for high milk yielding varieties. Reported cases and mortality rates are increasing worldwide. Hence, there is an urgent need to develop rapid, sensitive, and inexpensive diagnostic tests for mastitis at both high- and low-resource settings for conferring protection against the pathogenic strains of S. aureus. Early diagnosis plays a crucial role to forecast a warning of mastitis surveillance. In this context, leukotoxin LukM/F´-PV secreted by the pathogen playing an important role in the mastitis pathogenicity has recently attracted attention as a novel therapeutic target. The toxins belong to a ß-barrel pore forming toxin family are known to selectively kill the circulating immune molecules of the host system. The destroyed cells play a significant role in the regulation of the inflammatory reactions and the tissue breakdown induce resulting in inflamed mammary gland in bovine population. The staphylococcal strains producing high levels of the leukotoxin are strongly associated with severe disease. Considering these facts, the study aimed to characterize the LukM/F´-PV producing S. aureus through rapid conventional and molecular methods. The genomic DNA from the characterized strains was exploited for amplifying individual gene leukotoxin amplicons. These amplicons were cloned and expressed in Escherichia coli as a recombinant leukotoxin monomer with high purity. Polyclonal antibody against rLukM and rLukF was produced and purified using immune-affinity chromatography. The immune response was evaluated as diagnostic reagent for detecting and grading mastitis in bovine population. Additionally, neutralizing property of the antibodies was determined to block the pore forming activity of native toxin in vitro. As a proof of concept, the results obtained in the study showed that immunogenicity of S. aureus recombinant leukotoxin was efficient in probing undetected subclinical mastitis and neutralized the native toxin in vitro.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Bovine mastitis, Staphylococcus aureus, leukotoxin
Subjects:	500 Natural Sciences and Mathematics > 07 Life Sciences > 04 Microbiology
Divisions:	Food Microbiology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	06 Nov 2015 07:44
Last Modified:	06 Nov 2015 07:44
URI:	http://ir.cftri.res.in/id/eprint/11940

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