Journal of Fish Diseases 2010, 33, 455–458 doi:10.1111/j.1365-2761.2009.01137.
Short Communication
Amoebic gill disease in hatchery-reared ayu, Plecoglossus
altivelis (Temminck & Schlegel), in Japan is caused by
Neoparamoeba perurans
P B B Crosbie1, K Ogawa2, D Nakano3 and B F Nowak1
1 National Centre for Marine Conservation and Resource Sustainability, Australian Maritime College, University of
Tasmania, Launceston, Tasmania, Australia
2 Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan
3 Fukui Prefectural Fisheries Experimental Station, Urasoko, Tsuruga, Fukui Prefecture, Japan
Keywords: amoebic gill disease, hatchery-reared
ayu, in situ hybridisation, Japan, Neoparamoeba.
Amoebic gill disease (AGD) is a potentially fatal
infection in fish farmed in marine environments
and has been reported throughout the world in
numerous species, including salmonids (Kent,
Sawyer & Hedrick 1988; Munday, Foster, Roubal
& Lester 1990) and turbot (Dyková, Figueras &
Novoa 1995). Gill pathology, including epithelial
hyperplasia resulting in lamellar fusion, is usually
initiated by colonisation and proliferation of a
marine amoeba harbouring an endosymbiont, on
the gills of susceptible fish (Adams & Nowak 2003,
2004). Neoparamoeba perurans has been shown to
be the AGD-causing agent in Atlantic salmon from
Australia, the USA, Scotland and Ireland; rainbow
trout from Tasmania; chinook salmon from New
Zealand, and turbot from Spain (Young, Dyková,
Snekvik, Nowak & Morrison 2008a). The first
reported outbreak of AGD in farmed Atlantic
salmon in Norway was attributed to N. perurans
infection based on sequence analyses of 18S cDNA
derived from AGD-affected gill tissue (Steinum,
Correspondence B Nowak, Aquafin CRC, National Centre for
Marine Conservation and Resource Sustainability, University of
Tasmania, Locked Bag 1370, Launceston 7250, Tasmania,
Australia
(e-mail: bnowak@utas.edu.au)
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Blackwell Publishing Ltd 455
Kvellestad, Rønneberg, Nilsen, Asheim, Fjell,
Nygård, Olsen & Dale 2008).
Therefore, when mortalities were seen in sea
water-reared juvenile ayu, Plecoglossus altivelis
(Temminck & Schlegel), which displayed gross gill
pathology typical of AGD, we applied the available
molecular tools to determine whether N. perurans
caused this infection. This article reports on that
investigation and is the first reported incidence of
AGD in tank-reared ayu.
Ayu is an important commercial and recreational
species in Japan. It is a diadromous fish, which
naturally spawns in downstream stretches of rivers
in autumn and larvae enter the sea where they stay
until spring (Masuda, Amaoka, Araga, Uyeno &
Yoshino 1984). Young ayu swim upstream and
grow in the middle stretches of rivers. The life span
of ayu is 1 year. Ayu is farmed for stocking local
rivers and the rearing includes both freshwater and
marine phases. Thus, spawners are reared in fresh
water, where fertilised eggs hatch. Larvae spend
their early days in sea water and then are reared in
fresh water for the final stage of seed production.
A total of 1.89 million eggs of ayu were collected
from spawners and fertilised at the Fukui Prefec-
tural Inland Fisheries Centre, Fukui City, in
October 2007. Eyed eggs were transported to the
Fukui Prefectural Centre for Fish Stock Enhance-
ment, Obama City. Hatched larvae were fed rotifers
and subsequently formulated feed. In February
2008, ayu juveniles were transported to facilities
 Journal of Fish Diseases 2010, 33, 455–458
adjacent to the Mihama Fisheries Cooperative,
Mihama City for further growth. These fish were
maintained in circular tanks (6 or 7 m diameter; 23
or 30 tons in volume) using coastal and under-
ground sea water. Water temperature was kept at ca.
14 °C by mixing sea water from the two sources.
Fish were monitored daily for any mortality or
abnormality. Mortality was first noticed in early
April 2008. Some fish were inactive and unrespon-
sive to stimuli. Mortality had increased by 20 April.
Dead fish were removed from the tanks every
morning, and many newly dead fish were collected
in the evening. On 28 April, moribund fish
(76.9 mm in average total length and 2.4 g in
average body weight; n = 150) were sampled by a
hand net for disease inspection. Numerous amoeba-
like organisms were found on the hyperplastic gill
tissues in the wet mount preparations. Subse-
quently, survivors were transported to freshwater
facilities, and thereafter the mortality ceased and
amoebae disappeared from the gills of young ayu.
Cumulative mortality was estimated to be 49.4%.
During the episode, two recently dead fish
(approximately 10 g) were removed from the tank
and fixed in 70% ethanol. Gills were dissected from
a third dead fish and fixed in seawater DavidsonÕs
fixative for 24 h, then a single gill hemibranch was
processed for routine histology. This sample was
sequentially dehydrated in an ethanol series, cleared
with xylol and embedded in paraffin.
Five-micron paraffin sections were cut from the
fixed gill hemibranch and stained with haematoxy-
lin and eosin (H & E) for examination by light
microscopy. Additional 7-lm sections were cut and
placed onto Polysine slides (Menzel-Gläser) then
probed with digoxigenin (DIG)-labelled species-
specific oligonucleotides that hybridise to rRNA
within amoebae according to an in situ hybridisa-
tion (ISH) protocol of Young, Crosbie, Adams,
Nowak & Morrison (2007). All slides, including a
Neoparamoeba array (with representative Neopar-
amoeba strains that had been set in agarose and
processed for histology as described for gill samples;
Young et al. 2007), were probed with oligonucle-
otides specific for either N. pemaquidensis or
N. perurans or with the hybridisation buffer only
(i.e. oligonucleotides omitted). Hybridisations were
visualised after 1.5-h incubation in a premixed
BCIP/NBT solution (Sigma–Aldrich) and light
microscopy.
Gill tissue was removed from whole ethanol-fixed
fish, macerated with a scalpel and then total DNA
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P B B Crosbie et al. Amoebic gill disease in hatchery-reared ayu
extracted from 0.1 g portions using a DNeasy
Tissue Kit (Qiagen) by following the manufac-
turerÕs instructions. Polymerase chain reactions
(PCRs) were performed using extracted DNA as
template (approximately 10 ng) and N. perurans-
specific oligonucleotide primers and under condi-
tions detailed by Young, Dyková, Nowak &
Morrison (2008b). PCR products were electropho-
resed through a 2% agarose gel and visualised by
staining with ethidium bromide. Each reaction
included controls of DNA from N. perurans and
reaction mixture with no template.
Microscopic examination and application of
molecular tools confirmed that the gill pathology
seen in the ayu was caused by N. perurans.
Oligonucleotide primers specific for this species
(Young et al. 2007) amplified the 18S rRNA gene
from extracted genomic DNA from AGD-affected
gill tissue (Fig. 1). Histological examination
revealed extensive hyperplastic epithelium and
lamellar fusion typical of AGD pathology and
amoebae containing the symbiont were evident in
close association with these lesions (Fig. 2).
Observation of a symbiont adjacent to the nucleus
within amoebae is important as these endos-
ymbionts are characteristic of only three genera of
amoebae, two of which, Paramoeba and Neopar-
amoeba, include species which are pathogenic to
marine animals (Dyková, Fiala, Lom & Lukeš
2003). These histological observations are consis-
tent with those previously reported for AGD in
salmonids (Roubal, Lester & Foster 1989; Mun-
day et al. 1990; Adams & Nowak 2003) and
turbot (Dyková et al. 1995; Dyková, Figueras,
Novoa & Casal 1998).
In situ hybridisation probes, used on tissue
samples in paraffin sections, confirmed the identity
of the amoebae associated with the lesions as
N. perurans. The amoeba cells probed with
N. perurans-specific oligonucleotides were strongly
Genomic DNA from ayu Controls
NP NT
700 bp
600 bp
Figure 1 Polymerase chain reaction amplification of the 18s
rRNA gene using Neoparamoeba perurans-specific oligonucleo-
tide primers applied to templates of genomic DNA extracted
from gill tissue of ayu displaying typical amoebic gill disease
pathology. Control reactions were templates with genomic DNA
from N. perurans (NP) and reaction mix with no template (NT).
 Journal of Fish Diseases 2010, 33, 455–458 P B B Crosbie et al. Amoebic gill disease in hatchery-reared ayu

