Neoparamoeba perurans n. sp., an agent of amoebic gill disease of Atlantic salmon (Salmo salar)

Amoebic gill disease (AGD) is a potentially fatal disease of some marine fish. Two amphizoic amoebae Neoparamoeba pemaquidensis and Neoparamoeba branchiphila have been cultured from AGD-affected fish, yet it is not known if one or both are aetiological agents. Here, we PCR amplified the 18S rRNA gene of non-cultured, gill-derived (NCGD) amoebae from AGD-affected Atlantic salmon (Salmo salar) using N. pemaquidensis and N. branchiphila-specific oligonucleotides. Variability in PCR amplification led to comparisons of 18S rRNA and 28S rRNA gene sequences from NCGD and clonal cultured, gill-derived (CCGD) N. pemaquidensis and N. branchiphila. Phylogenetic analyses inferred from either 18S or 28S rRNA gene sequences unambiguously segregated a lineage consisting of NCGD amoebae from other members of the genus Neoparamoeba. Species-specific oligonucleotide probes that hybridise 18S rRNA were designed, validated and used to probe gill tissue from AGD-affected Atlantic salmon. The NCGD amoebae-specific probe bound AGD-associated amoebae while neither N. pemaquidensis nor N. branchiphila were associated with AGD-lesions. Together, these data indicate that NCGD amoebae are a new species, designated Neoparamoeba perurans n.sp. and this is the predominant aetiological agent of AGD of Atlantic salmon cultured in Tasmania, Australia.     

In vitro cultured Neoparamoeba perurans causes amoebic gill disease in Atlantic salmon and fulfils Koch's postulates

Amoebic gill disease (AGD) in marine farmed Atlantic salmon is of growing concern worldwide and remains a significant health issue for salmon growers in Australia. Until now the aetiological agent, Neoparamoeba perurans, has not been amenable to in vitro culture and therefore Koch's postulates could not be fulfilled. The inability to culture the amoeba has been a limiting factor in the progression of research into AGD and required the maintenance of an on-going laboratory-based infection to supply infective material. Culture methods using malt yeast agar with sea water overlaid and subculturing every 3-4 days have resulted in the establishment of a clonal culture of N. perurans, designated clone 4. Identity of the amoeba was confirmed by PCR. After 70 days in culture clone 4 infected Atlantic salmon, causing AGD, and was re-isolated from the infected fish. Diagnosis was confirmed by histology and the infectious agent identified by PCR and in situ hybridisation using oligonucleotide primers and probes previously developed and specific to N. perurans. This study has fulfilled Koch's postulates for N. perurans as a causative agent of AGD and illustrates its free-living and parasitic nature.     

18S ribosomal DNA-based PCR identification of Neoparamoeba pemaquidensis, the agent of amoebic gill disease in sea-farmed salmonids

Neoparamoeba pemaquidensis is a parasomal amoeboid protozoan identified as the agent of amoebic gill disease (AGD) in Atlantic salmon Salmo salar reared in sea-pens in Tasmania, Australia, and coho salmon Oncorhynchus kisutch farmed on the west coast of the USA. Outbreaks of AGD caused by immunologically cross-reactive paramoebae have also been reported in sea-farmed salmonids in several other countries. Complete 18S rDNA sequences were determined for respective paramoebae isolated from infected gills of salmon from Tasmania and Ireland, and N. pemaquidensis isolates from the USA and UK, including representative free-living isolates. Alignments over 2110 bp revealed 98.1 to 99.0% sequence similarities among isolates, confirming that paramoebae implicated in AGD in geographically distant countries were homologous and belonged to the same species, N. pemaquidensis. The results supported previous findings that N. pemaquidensis exists as a widely distributed, amphizoic marine protozoan. Partial 18S rDNA sequences were obtained for the ultrastructurally similar species, N. aestuarina, and for the morphologically similar but non-parasomal amoeba Pseudoparamoeba pagei. N. aestuarina had 95.3 to 95.7% sequence similarities with N. pemaquidensis strains, which distinguished 2 closely related but separate species. Neoparamoeba spp. were not analogous to P. pagei or to other marine Gymnamoebia. We designed 4 oligonucleotide primers based on elucidated 18S rDNA sequences and applied them to single-step and nested 2-step PCR protocols developed to identify N. pemaquidensis to the exclusion of apparently closely related and non-related protistan taxa. Nested PCR was able to detect the AGD parasite from non-purified, culture-enriched net microfouling samples from Atlantic salmon sea-pens in Tasmania, and confirmed that N. pemaquidensis was also responsible for AGD in chinook salmon O. tshawytscha in New Zealand. Our sequence and PCR analyses have now shown that AGD affecting 3 different salmonid species farmed in 4 countries are associated with N. pemaquidensis. A species-specific diagnostic PCR provides for the first time, a highly specific detection and identification assay for N. pemaquidensis that will facilitate future ecological and epidemiological studies of AGD.     

