Quantification of Defensive Proteins in Skin Mucus of Atlantic salmon Using Minimally Invasive Sampling and High-Sensitivity ELISA

SIMPLE SUMMARY: The external surfaces of fish, including the skin, are covered by mucus. Mucus is an important and multifunctional matrix of substantial complexity. The mucus is viscous and sticky, and adheres to the underlying epithelium, making the sampling of mucus challenging. To help define more standardized protocols for mucus sampling, we here compare three different sampling methods. The methods include scraping of mucus, wiping of mucus, and absorption of the liquid part of mucus. We compare the resulting damage to the fish skin and compare the content of two specific immune proteins in the three sample types. Using histological examination, we show that absorption leads to very limited damage to the skin epithelium while scraping causes substantial damage. Using Enzyme-Linked Immunosorbent Assay (ELISA) methods, we show that the mucus sample types contain similar amounts of antigen specific immunoglobulin M (IgM) and complement component 5 (C5), respectively. The levels of the IgM but not the C5 were moderately correlated between mucus and blood from the same fish, suggesting the importance of fish skin mucus for analyzing antigen-specific IgM after vaccination procedures. We conclude that absorption is an easily performed and minimally invasive sampling method that produces mucus samples with comparable contents of IgM and C5. ABSTRACT: Protocols used to collect fish skin mucus may inadvertently compromise the sampled fish or the resulting sample. Here, we used three methods (wiping, scraping, and absorption) to collect skin mucus from Atlantic salmon and compared their invasiveness on fish skin epithelium. We found that the absorption method was the least invasive. We also compared the abundance of antigen-specific immunoglobulin M subtype A antibodies (IgM-A Ab) and complement component 5 (C5) in mucus samples collected from vaccinated fish by the three methods. An enzyme-cascade-amplification strategy colorimetric immune assay was optimized and used to analyze IgM-A, and ELISA was used to analyze C5. The abundance of antigen-specific IgM-A in skin mucus was comparable between the three methods, but C5 was significantly lower in absorbed mucus in comparison to in the wiped or scraped mucus samples. Absorbed skin mucus samples collected from various body regions of salmon, levels of C5 were comparable, while specific IgM-A amounts varied between the regions. By comparing three mucus-absorbing materials (medical wipe, gauze, and cotton) for their ability to absorb and release IgM-A and C5, medical wipes proved to be ideal for IgM-A analysis, whereas gauze was the best for C5 analysis.