Methacarn preserves mucus integrity and improves visualization of amoebae in gills of Atlantic salmon (Salmo salar L.)

Abstract

Two aqueous fixation methods (modified Davidson's solution and modified Davidson's solution with 2% (w/v) Alcian blue) were compared against two non‐aqueous fixation methods (methacarn solution and methacarn solution with 2% (w/v) Alcian blue) along with the standard buffered formalin fixation method to (a) improve preservation of the mucous coat on Atlantic salmon, Salmo salar L., gills and (b) to examine the interaction between the amoebae and mucus on the gill during an infection with amoebic gill disease. Aqueous fixatives demonstrated excellent cytological preservation but failed to deliver the preservation of the mucus when compared to the non‐aqueous‐based fixatives; qualitative and semi‐quantitative analysis revealed a greater preservation of the gill mucus using the non‐aqueous methacarn solution. A combination of this fixation method and an Alcian blue/Periodic acid–Schiff staining was tested in gills of Atlantic salmon infected with amoebic gill disease; lectin labelling was also used to confirm the mucus preservation in the methacarn‐fixed tissue. Amoebae were observed closely associated with the mucus demonstrating that the techniques employed for preservation of the mucous coat can indeed avoid the loss of potential mucus‐embedded parasites, thus providing a better understanding of the relationship between the mucus and parasite.

Evaluation of Low Temperature and Salinity as a Treatment of Atlantic Salmon against Amoebic Gill Disease

“…For histological assessment, gills are fixed with formalin or Davidson's fixative and then processed with ethanol, xylene, and paraffin wax. Although the fixation process provides high quality preservation of cells, the mucus layer is generally lost, and this can result in the loss of associated amoebae [36]. This is more severe with aqueous fixatives such as Davidson's, compared to non-aqueous fixatives [36].…”

“…Although the fixation process provides high quality preservation of cells, the mucus layer is generally lost, and this can result in the loss of associated amoebae [36]. This is more severe with aqueous fixatives such as Davidson's, compared to non-aqueous fixatives [36]. On top of this, in this study only one gill arch was used, and only a thin section of this arch was sectioned and stained.…”

Comparison of bacterial diversity and distribution on the gills of Atlantic salmon ( Salmo salar L.): an evaluation of sampling techniques

“…1995; Fernandez et al . 2019). It is likely that gill tissue fixed in seawater Davidson’s fixative and those of a similar nature lose much of the resident bacterial community occupying the mucus layer.…”

Changes in distribution, morphology and ultrastructure of chloride cell in Atlantic salmon during an AGD infection

“…However, a disparity has previously been reported between diagnostic methods used for AGD; Atlantic salmon gills displaying grossly visible lesions did not show the presence of P. perurans at a histological level, with this discrepancy deemed likely to be due to the sampling technique during gill excision, that is only one section from one gill arch was examined for subsequent histological analysis (Clark & Nowak, ) and also loss of mucus and associated amoebae during fixation. It is indeed well established that conventional fixatives often wash away the overlaying mucus layers (Lee et al, ; Mays, Feldhoff, & Nettleton, ) and associated amoebae, hence the need for alternative methods to optimize fixation to enable mucus stabilization and allow a better understanding of parasite interactions with gill mucus during AGD infection (Fernandez et al, ). In the current study, gills for subsequent downstream molecular analysis of P. perurans load were taken from a section of gill displaying grossly visible AGD lesions which would most likely account for the correlation seen.…”