Investigating the impacts of H2O2 treatment on gills of healthy Atlantic salmon reveals potential changes to mucus production with implications on immune activity.

Current treatment strategies for relevant infectious diseases in Atlantic salmon (Salmo salar L.) include the use of low salinity or freshwater bathing. However, often availability is restricted, and hydrogen peroxide (H2O2) is used as an alternative. The potential impacts of H2O2 on fish mucosal tissues, especially the gills therefore need to be considered. In this study the mucosal and immunological effects of H2O2 treatment on the gills of healthy Atlantic salmon were examined by gene expression (qPCR) and immunohistochemistry (IHC) investigating T-cell, B-cell, and mucin activity. Healthy fish were treated with H2O2 and sampled at different times: 4 h, 24 h and 14 days post-H2O2 treatment (dpt) (total n = 18) to investigate the effect of holding time and H2O2 treatment. Treatment with H2O2 resulted in up-regulation of markers for T-cell activity and anti-inflammatory response and down-regulation of mucin expression in the gills at 14 dpt compared to fish sampled prior to treatment (0h; n = 5 fish). These findings were supported by IHC analysis, which despite being highly variable between samples, showed an increase in the number of CD3+ T cells at 14 dpt in 50% of treated fish compared to pre-treatment fish. The results from this study suggest that H2O2 treatment does not immune compromise healthy Atlantic salmon after 14 dpt (i.e., post-recovery) but modulates gill immune activity and disrupts the mucus covering of the gills. However, further studies are required to determine whether the effects observed are related to H2O2 treatment in isolation or other variables such as holding time or environmental factors.

A comparison of the use of different swab materials for optimal diagnosis of amoebic gill disease (AGD) in Atlantic salmon (Salmo salar L.).

Routine gill swabbing is a non-destructive sampling method used for the downstream qPCR detection and quantitation of the pathogen Neoparamoeba perurans, a causative agent of amoebic gill disease (AGD). Three commercially available swabs were compared aiming their application for timelier AGD diagnosis (Calgiswab® (calcium alginate fibre-tipped), Isohelix® DNA buccal and cotton wool-tipped). Calcium alginate is soluble in most sodium salts, which potentially allows the total recovery of biological material, hence a better extraction of target organisms' DNA. Thus, this study consisted of (a) an in vitro assessment involving spiking of the swabs with known amounts of amoebae and additional assessment of retrieval efficiency of amoebae from agar plates; (b) in vivo testing by swabbing of gill arches (second, third and fourth) of AGD-infected fish. Both in vitro and in vivo experiments identified an enhanced amoeba retrieval with Calgiswab® and Isohelix® swabs in comparison with cotton swabs. Additionally, the third and fourth gill arches presented significantly higher amoebic loads compared to the second gill arch. Results suggest that limiting routine gill swabbing to one or two arches, instead of all, could likely lead to reduced stress-related effects incurred by handling and sampling and a timelier diagnosis of AGD.

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

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.

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

Amoebic gill disease (AGD) is emerging as one of the most significant health challenges affecting farmed Atlantic salmon in the marine environment. It is caused by the amphizoic amoeba Neoparamoeba perurans, with infestation of gills causing severe hyperplastic lesions, compromising overall gill integrity and function. This study used histology, transmission electron microscopy (TEM), immunohistochemistry and transcript expression to relate AGD-associated pathological changes to changes in the morphology and distribution of chloride cells (CCs) in the gills of Atlantic salmon (Salmo salar L.) showing the progression of an AGD infection. A marked reduction in numbers of immunolabelled CCs was detected, and a changing pattern in distribution and morphology was closely linked with the level of basal epithelial hyperplasia in the gill. In addition, acute degenerative ultrastructural changes to CCs at the lesion site were observed with TEM. These findings were supported by the early-onset downregulation of Na+ /K+ -ATPase transcript expression. This study provides supportive evidence that histological AGD lesion assessment was a good qualitative tool for AGD scoring and corresponded well with qPCR genomic Paramoeba perurans quantification. Ultrastructural changes induced in salmon CCs as a result of AGD are reported here for the first time.

