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

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 (R 2 = 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 AGDassociated 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 postinfection 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. KEY WORDS: Fish gill disease · Salmonid · In-feed treatment · Oral therapeutant · Mucolytic drug · Mucus viscosity · Physiological effects Resale or republication not permitted without written consent of the publisherDis Aquat Org 66: [21][22][23][24][25][26][27][28] 2005 Roberts & Powell 2003a). It has been previously suggested that excessive branchial mucus impairs CO 2 excretion, leading to respiratory acidosis and subsequent death in AGD affected fish (Powell et al. 2000). The break-up and removal of mucus from the gills seems to be an important function of freshwater bathing as a treatment . Indeed, freshwater bathing has been shown to significantly decrease mucus viscosity of marine Atlantic salmon (Roberts 2004). Thinner and more removable mucus is thought to aid the significant lesion fragmentation and shedding of AGD lesion-associated hyperplastic gill tissue, as well as sloughing of gill-associated amoebae in soft, freshwater-bathed fish (Roberts & Powell 2003b).Therapeutic mucolytic drugs have been extensively used for the alleviation of respiratory diseases associated with excessive production and accumulation of mucus in humans and domestic animals. N-acetylcysteine (NAC) is a widely used mucolytic agent that by virtue of its free sulfhydryl group reacts w...

Section: Methodsmentioning

confidence: 99%

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

Roberts

Powell²

2005

“…Neoparamoeba sp. was identified among populations of amoebae based on the existence of a parasome, an endosymbiont (Dyková et al 2003), reactivity with a polyclonal antibody visualised by an immunofluorescent antibody test (Howard & Carson 1993) and detection, via polymerase chain reaction (PCR), of small subunit (SSU) rDNA originally thought to be specific to N. pemaquidensis (Wong et al 2004) but subsequently also found capable of detecting N. branchiphila. Sites were designated positive if one or both duplicate sediment samples revealed the pres- Site reduction/oxidation (redox) potential and sulphide assessment.…”

Section: Methodsmentioning

confidence: 99%

Distribution of Neoparamoeba sp. in sediments around marine finfish farming sites in Tasmania

Crosbie

MacLeod²,

Forbes³

et al. 2005

Marine sediment samples collected from various sites at 2 Atlantic salmon farms in Tasmania were analysed for the presence of Neoparamoeba sp., an amoeba associated with amoebic gill disease (AGD) in farmed Atlantic salmon. Environmental variables of the sediment layer at each site, including redox potential and sulphide concentration, were measured and the general biological condition assessed by video observation. Sediments and environmental data were collected on 4 occasions at each site over a 12 mo period. Neoparamoeba sp. was detected in populations of amoebae recovered by culture from all sites and in 50% of all sediment samples taken. There was evidence of a seasonal influence on the presence of the amoeba, but this was different at each farm. No Neoparamoeba sp. was recovered from any sites at Farm 1 during the winter of 2002 whereas at Farm 2 this was the case for the summer of 2003. There appeared to be no relationship between the presence of Neoparamoeba sp., salmon farming activities and environmental parameters. KEY WORDS: Neoparamoeba · Amoebic gill disease · AGD · Marine sediments · Environmental parametersResale or republication not permitted without written consent of the publisher

“…Many authors assumed that Neoparamoeba pemaquidensis was the sole aetiological agent of AGD of Atlantic salmon (Douglas-Helders et al 2000, 2001a,b, 2002, 2003a,b,c, 2005, Adams & Nowak 2001, Munday et al 2001, Tan et al 2002, Clark et al 2003, Powell & Clark 2003, Bowman & Nowak 2004, Wong et al 2004, Zilberg 2005. However, the isolation of N. branchiphila from the gills of AGD-affected fish (Dyková et al 2005) suggested that AGD may be a condition of mixed aetiology.…”

Section: Introductionmentioning

confidence: 99%

Neoparamoeba perurans is a cosmopolitan aetiological agent of amoebic gill disease

Young¹,

Dyková²,

Snekvik³

et al. 2008

Previously we described a new member of the Neoparamoeba genus, N. perurans, and showed that it is an agent of amoebic gill disease (AGD) of Atlantic salmon Salmo salar cultured in southeast Tasmania, Australia. Given the broad distribution of cases of AGD, we were interested in extending our studies to epizootics in farmed fish from other sites around the world. Oligonucleotide probes that hybridise with the 18S rRNA of N. perurans, N. branchiphila or N. pemaquidensis were used to examine archival samples of AGD in Tasmania as well as samples obtained from 4 host fish species cultured across 6 countries. In archival samples, N. perurans was the only detectable amoeba, confirming that it has been the predominant aetiological agent of AGD in Tasmania since epizootics were first reported. N. perurans was also the exclusive agent of AGD in 4 host species across 6 countries. Together, these data show that N. perurans is a cosmopolitan agent of AGD and, therefore, of significance to the global mariculture industry. KEY WORDS: Amoebic gill disease · Neoparamoeba perurans · In situ hybridisation · Aquaculture Resale or republication not permitted without written consent of the publisherDis Aquat Org 78: [217][218][219][220][221][222][223] 2008 cases of AGD reported elsewhere, it is not known what role N. perurans, N. pemaquidensis and/or N. branchiphila play. Therefore, our objective was to use species-specific molecular probes to determine the aetiological agent or agents of AGD in 4 host species in 6 countries. MATERIALS AND METHODSParaffin-embedded gill tissues were obtained from 4 fish species predominantly during or following epizootics at commercial fish farming operations in 6 countries (Table 1). An epizootic was not reported from Chinook salmon Oncorhynchus tshawytscha cultured in New Zealand; however, the smallest fish (runts) within the healthy population were observed to have gill lesions that corresponded with AGD and these fish were used in this study. Gill tissues were sectioned (3 to 7 μm), stained with haematoxylin and eosin (H&E) and examined with light microscopy. Alternatively, sections (7 μm) of gill tissues were placed onto Polysine glass slides (Menzel-Gläser) and dried overnight at 37°C. Sections were hybridised with a digoxigenin (DIG)-labelled 'universal' 18S rRNA oligonucleotide probe to verify the integrity of rRNA as previously described (Young et al. 2007). All gill tissues with suitable host and amoeba rRNA were serially sectioned, placed onto Polysine glass slides and incubated with Neoparamoeba perurans, N. branchiphila and N. pemaquidensis DIG-labelled oligonucleotide probes as previously described (Young et al. 2007). Positive and negative (no probe) controls were run in parallel with each in situ hybridisation experiment by hybridising each probe with a section containing representative strains of each Neoparamoeba species termed an 'amoebae array' as previously described (Young et al. 2007). Tissue sections were incubated for up to 1 h with in a premixed solution of 5-b...