Changes in the interbranchial lymphoid tissue of Atlantic salmon (Salmo salar) affected by amoebic gill disease.

The interbranchial lymphoid tissue (ILT) was recently described in the gills of salmonids. This study examined changes in the ILT during a parasitic infection in marine environment, using amoebic gill disease (AGD) as a model. Atlantic salmon (Salmo salar) experimentally infected with Neoparamoeba perurans were sampled at 0, 3, 7, 14 and 28 days post challenge. Transversal sections of three areas of the gills (dorsal, medial and ventral) were histologically assessed for morphological and cellular changes. AGD induced morphological changes and a cellular response in the ILT of affected fish. These changes included a significant increase in the ILT surface area in fish 28 days after AGD challenge, compared to control fish at the same time point. The length of the ILT increased significantly 28 days post exposure in the dorsal area of the gill arch in the fish affected by AGD. The lymphocyte density of the ILT increased after AGD challenge, peaking at 7 days post exposure; however, by 28 days post exposure, a reduction of lymphocyte density to values close to pre-infection levels was observed. PCNA immunostaining revealed that epithelial hyperplasia was the most likely factor contributing to the ILT enlargement in the affected fish.

Evidence of immune and inflammatory processes in the gills of AGD-affected Atlantic salmon, Salmo salar L.

Amoebic gill disease (AGD) is a disease caused by the ectoparasite Neoparamoeba perurans which affects several cultured marine fish worldwide. The characterisation of pro-inflammatory and immune related genes at the mRNA level in AGD-affected Atlantic salmon gills was performed at 10 days post-inoculation using 2D quantitative RT-PCR, a method of mapping transcriptional responses in tissues. The genes of interest were IL-1ß, TNF-α, TCR-α chain, CD8, CD4, MHC-IIα, MHC-I, IgM and IgT. A significant increase in expression of the mRNA of all the genes was observed in the gills of AGD-affected fish. Contrary to previous studies, our data suggest that the parasite, N. perurans, elicits a classical inflammatory response in the gills of AGD-affected fish and indicates that the mRNA expression of immune genes within gill lesions misrepresents the cellular immune response in the gills during AGD.

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

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.

Effects of immunostimulants on ranched southern bluefin tuna Thunnus maccoyii: immune response, health and performance.

Ranched southern bluefin tuna Thunnus maccoyii were fed baitfishes supplemented with vitamins (predominantly E and C) or vitamins and immunostimulants, nucleotides and ß-glucans, over 12 weeks after transfer and monitored for enhancement in immune response, health and performance through their 19 week grow-out period. Fish from two different tows were sampled separately at three different sampling points: at transfer to grow-out pontoons, at 8 weeks post-transfer and at harvest, 19 weeks post-transfer. Lysozyme activity was enhanced during vitamin supplementation compared to control fish. Performance (i.e. survival, condition index and crude fat), health (i.e. blood plasma variables including pH, osmolality, cortisol, lactate and glucose) and alternative complement activity were not commonly improved through diet supplementation. There were some tow-specific improvements in performance through vitamin supplementation including survival, selected parasite prevalence and intensity, and alternative complement activity. Immunostimulant supplementation also showed a tow-specific improvement in plasma cortisol level. Tow-specific responses may suggest that life history, previous health condition and husbandry can affect the success of vitamin and immunostimulant enhancement of immune response, health and performance of ranched T. maccoyii.

Metabolic effects of amoebic gill disease (AGD) and chloramine-T exposure in seawater-acclimated Atlantic salmon Salmo salar.

Our aim was to determine possible metabolic effects amoebic gill disease (AGD) on Atlantic salmon Salmo salar. Standard (R(S)) and routine (R(ROU)) metabolic rates were evaluated by continually measuring oxygen consumption in 2 independent tanks of fish (18.69 +/- 1.01 kg m(-3), mean +/- SE). Active metabolic rate (R(ACT)) and metabolic scope (R(ACT) - R(S)) were assessed using a chasing protocol and determined at 3 time periods: (1) pre-infection, (2) 3 d post-infection, and (3) 2 d post-treatment. On Day 3 of the study, the fish were infected with amoebae isolated from the gills of AGD-affected salmon (2300 cells l(-1)). No significant elevations in R(ACT) or metabolic scope were detected 3 d post-infection and 2 d post-treatment; however, significant elevations in R(S) and R(ROU) were detected 3 d post-infection and 2 d post-treatment. Assessment of R(ROU) data, especially for the light period, also indicated a rise in oxygen consumption rate over the course of the experiment. Treatment of AGD-affected Atlantic salmon with chloramine-T (CL-T) appeared to briefly mitigate the rise in R(S), as there was a 30% drop (though non-significant) in R(S) following treatment. Despite this, R(S) continued the upward trend 1 d following treatment. These results suggest that over the course of AGD development, R(S) in Atlantic salmon increases. Therefore, considering the physical conditions which constrain R(ACT), we expect that metabolic scope would become compromised in fish more heavily affected with AGD. Treatment with CL-T shows promise for mitigating the respiratory effects of AGD and potentially minimising the loss of metabolic scope.

