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Abstract

Neoparamoeba spp. is a marine amphizoic protozoan parasite which infects the
gills of marine cultured Atlantic salmon, Salmo salar, worldwide causing amoebic
gill disease (AGD). Amoebic gill disease is a significant health issue affecting the
production of sea-caged Atlantic salmon in Australia with farms experiencing
outbreaks regularly throughout the year. It accounts for 10-20% of the gross value
of production due not only to the cost of treating and managing the disease, but also
to loss of fish condition, increased feed conversion ratio (FCR), lost growth and
sometimes mortalities. The current mitigation strategy for AGD is the
administration of a freshwater bath to affected sea-caged fish. However, this
method is becoming less effective with an apparent increase in bathing frequency
over the past few years. The increase in baths has fuelled a rise in already high
production costs to the Australian Atlantic salmon industry to approximately 20%
annually. This thesis aims to identify an improved method of treatment for AGD
either through development of a stand alone in-feed treatment or an in-feed
treatment used in conjunction with the current freshwater treatment strategy.
This thesis investigates in vitro and in vivo effects of bithionol and bithionol
sulphoxide on both Neoparamoeba spp. and Atlantic salmon. Initially, toxicity to
Neoparamoeba spp. was examined in vitro using isolated gill amoeba and exposing
them to seawater, freshwater, alumina (10 mg L-1), bithionol and bithionol
sulphoxide at 10, 5, 1, 0.5 and 0.1 mg L. The assays were observed for 72 h with
viable amoeba counts using trypan blue exclusion conducted at 0, 24, 48 and 72 h.
Both bithionol and bithionol sulphoxide were toxic to Neoparamoeba spp. in vitro at all concentrations examined. A similar toxicity to freshwater water was observed with bithionol and bithionol sulphoxide at 10 and 5 mg L-1 following a 72 h
treatment. However, freshwater was the most effective with only 6% viable
amoebae seen after 24 h and no viable amoeba observed a further 24 h later.
Once identified as toxic to Neoparamoeba spp. in vitro, an assessment of the
toxicity of bithionol to Atlantic salmon and the efficacy as an AGD treatment was
evaluated. This was conducted via a bath treatment to Atlantic salmon and rainbow
trout, Oncorhynchus mykiss, held in either fresh or seawater using concentrations
between 1 and 35 mg L-1 to examine toxicity. To examine efficacy, a bath treatment
of AGD-affected Atlantic salmon and rainbow trout at 1 to 25 mg L -1 was also
evaluated. To examine toxicity, fish were bathed for 1, 3 and 6 h in bithionol, an
anti-protozoal at 0, 1, 5, 10, 25 and 35 mg L -1 , with toxicity determined by time to
morbidity and histological examination of internal organs. Efficacy was examined
by bathing AGD-affected Atlantic salmon and rainbow trout for I h at bithionol
concentrations of 1 to 25 mg L -1 . Efficacy was determined by examining gill
amoeba counts and identifying percent lesioned gill filaments at I and 24 h after
bath exposure to bithionol. Only bithionol at 1 mg L-1 was considered non-toxic
with no signs of morbidity. Bithionol appeared to be more toxic in seawater than
freshwater, exhibiting a higher rate of morbidity, and had no acute effects on gill
Na+/K+ ATPase and succinic dehydrogenase, or plasma osmolality and chloride
concentration. Bithionol reduced the percentage of lesioned gill filaments to the
same level as freshwater.
Bithionol was examined as an in-feed treatment for AGD with and without the
administration of a freshwater bath. Bithionol when fed as a two week prophylactic
or therapeutic treatment at 25 mg kg' feed delayed the onset of AGD pathology and
reduced the percent lesioned gill filaments. Administration of a 3 h freshwater bath
at 28 days post-exposure significantly reduced amoebae numbers to a similar level
across all treatments; in contrast gross gill score and percent lesioned filaments
were reduced proportionally. Hence, the control was significantly higher than both
bithionol treatments. Following the freshwater bath, clinical signs of AGD recurred
at a similar level across all treatments although controls clinical signs were
significantly higher than the bithionol treatments to begin with. Palatability was not
a problem with mean feed intake of bithionol over the trial duration higher
compared to both the oil and plain controls.
This thesis has identified that bithionol at 25 mg kg-I feed, when fed as a two
week prophylactic or a therapeutic treatment, delayed and reduced the intensity of
AGD pathology. Such findings as the identification of bithionol as a possible infeed
treatment for AGD and its effectiveness against numerous other parasites
suggests that bithionol could be worth examining in other aquatic animal diseases.
Furthermore, bithionol warrants further investigation as a potential in-feed
treatment for AGD in Atlantic salmon especially in regards to a combination
therapy with the current freshwater mitigation.

Item Type:	Thesis - PhD
Authors/Creators:	Louwen-Skovdam, RL
Keywords:	Atlantic salmon, Fishes, Atlantic salmon fisheries, Gills, Salmon farming
Copyright Holders:	The Author
Copyright Information:	Copyright 2008 the author - The University is continuing to endeavour to trace the copyright owner(s) and in the meantime this item has been reproduced here in good faith. We would be pleased to hear from the copyright owner(s).
Additional Information:	Chapter 2 appears to be the equivalent of a post print article published as: Florent, R.L., Becker, J.A., Powell, M. D., 2010, In vitro efficacy of bithionol and bithionol sulphoxide to Neoparamoeba spp., the causative agent of amoebic gill disease (AGD), Diseases of aquatic organisms, 91(3), 257-262 Chapter 3 appears to be the equivalent of a post print article published as: Florent, R.L., Becker, J.A., Powell, M. D., 2007, Evaluation of bithionol as a bath treatment for amoebic gill disease caused by Neoparamoeba spp., Veterinary parasitology, 144(3-4), 197-207. Chapter 4 appears to be the equivalent of a post print article published as: Florent, R.L., Becker, J.A., Powell, M. D., 2007, Efficacy of bithionol as an oral treatment for amoebic gill disease in Atlantic salmon, Salmo salar, Aquaculture, 270(1-4), 15-22. Chapter 4 appears to be the equivalent of the peer reviewed version of the following article: Florent, R.L., Becker, J.A., Powell, M. D., 2009, Further development of bithionol therapy as a treatment for AGD, Journal of fish diseases, 32(5), 391-400, which has been published in final form at 0.1111/j.1365-2761.2008.01001.x This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
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