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

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.