Changes in antigenic profile during culture of Neoparamoeba sp., causative agent of amoebic gill disease in Atlantic salmon

Amoebic gill disease (AGD), the most serious infectious disease affecting farmed salmon in Tasmania, is caused by free-living marine amoeba Neoparamoeba sp. The parasites on the gills induce proliferation of epithelial cells initiating a hyperplastic response and reducing the surface area available for gaseous exchange. AGD can be induced in salmon by exposure to freshly isolated Neoparamoeba from AGD infected fish, however cultured Neoparamoeba are non-infective. We describe here antigenic differences between freshly isolated and in vitro cultured parasites, and within individual isolates of the parasite cultured under different conditions. Immunoblot analysis using polyclonal antisera, revealed differences in the antigen profiles of two cultured isolates of Neoparamoeba sp. when they were grown on agar versus in liquid medium. However, the antigen profiles of the two isolates were very similar when they were grown under the same culture conditions. Comparison of these antigen profiles with a preparation from parasites freshly isolated from infected gills revealed a very limited number of shared antigens. In addition monoclonal antibodies (mAbs) raised against surface antigens of cultured parasites were used in an indirect immunofluorescence assay to assess the expression of specific surface antigens of Neoparamoeba sp. after various periods in culture. Significant changes in antigen expression of freshly isolated parasites were observed after 15 days of in vitro culture. The use of mAb demonstrated progressive exposure/expression of individual antigens on the surface of the freshly isolated parasites during the period in culture.     

Searching for the sweet spot of amoebic gill disease of farmed Atlantic salmon: the potential role of glycan-lectin interactions in the adhesion of Neoparamoeba perurans

One of the first critical steps in the pathogenesis of amoebic gill disease (AGD) of farmed salmon is the adhesion of the causative amoeba to the host. The current study aimed to investigate the potential involvement of glycan-binding proteins expressed on the extracellular surface of Neoparamoeba perurans in gill tissue recognition and binding. The glycan-binding properties of the surface membrane of N. perurans and the carbohydrate binding profile of Atlantic salmon gill-derived epithelial cells were identified through the use of glycan and lectin microarrays, respectively. The occurrence of specific carbohydrate-mediated binding was then further assessed by in vitro attachment assays using microtitre plates pre-coated with the main glycan candidates. Adhesion assays were also performed in the presence of exogenous saccharides with the aim of blocking glycan-specific binding activity. Comparative analysis of the results from both lectin and glycan arrays showed significant overlap, as some glycans to which binding by the amoeba was seen were reflected as being present on the gill epithelial cells. The two main candidates proposed to be involved in amoeba attachment to the gills are mannobiose and N-acetylgalactosamine (GalNAc). Adhesion of amoebae significantly increased by 33.5 and 23% when cells were added to α1,3-Mannobiose-BSA and GalNAc-BSA coated plates. The observed increased in attachment was significantly reduced when the amoebae were incubated with exogenous glycans, further demonstrating the presence of mannobiose- and GalNAc-binding sites on the surfaces of the cells. We believe this study provides the first evidence for the presence of a highly specific carbohydrate recognition and binding system in N. perurans. These preliminary findings could be of extreme importance given that AGD is an external parasitic infestation and much of the current research on the development of alternative treatment strategies relies on either instant amoeba detachment or blocking parasite attachment.     

