Eignung des indirekten Immuno-Dotblot-Verfahrens zum Nachweis von Aeromonas salmonicida in Forellengeweben

Abstract

Using an indirect immuno-dotblot technique it was possible to detect Aeromonas salmonicida in kidney, liver, spleen, and heart tissues of experimentally infected trout with acute furunculosis. Tissues of gills and gut could not be assessed because of their intense background staining. The average sensitivity of the test was 2 x 106 cfu per gramme tissue. The frequency of positive results in spleen samples was higher with the immuno- dotblot than with a bacteriological examination, and identical in kidney and liver tissues.

The method distinguished between infections with Aeromonas salmonicida and Yersinia ruckeri. It was cheap, easy to perform and read, and allowed simultaneous testing of large numbers of samples for different bacterial antigens. Results could be obtained after one and a half day.

In contrast, an indirect fluorescence assay revealed such an amount of specific crossreactivity between both Yersinia ruckeri and Aeromonas salmonicida, especially A-protein-positive isolates, and the respective opposite antiserum that it was impossible to distinguish the bacteria in tissue samples.

On nitrocelluose membranes, all tissues exhibited a strong peroxidase activity and pigmentation according to the organ examined and the fish´ health status. The resulting background staining made it difficult to decide whether or not a sample showed a positive reaction.

Comparing several methods to block endogenous peroxidase activity and minimize pigmentation, heated and centrifugated samples of infected fish showed residual peroxidase activity and pigments on the membrane spots. Peroxidate was even more unable to completely bleach pigments and inhibit the tissue peroxidase activitiy although it took effect in an overnight incubation. An oxidization of membrane spots with potassium permanganate and oxalic acid resulted in colourless backgrounds, even with the intensely pigmented and peroxidase rich samples of spleen and kidney. Their effect depended on the pH value of their solutions and the incubation time, which could be shortened by repeating the procedure.

The best results were obtained with a combination of permanganate and peroxide permitting a further reduction of permanganate incubation time.

Sensitivity and specificity of the test depended to a high degree on the sample processing technique. In centrifugated homogenates, up to 99% of antigen were missing in the supernatant, independent of time and speed. Boiling of samples enhanced the reactivity of A.salmonicida only slightly, but the cross reactivity of different gram-negative bacteria up to a hundred times. A measurable but much lesser effect on cross reactivity inflicted trypine and permanganate. In contrast to heating, their negative influence was stronger on pure bacteria suspensions than on bacteria mixed with tissues, presumably because of the higher tissue content of other oxidizable substances like pigment. Peroxidase alone did not visibly change cross reactivity at any concentrations examined. The combination of permanganate and peroxide effected only negligible alterations of bacterial epitopes, especially in richly pigmented tissue samples.

Therefore, the indirect immuno-dotblot with the described procedure of tissue processing seems to be efficient to identify bacteria in trout tissues as long as specific and sensitive antisera are available.