Leucotoxic Activities of Staphylococcus aureus Strains Isolated from Cows, Ewes, and Goats with Mastitis: Importance of LukM/LukF′-PV Leukotoxin

Abstract

Among the toxins that Staphylococcus aureus is able to secrete, bi-component toxins named leukotoxins target specifically leukocytes, mainly phagocytic cells. Isolates from cows, goats and ewes with mastitis were selected on the basis of the presence or not of the genes encoding the recently described LukM/LukF-PV leukotoxin. Of the 128 isolates tested, 126 had moderate to high leukotoxic activity to bovine polymorphonuclear cells (PMN). The supernatants of lukM-positive isolates were much more leukotoxic than the supernatants of lukM-negative isolates: mean leukotoxic titers were 122 versus 20 and 581 versus 26 for isolates of bovine and caprine origin, respectively. Among lukM/lukF-PV positive isolates, those of caprine and ovine origins were more leukotoxic than were isolates of bovine origin (P < 0.01). The two most abundant proteins in the culture supernatant of a highly toxic isolate were purified and identified as the two components of LukM (LukM and LukF-PV) on the basis of their molecular mass, N-terminal amino acid sequence, and high synergistic activity. LukM/LukF-PV induced the flattening of bovine PMN at a concentration as low as 3.6 ng/ml (0.1 nM). A higher concentration (18 ng/ml) was necessary to produce the same effect on caprine or ovine PMN. Affinitypurified antibodies to LukM or to LukF-PV neutralized the leukotoxic effect of all the culture supernatants. They neutralized with the same efficiency the toxic activity of supernatants from lukM/lukF-PV positive or negative isolates. These results establish that LukM/LukF-PV is very active on PMN of ruminants and suggest that this leukotoxin could be the most active leukotoxin produced by mastitis isolates. They prompt further studies to delineate the contribution of LukM/LukF-PV to the pathogenesis of mastitis in ruminants and the protective effect of antibodies to this leukotoxin.

Staphylococcus aureusleucotoxin LukM/F' is secreted and stimulates neutralising antibody response in the course of intramammary infection

“…This strain was selected from our strain collection on the basis of high production of LukM/F' leucotoxin [16]. It harbors the genes encoding the LukM/F', LukE/D and Hlg leucotoxins, but produces essentially LukM and LukF' when grown in vitro in conventional bacteriological media [16].…”

“…The assay is based on the shape change (spreading) induced on PMN by staphylococcal leucotoxins, as described by [1,16]. PMN were isolated from goat blood as described [1], and adjusted to 2 × 10 6 /mL in RPMI 1640 without bicarbonate, with 20 mM Hepes (Sigma) and 1 mg/mL bovine serum albumin (low endotoxin, Uptima Interchim, Montluçon, France).…”

“…Neutralization of the leucotoxic activity of milk samples was achieved by adding affinity-purified rabbit Ab against LukM and LukF' [16], and incubating for 4 h at 4 • C before performing the PMN spreading assay. Control of specificity was carried out by incubating the milk samples with the same concentration (see Results) of affinity-purified rabbit Ab to hen ovalbumin.…”

“…Cattle mastitis field isolates are almost all equipped with genes of the leucotoxins LukE/D and hemolysin γ, and a proportion (50 to 80%) possess the genes encoding the LukM/F' leucotoxin [6,21]. Data concerning mastitis isolates of goat origin are few, but indicate that LukM/F' is also well represented [16]. The most active leucotoxin on bovine neutrophils is LukM/F' [1].…”

A 3-year long study of Staphylococcus aureus isolates from subclinical mastitis in three Azawak zebu herds at the Sahelian experimental farm of Toukounous, Niger

“…less than one third of them, similarly to other published results. The most noticeable difference is the much higher prevalence of the lukS-PV (39 %) and lukF-PV (31 %) genes coding for the Panton-Valentine leukocidin (PVL) compared to the results of other studies in Europe and Africa (Rainard et al 2003;Fueyo et al 2005;Karahan et al 2009;Ikawaty et al 2010;Kadja et al 2010;Ote et al 2011;Issa et al 2013). Since the PVL toxin is strongly associated with human S. aureus isolates causing skin and soft-tissue infections (Shallcross et al 2013), the lukS-PV-and/or lukF-PV-positive isolates in Toukounous might therefore be of human rather than of cattle origin, transferred during the manual milking process.…”