In vitro toxicity of bithionol and bithionol sulphoxide to Neoparamoeba spp., the causative agent of amoebic gill disease (AGD)

The objective of the present study was to evaluate the in vitro toxicity of bithionol and bithionol sulphoxide to Neoparamoeba spp., the causative agent of amoebic gill disease (AGD). The current treatment for AGD-affected Atlantic salmon involves bathing sea-caged fish in freshwater for a minimum of 3 h, a labour-intensive and costly exercise. Previous attempts to identify alternative treatments have suggested bithionol as an alternate therapeutic, but extensive in vitro efficacy testing has not yet been done. In vitro toxicity to Neoparamoeba spp. was examined using amoebae isolated from the gill of AGD-affected Atlantic salmon and exposing the parasites to freshwater, alumina (10 mg l(-1)), seawater, bithionol or bithionol sulphoxide at nominal concentrations of 0.1, 0.5, 1, 5 and 10 mg l(-1) in seawater. The numbers of viable amoebae were counted using the trypan blue exclusion method at 0, 24, 48 and 72 h. Both bithionol and bithionol sulphoxide demonstrated in vitro toxicity to Neoparamoeba spp. at all concentrations examined (0.1 to 10 mg l(-1) over 72 h), with a comparable toxicity to freshwater observed for both chemicals at concentrations > 5 mg l(-1) following a 72 h treatment. Freshwater remained the most effective treatment, with only 6% viable amoebae seen after 24 h and no viable amoebae observed after 48 h.     

Cultured gill-derived Neoparamoeba pemaquidensis fails to elicit amoebic gill disease (AGD) in Atlantic salmon Salmo salar

Amoebic gill disease (AGD) affects the culture of Atlantic salmon Salmo salar in the southeast of Tasmania. The disease is characterised by the presence of epizoic Neoparamoeba spp. in association with hyperplastic gill tissue. Gill-associated amoebae trophozoites were positively selected by plastic adherence for culture in seawater, where they proliferated using heat-killed E. coli as a nutrient source. One isolate of gill-harvested amoebae designated NP251002 was morphologically consistent to N. pemaquidensis under light, fluorescence and transmission electron microscopy. Rabbit anti-N. pemaquidensis antiserum bound to NP251002, and N. pemaquidensis small subunit (SSU) ribosomal DNA (18S rDNA) was detected in NP251002 genomic DNA preparations using PCR. A high degree of similarity in the alignment of the NP251002 18S rDNA PCR amplicon sequence with reference isolates of N. pemaquidensis suggested conspecificity. While short-term culture (72 h) of gill-harvested amoebae does not affect the capacity of amoebae to induce AGD, Atlantic salmon challenged with NP251002 after the trophozoites had been 34 and 98 d in culture exhibited neither gross nor histological evidence of AGD. It is not known if NP251002 were avirulent at the time of isolation, had down-regulated putative virulence factors or virulence was inhibited by the culture conditions. Therefore, the time in culture could be a limiting factor in maintaining virulence using the culture technique described here.     

