Causative Agent Of Amoebic Gill Disease Research Articles

Regulation of the molecular repertoires of oxidative stress response in the gills and olfactory organ of Atlantic salmon following infection and treatment of the parasite Neoparameoba perurans

The present study investigated the involvement of key molecular regulators of oxidative stress in amoebic gill disease (AGD), a parasitic infestation in Atlantic salmon. In addition, the study evaluated how these molecular biomarkers responded when AGD-affected fish were exposed to a candidate chemotherapeutic peracetic acid (PAA). Atlantic salmon were experimentally infected with the parasite Neoparameoba perurans, the causative agent of AGD, by bath exposure and after 2 weeks, the fish were treated with three commercial PAA products (i.e., Perfectoxid, AquaDes and ADDIAqua) at a dose of 5 ppm. Two exposure durations were evaluated – 30 min and 60 min. Sampling was performed 24 h and 2 weeks after PAA treatment (equivalent to 2- and 4-weeks post infection). At each sampling point, the following parameters were evaluated: gross gill pathology, gill parasitic load, plasma reactive oxygen species (ROS) and total antioxidant capacity (TAC), histopathology and gene expression profiling of genes with key involvement in oxidative stress in the gills and olfactory organ. AGD did not result in systemic oxidative stress as ROS and TAC levels remained unchanged. There were no clear patterns of AGD-mediated regulation of the oxidative stress biomarkers in both the gills and olfactory organ; significant changes in the expression were mostly related to time rather than infection status. However, the expression profiles of the oxidative stress biomarkers in AGD-affected salmon, following treatment with PAA, revealed that gills and olfactory organ responded differently – upregulation was prominent in the gills while downregulation was more frequent in the olfactory organ. The expression of catalase, glutathione S-transferase and thioredoxin reductase 2 was significantly affected by the treatments, both in the gills and olfactory organ, and these alterations were influenced by the duration of exposure and PAA product type. Parasitic load in the gills did significantly increase after treatment regardless of the product and exposure duration; the parasite was undetectable in some fish treated with AquaDes for 30 mins. However, PAA treated groups for 30 min showed lower macroscopic gill scores than the infected-untreated fish. Histology disclosed the classic pathological findings such as multifocal hyperplasia and increased number of mucous cells in AGD-affected fish. Microscopic scoring of gill injuries showed that AGD-infected-PAA-treated fish had lower scores, however, an overall trend could not be established. The morphology and structural integrity of the olfactory organ were not significantly altered by parasitism or PAA treatment. Collectively, the results indicate that AGD did not affect the systemic and mucosal oxidative status of Atlantic salmon. However, such a striking profile was changed when AGD-affected fish were exposed to oxidative chemotherapeutics. Moreover, the gills and olfactory organ demonstrated distinct patterns of gene expression of oxidative stress biomarkers in AGD-infected-PAA-treated fish. Lastly, PAA treatment did not fully resolve the infection, but appeared not to worsen the mucosal health either.

Parasitic isopods Ceratothoa banksii (Leach, 1818) and Nerocila orbignyi (Guérin-Méneville, 1832) of farmed Atlantic salmon and their potential as vectors of Neoparamoeba perurans (Young et al. 2007) in Tasmania

Reiterative freshwater bathing is the main treatment to control amoebic gill disease (AGD) of farmed Atlantic salmon in Tasmania, Australia. Regular freshwater exposure appears to control ectoparasitic cymothoid isopods, which were only seen at high prevalence and intensity in summer when fish had not been treated for over 100 days. With the potential advent of non-freshwater AGD treatments or increased periods between freshwater bathing due to selective breeding for AGD resistance, it is possible that cymothoid parasitism will become an increasing threat on Tasmanian salmon farms. In order to establish whether isopods could be vectors of Neoparamoeba perurans Young et al. 2007 (the causative agent of AGD), gill isopods were collected from salmon that had not been bathed for seven months and carried a 95% prevalence of isopods, including Ceratothoa banksii (Leach, 1818) and Nerocila orbignyi (Guérin-Méneville, 1832). PCR analyses of gill swabs indicated that 82% of salmon were positive for N. perurans while 41% of the sampled isopods were positive for N. perurans on external surfaces. When internal material was analysed, only 9% of the isopods were positive for the amoeba, but in very low concentration. Quantitative analysis showed no correlation between the concentrations of N. perurans from gill swabs and the isopods from the same individual fish. Thus, it is unlikely that these isopods act as a significant reservoir or vector for N. perurans.

