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

Abstract

Amoebic gill disease (AGD) is a proliferative gill disease of marine cultured Atlantic salmon Salmo salar, with the free-living protozoan Neoparamoeba perurans being the primary aetiological agent. The increased incidence of AGD in recent years presents a significant challenge to the Atlantic salmon farming industry in Europe. In this study, a real-time TaqMan ® PCR assay was developed and validated to detect Neoparamoeba perurans on Atlantic salmon gills and further used to monitor disease progression on a marine Atlantic salmon farm in Ireland in conjunction with gross gill pathology and histopathology. The assay proved specific for N. perurans, with no cross-reactivity with the related species N. pemaquidensis, N. branchiphila or N. aestuarina, and was capable of detecting 2.68 copies of N. perurans DNA µl −1. Although the parasite was detected throughout the 18 mo period of this study, mortality peaks associated with clinical AGD were only recorded during the first 12 mo of the marine phase of the production cycle. The initial AGD outbreak resulted in peak mortality in Week 17, which was preceded by PCR detections from Week 13 onwards. Freshwater treatments were an effective method for controlling the disease, resulting in a reduction in the weekly mortality levels and also a reduction in the number of PCR-positive fish. In comparison to traditional diagnostic methods, our PCR assay proved to be highly sensitive and a valuable tool to monitor disease progression and, therefore, has the potential to provide information on the timing and effectiveness of treatments.

Oxidative stress is associated with late‐stage amoebic gill disease in farmed Atlantic salmon (Salmo salar L.)

“…Samples were snap‐frozen in dry ice and stored at −80°C. In addition, histology samples and gill swabs for N. perurans real‐time PCR (Downes et al., ) were taken to confirm AGD and the presence of N. perurans . Average weight and water temperature for the three sampling points were 85.2g, 312.1g and 375.3g and 9°C, 15.0°C and 15.2°C, respectively.…”

Evaluation of Non-destructive Molecular Diagnostics for the Detection of Neoparamoeba perurans

“…In order to conduct an infection trial for the comparison of the preferred assay with traditional screening methods, N. perurans was isolated from farmed Atlantic salmon affected by AGD in the west of Ireland using a method described in Downes et al (2015), adapted from Morrison et al (2004). The amoeba culture was established and maintained according to Crosbie et al (2012).…”

“…The sensitivity, specificity, linearity and correlation to gill score of each assay were also analyzed. In order to investigate the sensitivity of the assays, a plasmid was created and its concentration determined as previously described (Downes et al, 2015). A dilution series was generated and analyzed by the three real-time assays (protocols 1, 2, and 4) to assess the lowest copy numbers detectable.…”

“…Protocol 1 is a TaqMan R qPCR targeting the 18S rRNA gene sequence of N. perurans generating an amplicon of 70 bp (Downes et al, 2015).…”

“…The identification of N. perurans as the causal agent of AGD (Young et al, 2007) has allowed the development of specific DNA based molecular diagnostic assays for the detection of the amoeba. Currently there are two conventional polymerase chain-reaction (PCR) assays published for the detection of N. perurans, (Young et al, 2008;Rozas et al, 2011), while three real-time PCR assays were developed based on SYBR R green (Bridle et al, 2010) and TaqMan R chemistries (Fringuelli et al, 2012;Downes et al, 2015).…”