Identification and phase inversion of Salmonella flagellar antigens, using immuno-discs

Abstract

Disc immuno-immobilization is a simple method for typing the flagellar phase of Salmonella enterica. We re-examined this method using commercial antisera, which contains the preservative sodium azide. Originally prepared motility agar activates bacterial motility and renders S. enterica resistant to sodium azide, resulting in the formation of immuno-immobilization lines around reactive immuno-discs. Though disc immuno-immobilization serves both serotyping and phase inversion, this method is insufficient for the strains in which phase variation rarely occurs. Here, we devised a novel immuno-disc phase inversion method, and all S. enterica strains tested were identically typed. These methods would drastically simplify the task of S. enterica typing in clinical laboratories.

Keywords: Salmonella enterica; flagellar antigen; immuno-disc.

Fig. 1.

Growth behaviors on each motility agar. S. Abortusequi Ne-ae, S. Dublin Ab-du and S. Typhimurium Ab-ty were inoculated at the center of each motility agar. Immuno-discs contained H-G, i, en, p, x, 1, and 2 antisera in a clockwise fashion from top. Arrows show immuno-immobilization lines.

Fig. 2.

Results of disc immuno-immobilization. Immuno-discs on motility agar D contained H-b, c, G, i, m, 1, 2 and 5 antisera in a clockwise fashion from top. Arrows show immuo-immobilization lines. (A) S. Enteritidis Ab-en was completely immobilized by the H-G and m immuno-discs. (B) S. Typhimurium Ab-ty formed immuno-immobilization lines around H-i, 1 and 2 immuno-discs. (C) S. Paratyphi B Ne-pb formed immuno-immobilization lines only around H-1 and 2 immuno-discs. (D) S. Paratyphi B picked from the inside of the immuno-immobilization lines formed by H-1 immuno-disc, formed immuno-immobilization lines around H-b.

Fig. 3.

Serotyping of S. Choleraesuis Ab-ch. Immuno-discs on motility agar D contained H-b, c, G, i, m, 1, 2 and 5 antisera in a clockwise fashion from top (A and C). (A) S. Choleraesuis was completely immobilized by H-1 and 5 immuno-discs. It did not form immuno-immobilization line around other immuno-discs including H-c by disc immuno-immobilization. (B) S. Choleraesuis was spot-inoculated under the inner H-1 immuno-discs. After incubation, S. Choleraeusuis reached the outer H-1 immuno-discs. Bacterial cells around outer immuno-discs are considered immuno-disc phase inverted. On the lower left section of the dish, S. Choleraesuis was completely immobilized and formed inhibition zones around outer immuno-disc. It was considered not to be phase inverted. (C) Immuno-disc phase inverted cells were completely immobilized by H-c immuno-disc.

Fig. 4.

The principle of immuno-disc phase inversion. (A) S. Choleraesuis was spot-inoculated on motility agar and (B) overlaid with a reactive immuno-disc. Another immuno-disc was separately placed. (C) S. Choleraesuis cells expressing phase II antigen are trapped by antibody. During growth, phase inverted S. Choleraesuis appeared and starts migration, (D) resulting to reach another immuno-disc. Bacterial cells expressing phase II antigen also starts to migrate after cell counts exceeded the titer of the antibody; however, they were immobilized by another immuno-disc.