Neoparamoeba pemaquidensis引起的海水鱼阿米巴鳃病

海水养殖的鲑鱼及鲽鱼(Scophthalmusmaximus)的阿米巴鳃病是由Neoparamoebapemaquidensis引起的。在西班牙,该病对鲑鱼的海水养殖造成巨大损失,同时也正威胁着鲽鱼的养殖。组织病理损伤主要是鱼鳃上皮细胞的增生和肥大。该虫仅寄生在鱼鳃表面。现已有证据证明,非特异免疫参与鱼类抵御该病,但还没有证明特异性免疫在此过程中发挥作用的相似证据。对鲑鱼来说,治疗该病惟一有效的方法就是用淡水浸泡     

Influence of salmonid gill bacteria on development and severity of amoebic gill disease

The relationship between salmonid gill bacteria and Neoparamoeba sp., the aetiological agent of amoebic gill disease (AGD) was determined in vivo. Fish were divided into 4 groups and were subjected to following experimental infections: Group 1, amoebae only; Group 2, Staphylococcus sp. and amoebae; Group 3, Winogradskyella sp. and amoebae; Group 4, no treatment (control). Fish (Groups 1, 2 and 3) were exposed to potassium permanganate to remove the natural gill microflora prior to either bacterial or amoebae exposure. AGD severity was quantified by histological analysis of gill sections to determine the percentage of lesioned filaments and the number of affected lamellae within each lesion. All amoebae infected groups developed AGD, with fish in Group 3 showing significantly more filaments with lesions than other groups. Typically lesion size averaged between 2 to 4 interlamellar units in all AGD infected groups. The results suggest that the ability of Neoparamoeba sp. to infect filaments and cause lesions might be enhanced in the presence of Winogradskyella sp. The possibility is proposed that the prevalence of more severe AGD is due to the occurrence of Winogradskyella sp. at high concentrations on the gills.     

Effects of graded hypoxia on Atlantic salmon infected with amoebic gill disease

Atlantic salmon Salmo salar with amoebic gill disease (AGD) were exposed to a graded hypoxia (135–40 mmHg water P O₂) and blood samples analysed for respiratory gases and pH at 119, 79·5 and 40 mmHg water P O₂. There were no differences in the rate of oxygen uptake between infected and control fish. However, arterial P O₂, and pH were significantly lower in the infected fish whereas P CO₂ was significantly higher in infected fish compared with controls prior to hypoxia and at 119 mmHg water P O₂. At 79·5 and 40 mmHg water P O₂ saturation, there were no significant differences in blood P O₂ or pH although blood P CO₂ was elevated in AGD affected fish at 50% hypoxia (79·5 mmHg water P O₂). The elevated levels of P CO₂ in fish affected by AGD resulted in a persistent respiratory acidosis even during hypoxic challenge. These data suggest that even though the fish were severely affected by AGD, the presence of AGD while impairing gas transfer under normoxic conditions, did not contribute to respiratory failure during hypoxia.     

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.     

Concentration effects of Winogradskyella sp. on the incidence and severity of amoebic gill disease

To study the concentration effects of the bacterium Winogradskyella sp. on amoebic gill disease (AGD), Atlantic salmon Salmo salar were pre-exposed to 2 different doses (10(8) or 10(10) cells 1(-1)) of Winogradskyella sp. before being challenged with Neoparamoeba spp. Exposure of fish to Winogradskyella sp. caused a significant increase in the percentage of AGD-affected filaments compared with controls challenged with Neoparamoeba only; however, these percentages did not increase significantly with an increase in bacterial concentration. The results show that the presence of Winogradskyella sp. on salmonid gills can increase the severity of AGD.     

Isolation and characterization of an aetiological agent in Whipple's disease.

A cell wall deficient form of an alpha-haemolytic streptococcus was grown from a prolonged monolayer cell culture of a lymph node taken from a patient with Whipple''s disease. Serological cross reactivity was shown between the organism and the material within Whipple''s disease macrophages positive for diastase-resistant periodic acid-Schiff (D./P.A.S.). In vitro studies characterized the organism as a facultative intracellular parasite which caused the accumulation within cells of D./P.A.S.-positive material. These results suggest that a pathogenic bacterium is the essential aetiological agent and that the culture of Whipple''s disease tissues in hypertonic media may have practical value.     

