isolated from atopic dogs with pyoderma induces mast cell degranulation.

Abstract

AIMS: First, to determine via whole genome sequencing the sequence of thegene that encodes δ-toxin and elements of the accessory gene regulator () locus that encode quorum sensing in fourisolates from atopic dogs; second, to assess degranulation of mast cells by synthetic δ-toxinand by culture filtrate containing δ-toxin from theisolates in canine skin; and third, to determine whether the genetic region () encoding the δ-toxin gene is upregulated in response to increasing bacterial density (quorum sensing) in the isolates. METHODS: Four isolates ofwere obtained from four dogs with pyoderma and canine atopic dermatitis (cAD). All four isolates were sequenced to compare their genomes and the sequences of theandelements. Syntheticδ-toxin was applied to a mast cell culture from murine fetal liver cells. Degranulation was assessed using a β-hexosaminidase assay. Filtered supernatants from cultures of the fourisolates were tested by mass spectrometry to detect δ-toxin. These filtrates were then injected into the skin of five normal dogs. The injection sites were biopsied 15 minutes later. Degranulation of canine mast cells was assessed and quantified histologically. To assess up-regulation of the genetic region encoding the δ-toxin gene in response to increasing bacterial density in the fourisolates, relative expression ofwas assayed using quantitative PCR after 1, 2, 4, 7 and 8 hours of culture. RESULTS: Syntheticδ-toxin caused comparable degranulation of MC/9 cells to δ-toxin of. Mast cell degranulation was demonstrated in the skin of all five normal dogs following intradermal injection of a purified supernatant that containedδ-toxin. The genetic elements of the δ-toxins were described. As the cell density of cultures of theisolates from atopic dogs increased,expression increased relative to the reference gene (), suggesting thatexpression may be controlled by a quorum-sensing mechanism. CONCLUSIONS AND CLINICAL RELEVANCE: isolates from atopic dogs carry genes encoding δ-toxin, a staphylococcal exotoxin that can degranulate murine mast cells. An agent in filteredculture known to contain δ-toxin causes degranulation of dermal mast cellsand may play a role in the initiation and/or exacerbation of cAD.