Evaluation of fixation methods for demonstration of Neoparamoeba perurans infection in Atlantic salmon, Salmo salar L., gills

Abstract

Formaldehyde-based fixatives are generally employed in histopathology despite some significant disadvantages associated with their usage. Formaldehyde fixes tissue by covalently cross-linking proteins, a process known to mask epitopes which in turn can reduce the intensity of immunohistochemical stains widely used in disease diagnostics. Additionally, formaldehyde fixation greatly limits the ability to recover DNA and mRNA from fixed specimens to the detriment of further downstream molecular analyses. Amoebic gill disease (AGD) has been reliably diagnosed from histological examination of gills although complementary methods such as in situ hybridization (ISH) and polymerase chain reaction (PCR) are required to confirm the presence of Neoparamoeba perurans, the causative agent of AGD. As molecular techniques are becoming more prevalent for pathogen identification, there is a need to adapt specimen collection and preservation so that both histology and molecular biology can be used to diagnose the same sample. This study used a general approach to evaluate five different fixatives for Atlantic salmon, Salmo salar L., gills. Neutral-buffered formalin and seawater Davidson's, formaldehyde-based fixatives commonly used in fish histopathology, were compared to formalin-free commercial fixatives PAXgene®, HistoChoice™MB* and RNAlater™. Each fixative was assessed by a suite of analyses used to demonstrate AGD including routine histochemical stains, immunohistochemical stains, ISH and DNA extraction followed by PCR. All five fixatives were suitable for histological examination of Atlantic salmon gills, with seawater Davidson's providing the best quality histopathology results. Of the fixatives evaluated seawater Davidson's and PAXgene® were shown to be the most compatible with molecular biology techniques. They both provided good DNA recovery, quantity and integrity, from fixed and embedded specimens. The capacity to preserve tissue and cellular morphology in addition to allowing molecular analyses of the same specimens makes seawater Davidson's and PAXgene® appear to be the best fixation methods for diagnosis and research on AGD in Atlantic salmon gills.

Inactivation ofInfluenza A virus,Adenovirus,andCytomegaloviruswith PAXgene Tissue Fixative and Formalin

“…However, previously published articles show that histological techniques as well as proteomic and genomic techniques work with PAXgene-fixed tissues. [7][8][9][10] The possibility of detecting parasite DNA in DNA extracted from PFPE salmon gills 11 suggests that in future experiments, virus RNA, DNA, and proteins may be easily detected in PFPE tissues.…”

“…In proteomic and genomic assays, proteins and nucleic acids derived from PAXgene-fixed, paraffin-embedded (PFPE) tissues react as those derived from fast frozen tissues. [7][8][9][10] Recently, Cadoret et al 11 showed that DNA isolated from PAXgene-fixed tissue of Atlantic salmon gills could be used to detect Neoparamoeba perurans DNA by polymerase chain reaction (PCR). Also, conventional histological staining and immunohistochemistry (IHC) could be performed on PAXgene-fixed tissue to visualize the amoeboid parasite in salmon gills.…”

A longitudinal study of amoebic gill disease on a marine Atlantic salmon farm utilising a real-time PCR assay for the detection of Neoparamoeba perurans

“…It is recognised that the histological gill score may have been influenced by the buffered formalin fixative used in this study. The use of an alternative fixa- tive such as Davidson's may have resulted in higher retention of amoebae on the gills sampled (Cadoret et al 2013); however, it was more practical to use buffered formalin which is also routinely used in fish histopathology. The sensitivity of the real-time assay was demonstrated by the fact that positive fish were already detected by Week 12 (10% of fish tested were positive), and by Week 16, all fish tested were positive for N. perurans.…”

Integration of Transcriptome, Gross Morphology and Histopathology in the Gill of Sea Farmed Atlantic Salmon (Salmo salar): Lessons From Multi-Site Sampling

“…Immediately after PGD scoring, fish were bled and the gill arch with the highest PGD score was excised for both transcriptome profiling and histopathological examination. For transcriptomic profiling, 3 transverse sections (gill arch ∼3 mm long with 3-4 pairs of filaments) from dorsal, medial and ventral regions of the gill (Figure 1) were transferred to RNAlater R (Sigma-Aldrich, St. Louis, MO, United States), kept at (Cadoret et al, 2013) for 24 h, followed by a short-term exposure to 10% neutral buffered formalin before tissue processing for histopathological examination.…”