Effects of anaesthetics containing eugenol on Neoparamoeba perurans

Oil extracted from cloves, Syzygium aromaticum, consists mainly of eugenol, which has been documented to affect protozoa attachment, viability and growth. Isoeugenol (sold under the trade name AQUI-S®) is a commercially available fish anaesthetic produced by isomerisation of eugenol. Both are anaesthetics commonly employed during the husbandry and euthanasia of Atlantic salmon, Salmo salar L., used for amoebic gill disease (AGD) research. Neoparamoeba perurans (the causative agent of AGD) trophozoites were exposed to clove oil at 10, 20, 40 and 80μLL−1 for 10min and 5, 10, 20 and 40μLL−1 for 120min; AQUI-S® at 5, 10, 20 and 40μLL−1 for 10min and 2.5, 5, 10 and 20μLL−1 for 120min There were no significant differences in viability and survival of trophozoites after exposure to the anaesthetics. When trophozoites were exposed to clove oil at 80μLL−1 for 10min there was significantly (P<0.05) more detachment from a plastic surface compared to the control. Continued use of clove oil and AQUI-S® at 40μLL−1 or less for up to 120min is unlikely to have a detrimental impact on amoebae that are isolated and collected from salmon with AGD to be used for downstream research such as projects involving disease challenges and in vitro screening of anti-N. perurans compounds. Statement of relevanceWe believe that the study has relevance because it establishes that there were no anti-amoeba properties of eugenol observed at concentrations commonly used and therefore interpretation of previous in vitro and in vivo work unlikely to be affected as will future work.

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.

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 3–4days have resulted in the establishment of a clonal culture of N. perurans, designated clone 4. Identity of the amoeba was confirmed by PCR. After 70days 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.

Quantitation of immune response gene expression and cellular localisation of interleukin-1β mRNA in Atlantic salmon, Salmo salar L., affected by amoebic gill disease (AGD)

The characterisation of selected immune response genes during amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L., was performed using semi-quantitative RT-PCR, quantitative real-time RT-PCR (qRT-PCR), and in situ hybridisation (ISH). The immune response genes of interest were interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), serum amyloid A (SAA), and serum amyloid P-like pentraxin (SAP). Atlantic salmon were inoculated with the ectoparasite Neoparamoeba sp., the causative agent of AGD, and gill, liver and anterior kidney tissue sampled at 0, 7 and 14 d post-inoculation (p.i.). Semi-quantitative RT-PCR was performed on the tissue samples to identify up/down-regulated mRNA expression relative to uninfected control fish and normalised to the housekeeping gene, β-actin. Interleukin-1β (IL-1β) was the only immune response gene of those investigated whose mRNA was differentially regulated in any of the tissues and was found to be up-regulated in the gills by semi-quantitative RT-PCR. Increased gill IL-1β mRNA expression was then accurately quantitated and confirmed using probe-based qRT-PCR. The cellular localisation of the IL-1β mRNA expression in the gills of uninfected and infected fish was then determined by ISH using an IL-1β-specific biotinylated cRNA probe. Expression of IL-1β mRNA was localised to filament and lamellar epithelium pavement cells in gills of uninfected and infected Atlantic salmon. These data implicate the involvement of IL-1β at the site of infection, the gills, of Atlantic salmon during AGD. This work supports previous studies that suggest IL-1β is important in the regulation of the fish immune response to parasitic infection but additionally shows the cellular localisation of fish IL-1β mRNA expression during infection.

Efficacy and toxicity of oxidative disinfectants for the removal of gill amoebae from the gills of amoebic gill disease affected Atlantic salmon (Salmo salar L.) in freshwater

Amoebic gill disease (AGD) of Atlantic salmon is treated commercially by bathing aiected ¢sh in freshwater. Recently, the e⁄cacy of freshwater bathing has been questioned, and the aim of this study was to examine the potential for improving bathing e⁄cacy using additives to the freshwater bath. AGDaiected Atlantic salmon were bathed in 350 L tanks containing oxygenated freshwater to which chlorine dioxide (0^50 mg L � 1 ), chloramine-T (0^50 mg L � 1 ) or hydrogen peroxide (0^100m LL � 1 ) was added. Before and following a 3-h exposure to the freshwater and chemical additive, the gills were removed from a sub-sample of ¢sh and the number of live amoebae on the gills were counted and smears made for con¢rmation of the presence of Neoparamoeba pemaquidensis, the causative agent of AGD. Following a further 3h exposure, a sub-sample of ¢sh was bled from the caudal vein and the gills were removed for histological examination. Chlorine dioxide and chloramine-T at 25^50 and 10^50 mg L � 1 , respectively, reduced the number of amoebae on the gills by approximately 50% compared with pre-exposure numbers. The results from hydrogen peroxide treatment were equivocal and the toxicity of hydrogen peroxide was high. The toxicity of chlorine dioxide varied with freshwater hardness and/or suspended solid load, whereas chloramine-T toxicity was low, with mortalities attributable only to elevated temperatures at the highest concentration tested. In conclusion, chlorine dioxide and chloramine-T show promise as potential freshwater additives for the improved removal of N. pemaquidensis and possibly, other amoebae from the gills of commercially farmed Atlantic salmon.

Production of salmonid amoebic gill disease by exposure to Paramoeba sp. harvested from the gills of infected fish

Atlantic salmon, Salmo salar L., were exposed to different concentrations of Paramoeba sp. The lowest concentration which induced amoebic gill disease (AGD) was 230 Paramoeba sp. L–1 and the severity of infection increased with increasing concentration. The concentration of Paramoeba sp. positively correlated with the number of gill lesions (R2 &gt; 0.7). This study provides evidence that Paramoeba sp. is the causative agent of AGD and describes an experimental model that enables the severity of the induced disease to be controlled.