Assessment of Marine Gill Disease in Farmed Atlantic Salmon (Salmo salar) in Chile Using a Novel Total Gross Gill Scoring System: A Case Study.

Gill disorders have become more prevalent and widespread in finfish aquaculture in recent years. Their aetiology is often considered to be multifactorial. Effective diagnosis, control and prevention are hindered by the lack of standardised methodologies to characterise the aetiological agents, which produce an array of clinical and pathological presentations. The aim of this study was to define a novel gross pathological scoring system suitable for field-based macroscopic assessment of complex or multifactorial gill disease in farmed Atlantic salmon, using samples derived from a gill disease outbreak in Chile. Clinical assessment of gross gill morphology was performed, and gill samples were collected for qPCR and histology. A novel total gill scoring system was developed, which assesses gross pathological changes combining both the presumptive or healed amoebic gill disease (AGD) and the presence of other types of gill lesions. This scoring system offers a standardised approach to characterise the severe proliferative pathologies in affected gills. This total gill scoring system can substantially contribute to the development of robust mitigation strategies and could be used as an indicator trait for incorporating resistance to multifactorial gill disease into breeding goals.

Confocal scanning laser microscopy with complementary 3D image analysis allows quantitative studies of functional state of ionoregulatory cells in the Nile tilapia (Oreochromis niloticus) following salinity challenge.

The development of a novel three-dimensional image analysis technique of stacks generated by confocal laser scanning microscopy is described allowing visualization of mitochondria-rich cells (MRCs) in the seawater-adapted Nile tilapia in relation to their spatial location. This method permits the assessment and classification of both active and nonactive MRCs based on the distance of the top of the immunopositive cell from the epithelial surface. In addition, this technique offers the potential for informative and quantitative studies, for example, densitometric and morphometric measurements based on MRC functional state. Confocal scanning laser microscopy used with triple staining whole-mount immunohistochemistry was used to detect integumental MRCs in the yolk-sac larvae tail of the Nile tilapia following transfer from freshwater to elevated salinities, that is, 12.5 and 20 ppt. Mean active MRC volume was always significantly larger and displayed a greater staining intensity (GLM; P<0.05) than nonactive MRCs. Following transfer, the percentage of active MRCs was seen to increase as did MRC volume (GLM; P<0.05).

Morphological and ultrastructural characterization of ionoregulatory cells in the teleost Oreochromis niloticus following salinity challenge combining complementary confocal scanning laser microscopy and transmission electron microscopy using a novel prefixation immunogold labeling technique.

Aspects of ionoregulatory or mitochondria-rich cell (MRC) differentiation and adaptation in Nile tilapia yolk-sac larvae following transfer from freshwater to elevated salinities, that is, 12.5 and 20 ppt are described. Investigations using immunohistochemistry on whole-mount Nile tilapia larvae using anti- Na⁺/K⁺-ATPase as a primary antibody and Fluoronanogold™ (Nanoprobes) as a secondary immunoprobe allowed fluorescent labeling with the high resolution of confocal scanning laser microscopy combined with the detection of immunolabeled target molecules at an ultrastructural level using transmission electron microscopy (TEM). It reports, for the first time, various developmental stages of MRCs within the epithelial layer of the tail of yolk-sac larvae, corresponding to immature, developing, and mature MRCs, identifiable by their own characteristic ultrastructure and form. Following transfer to hyperosmotic salinities the density of immunogold particles and well as the intricacy of the tubular system appeared to increase. In addition, complementary confocal scanning laser microscopy allowed identification of immunopositive ramifying extensions that appeared to emanate from the basolateral portion of the cell that appeared to be correlated with the localization of subsurface tubular areas displaying immunogold labeled Na⁺/K⁺-ATPase. This integrated approach describes a reliable and repeatable prefixation immunogold labeling technique allowing precise visualization of NaK within target cells combined with a 3D imaging that offers valuable insights into MRC dynamics at an ultrastructural level.