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.     

Acid–base and respiratory effects of confinement in Atlantic salmon affected with amoebic gill disease

Amoebic gill disease (AGD) in cultured salmonids causes severe multifocal hyperplastic lesions in the gills with the potential to influence respiratory and acid–base physiology. Atlantic salmon Salmo salar affected with AGD were surgically implanted with dorsal aortic catheters and, following recovery, were confined for 5 min ( n  = 16) or left undisturbed ( n  = 8). Confinement caused an acute extracellular acidosis that was corrected in 6 h amongst surviving fish. There was a gradual increase in plasma lactate concentrations that peaked at 1 h post-confinement then declined by 9 h recovery. In a second experiment, AGD-affected fish were confined then recovered either in a tank of static water ( n  = 9) or while being forced to swim at 1·5 body lengths s⁻¹ ( n  = 6). There was no significant difference between fish recovered by swimming and those in static water in terms of recovery of the acute extracellular acidosis and lactate accumulations coincident with exhaustive exercise. Confinement severely compromised the survival of AGD-affected Atlantic salmon, although survivors appeared to recover similarly to other studies. Forced swimming of AGD-affected Atlantic salmon following confinement did not facilitate recovery and is unlikely to be a useful strategy for mitigating the effects of stressful episodes such as crowding and fish movement and commercial handling.     

Effects of gill abrasion and experimental infection with Tenacibaculum maritimum on the respiratory physiology of Atlantic salmon Salmo salar affected by amoebic gill disease

The effects of gill abrasion and experimental infection with Tenacibaculum maritimum were assessed in Atlantic salmon Salmo salar with underlying amoebic gill disease. The respiratory and acid-base parameters arterial oxygen tension (P(a)O2), arterial whole blood oxygen content (C(a)O2), arterial pH (pHa), haematocrit and haemoglobin concentrations were measured at intervals over a 48 h recovery period following surgical cannulation of the dorsal aorta. Mortality rates over the recovery period were variable, with gill abrasion and inoculation with T. maritimum causing the highest initial mortality rate and unabraded, uninoculated controls showing the lowest overall mortality rate. Fish with abraded gills tended to show reduced P(a)O2 and lower C(a)O2 compared with unabraded fish. Infection with T. maritimum had no effect on P(a)O2 or C(a)O2. All fish showed an initial alkalosis at 24 h post-surgery/inoculation which was more pronounced in fish inoculated with T. maritimum. There were no significant effects of gill abrasion or infection upon the ratio of oxygen specifically bound to haemoglobin or mean cellular haemoglobin concentration. Histologically, 48 h following surgery, abraded gills showed multifocal hyperplastic lesions with pronounced branchial congestion and telangiectasis, and those inoculated with T. maritimum exhibited focal areas of branchial necrosis and erosion associated with filamentous bacterial mats. All fish examined showed signs of amoebic gill disease with multifocal hyperplastic and spongious lesions with parasome-containing amoeba associated with the gill epithelium. The results suggest that respiratory compromise occurred as a consequence of gill abrasion rather than infection with T. maritimum.     

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.     

Major histocompatibility polymorphism associated with resistance towards amoebic gill disease in Atlantic salmon (Salmo salar L.)

The association between major histocompatibility (MH) polymorphism and the severity of infection by amoebic gill disease (AGD) was investigated across 30 full sibling families of Atlantic salmon. Individuals were challenged with AGD for 19days and then their severity of infection scored by histopathological examination of the gills. Fish were then genotyped for the MH class I (Sasa-UBA) and MH class II alpha (Sasa-DAA) genes using polymorphic repeats embedded within the 3' untranslated regions of the Sasa-UBA and Sasa-DAA genes. High variation in the severity of infection was observed across the sample material, ranging from 0% to 85% gill filaments infected. In total, seven Sasa-DAA-3UTR and ten Sasa-UBA-3UTR marker alleles were identified across the 30 families. A significant association between the marker allele Sasa-DAA-3UTR 239 and a reduction in AGD severity was detected. There was also a significant association found between AGD severity and the presence of two Sasa-DAA-3UTR genotypes. While the associations between MH allele/genotypes and AGD severity reported herein may be statistically significant, the small sample sizes observed for some alleles and genotypes means these associations should be considered as suggestive and future research is required to verify their biological significance.     

