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► Koch’s postulates have been fulfilled for Neoparamoeba perurans and amoebic gill disease. ► For the first known time the causative agent of amoebic gill disease in farmed Atlantic ...salmon was cultured in vitro. ► Neoparamoeba perurans retains the ability to cause amoebic gill disease after 70days in culture.
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.
The protozoan parasite
Neoparamoeba perurans is the causative agent of amoebic gill disease (AGD) and an emerging threat to the aquaculture of marine finfish species worldwide. Despite several years ...of research and continuing efforts the culture of
N. perurans remains elusive. As a result current detection methods rely on molecular techniques namely, polymerase chain reaction (PCR) and in situ hybridization (ISH). In this study, a total DNA extraction technique combined with a highly sensitive real-time PCR assay using primers specific for
N. perurans was developed and validated. Using this method we were able to detect a single 18S rRNA gene copy and readily detected
N. perurans with the lowest detection limit for
N. perurans cells spiked in sea water being one cell (100% detection rate). The genome of
N. perurans contains multiple copies of the 18S rRNA gene, and an estimate of 2880 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. The developed method was applied to seawater samples collected from both an experimental AGD infection tank and a variety of environmental sites including those used to culture Atlantic salmon (
Salmo salar L.) in Tasmania, Australia. Detectable populations were highly abundant from sites in and closely surrounding cage culture of Atlantic salmon. Furthermore, the method when applied to gill swabs from an on-farm gill pathology assessment demonstrated that non-destructive semi-quantitative analysis of amoebae loads from these fish was possible. Not only does this study provide evidence that
N. perurans is a free-living amoeba but the quantitative nature of this novel assay clearly demonstrates the impact of marine cage aquaculture on the prevalence of this fish pathogen and is a step towards establishing the distribution of
N. perurans in the marine environment and its relationship with AGD outbreaks.
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.
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.
We 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.
•Anti-protozoa properties of eugenol were not observed at concentrations commonly used.•Interpretation of previous in vitro work unlikely to be affected•Anaesthetic concentrations suitable for future work established
Chlamydial infections of fish are emerging as an important cause of disease in new and established aquaculture industries. To date, epitheliocystis, a skin and gill disease associated with infection ...by these obligate intracellular pathogens, has been described in over 90 fish species, including hosts from marine and fresh water environments. Aided by advances in molecular detection and typing, recent years have seen an explosion in the description of these epitheliocystis-related chlamydial pathogens of fish, significantly broadening our knowledge of the genetic diversity of the order Chlamydiales. Remarkably, in most cases, it seems that each new piscine host studied has revealed the presence of a phylogenetically unique and novel chlamydial pathogen, providing researchers with a fascinating opportunity to understand the origin, evolution and adaptation of their traditional terrestrial chlamydial relatives. Despite the advances in this area, much still needs to be learnt about the epidemiology of chlamydial infections in fish if these pathogens are to be controlled in farmed environments. The lack of in vitro methods for culturing of chlamydial pathogens of fish is a major hindrance to this field. This review provides an update on our current knowledge of the taxonomy and diversity of chlamydial pathogens of fish, discusses the impact of these infections on the health, and highlights further areas of research required to understand the biology and epidemiology of this important emerging group of fish pathogens of aquaculture species.
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-1β, 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.
•2D quantitative RT-PCR was used to map the transcriptional responses in AGD-affected gills of Atlantic salmon.•Neoparamoeba perurans elicits a classical inflammatory response in the gills of AGD-affected A. salmon.•Expression of immune genes within gill lesions misrepresents the cellular immune response in the gills during AGD.•AGD elicited an increased expression of cellular markers, most notably antigen presenting cells, B-cells and T-cells.•T-cells within the AGD-affected gills are mainly constituted of CD8+ cells and not CD4+ T-cells.
Currently, the only effective and commercially used treatment for amoebic gill disease (AGD) in farmed Tasmanian Atlantic salmon is freshwater bathing. Hydrogen peroxide (H2O2), commonly used ...throughout the aquaculture industry for a range of topical skin and gill infections, was trialled in vitro and in vivo to ascertain its potential as an alternative treatment against AGD. Under in vitro conditions, trophozoites of Neoparamoeba perurans were exposed to three concentrations of H2O2 in sea water (500, 1000 and 1500 mg L−1) over four durations (10, 20, 30 and 60 min) each at two temperatures (12 and 18 °C). Trophozoite viability was assessed immediately post‐exposure and after 24 h. A concentration/duration combination of 1000 mg L−1 for >10 min demonstrated potent amoebicidal activity. Subsequently, Atlantic salmon mildly affected with experimentally induced AGD were treated with H2O2 at 12 and 18 °C for 15 min at 1250 mg L−1 and their re‐infection rate was compared to freshwater‐treated fish over 21 days. Significant differences in the percentage of filaments affected with hyperplastic lesions (in association with amoebae) and plasma osmolality were noted between treatment groups immediately post‐bath. However, the results were largely equivocal in terms of disease resolution over a 3‐week period following treatment. These data suggest that H2O2 treatment in sea water successfully ameliorated a clinically light case of AGD under laboratory conditions.
A weekly monitoring project was designed to improve our understanding of the dynamics of southern bluefin tuna health, immune response, and performance during the first two months of ranching. In ...addition, results were compared to the data for wild fish to highlight any effects of captivity. Weight, length, condition index, hemoglobin concentration, and immune response were all found to change significantly over this period. SBT were found to be relatively healthy prior to a mortality event which resulted in a cumulative mortality of 8.5%. The mortality event was associated with decreased hemoglobin concentrations and changes in immune response.
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