Increased systemic vascular resistance in Atlantic salmon, Salmo salar L., affected with amoebic gill disease.

Previous investigations into the pathophysiology of amoebic gill disease (AGD) have suggested that there are probable cardiovascular effects associated with this disease. In the present study Atlantic salmon, Salmo salar L., were experimentally infected by cohabitation with diseased individuals. Two commonly used vasodilators, sodium nitroprusside (SNP) and captopril, the angiotensin-converting enzyme (ACE) inhibitor, were used as tools to investigate possible vasoconstriction and/or renin-angiotensin system (RAS) dysfunction in AGD-affected animals. Within the SNP trial, results showed that AGD-affected fish exhibited lowered cardiac output (Q), lowered cardiac stroke volume (V(S)) and a significantly elevated systemic vascular resistance (R(S)) compared with non-affected naïve counterparts. These effects were totally abolished following SNP administration (40 microg kg(-1)), however significant cardiovascular effects associated with SNP were not observed. Within the captopril trial, where AGD-affected fish were more diseased compared with the SNP trial, a significant hypertension was observed in AGD-affected fish. Captopril administration (10(-4) mol L(-1) at 1 mL kg(-1)) resulted in a significant drop in dorsal aortic pressure (P(DA)) for both AGD-affected and naïve control fish. In terms of peak individual responses, captopril administration effectively lowered P(DA) in both AGD-affected and naïve control groups equally. The drop in P(DA) following SNP administration however was significantly greater in AGD-affected fish potentially suggesting disease-related vasoconstriction. The lack of significant cardiovascular effects directly associated with both SNP and captopril administrations possibly relate to the 6 h recovery period following surgical procedures. However, while variable, these results do suggest that there are significant cardiovascular effects including vasoconstriction and hypertension associated with AGD.

Respiratory pathogenesis of amoebic gill disease (AGD) in experimentally infected Atlantic salmon Salmo salar.

The aim of this study was to investigate the respiratory responses of Atlantic salmon, Salmo salar, experimentally affected with amoebic gill disease (AGD). In Series I, arterial blood samples were taken over a 96 h period following amoebae addition to examine potential respiratory effects associated with initial exposure. No major significant treatment effects were found between fish exposed to amoebae and control (non-exposed) fish. Arterial pH (pHa) was seen to be significantly elevated at 48 h in AGD fish relative to the 0 h time point. To investigate the long-term respiratory effects associated with infection, fish were similarly exposed to amoebae and sampled over a 16 d period. As for Series I, caudal blood pH was significantly elevated by Day 2 (48 h) compared to the pre (Day 0)-time point, suggesting that initial exposure to amoebae and/or amoebae attachment may have induced an initial respiratory alkalosis via increased ventilation frequency and/or amplitude. From Day 7 onwards, and coinciding with a significant increase in the percentage of affected gill filaments, blood pH decreased significantly, possibly indicating the onset of the characteristic respiratory acidosis that has previously been described for experimentally AGD-affected Atlantic salmon. Although fish in this study showed up to 90% AGD-affected filaments, the corresponding respiratory results do not reflect a major acid-base disturbance. Therefore, the findings from the present study support the contention that, although AGD only affects the gill, AGD-associated mortality in Atlantic salmon may not be primarily associated with respiratory failure.

Cardiovascular responses of three salmonid species affected with amoebic gill disease (AGD).

The cardiovascular effects of amoebic gill disease (AGD) were investigated immediately following surgery in three salmonid species; Atlantic salmon (Salmo salar L.), brown trout (Salmo trutta L.) and rainbow trout (Oncorhynchus mykiss Walbaum). Fish, both naïve (control) and infected (AGD-affected) of each species, were fitted with dorsal aorta catheters and cardiac flow probes. Cardiac output and dorsal aortic pressures were then continuously measured over a 6-h period following surgery. Results showed that Atlantic salmon, brown trout and rainbow trout displayed similar dorsal aortic pressure, cardiac output, and systemic vascular resistance (mean dorsal aotic pressure divided by cardiac output) values. However, the only significant differences relating to disease status i.e. infected or control, were found in Atlantic salmon. Although no significant differences were seen in dorsal aortic pressure values, AGD-affected salmon displayed significantly elevated systemic vascular resistance at 4 and 6 h post surgery. Cardiac output was also approximately 35% lower in AGD-affected salmon compared to the non-affected control counterparts. These results comparatively examine cardiac function in response to AGD across three salmonid species and highlight species-specific cardiovascular responses that occur in association with disease. It is suggested that the apparent cardiac dysfunction seen in AGD-affected Atlantic salmon could, under stressful conditions, become exacerbated. Cardiac failure is therefore suggested to be a possible physiological mechanism by which AGD causes or contributes to mortality in Atlantic salmon.