The expression of immune-regulatory genes in rainbow trout, Oncorhynchus mykiss, during amoebic gill disease (AGD)

Amoebic gill disease (AGD) is an ectoparasitic disease caused by infection with the protozoan Neoparamoeba sp. and is characterised by epithelial hyperplasia that manifests as gill lesions. In order to examine the nature of the immune response to AGD, the expression of a range of immune-regulatory genes was examined in naïve uninfected rainbow trout, Oncorhynchus mykiss, and naïve rainbow trout subjected to a laboratory-induced AGD infection. The immune-regulatory genes examined were interleukin-1 beta isoform 1 (IL-1β1), tumour necrosis factor alpha isoforms 1 and 2 (TNF-α1, TNF-α2), interleukin-8 (IL-8), transforming growth factor beta isoform 1 (TGF-β1), inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), major histocompatibility complex beta chain (MHC-II β-chain) and T-cell receptor beta chain (TCR β-chain). Immune-regulatory genes that were up/down-regulated in AGD-infected trout compared to uninfected controls at 0, 7, and 14 days post-inoculation (p.i.) in gill, liver and anterior kidney tissue were initially identified by means of semi-quantitative RT–PCR. Up/down-regulated immune-regulatory genes were subsequently quantitated and validated by real-time RT–PCR (qRT–PCR). The extent of AGD-associated pathology was consistent amongst all AGD-infected trout at 7 days p.i. and increased considerably by 14 days p.i. At both 7 and 14 days p.i. IL-1β1 and iNOS gene expression was significantly up-regulated in the gills, and IL-8 was significantly up-regulated in the liver of AGD-infected trout at 7 days p.i. These data demonstrate the involvement of the immune response to AGD at the molecular level, and indicate the importance of this response at the site of infection and the possible involvement of a systemic immune response.     

Oral L-cysteine ethyl ester (LCEE) reduces amoebic gill disease (AGD) in Atlantic salmon Salmo salar

There is a need for the development of alternative therapeutic treatments for amoebic gill disease (AGD) in Atlantic salmon Salmo salar L. to maintain the sustainability of the Tasmanian Atlantic salmon aquaculture industry. This study aimed to assess the effects of the mucolytic drug L-cysteine ethyl ester (LCEE) on marine Atlantic salmon mucus and whether or not it may have a therapeutic advantage for the alleviation of AGD when administered orally. We also aimed to document any physiological consequences of LCEE. Results showed that LCEE significantly decreased the viscosity of marine Atlantic salmon mucus both in vitro, where LCEE concentration showed a negative relationship to mucus viscosity (R2 = 0.95 at 11.5 s(-1)), and in vivo. Oral administration of LCEE at 52.7 mg LCEE kg(-1) fish d(-1) over 2 wk significantly delayed the progression of AGD-associated pathology during an aggressive, cohabitation induced, laboratory infection. Medicated fish had approximately 50% less gill filaments affected by AGD than control fed fish at 3 d post-infection when assessed using histology. Palatability of medicated feed was shown to be approximately 65% of control feed. No osmoregulatory disturbance was seen in medicated fish, although blood and whole body flux data indicated a slight acidosis coinciding with an increased plasma total ammonia concentration. However, both variables were within a tolerable physiological range and returned to control levels 3 d post-cessation of medicated feed. LCEE holds potential as an in-feed additive when administered over 2 wk prior to infection to delay the progression of AGD associated pathology. From the parameters measured, LCEE seems to have minimal physiological consequences after 2 wk of administration.