(a) (b)

(c)

Figure 2 Haematoxylin and eosin-stained sections of ayu gill tissue demonstrating gill hyperplastic responses to Neoparamoeba infection.
(a) Gill sections with arrows indicating amoebae (bar = 100 lm). (b) Same section with arrows indicating amoebae (bar = 50 lm). (c)
Same section with horizontal arrow indicating amoeba nucleus and vertical arrow indicating the parasome (bar = 25 lm).

(a) (c) (e)

(b) (d) (f)

Figure 3 In situ hybridisation using probes that hybridise to either Neoparamoeba perurans or N. pemaquidensis 18s rRNA. (a, b)
N. perurans probe with arrows showing probe-positive amoebae [bars = 50 lm (a) & 25 lm (b)]. (c, d) N. pemaquidensis probe with
arrows showing probe-negative amoebae [bars = 50 lm (c) & 25 lm (d)]. (e, f) No probe controls with arrows showing non-signalling
amoebae [bars = 50 lm (e) & 25 lm (f)].

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 Journal of Fish Diseases 2010, 33, 455–458
positive, indicative of hybridisation of the probe to
the target RNA, whereas no signals were seen in the
control sections probed with N. pemaquidensis-
specific oligonucleotides or with hybridisation
buffer with oligonucleotides omitted (Fig. 3). Both
the N. perurans and N. pemaquidensis probes
hybridised with their respective representatives on
the Neoparamoeba array, and again no signals were
seen when oligonucleotides were omitted from the
hybridisation experiment.
We report AGD from a new host and a new
location. The association of this AGD case with an
infection with N. perurans is consistent with all
previous cases of AGD outbreaks in fish farmed in
the marine environment. More information is
needed about the worldwide distribution of
N. perurans and its biology to determine the risk
of AGD outbreaks in new host species and at new
locations. The biology of ayu requires changes in
salinity during the farming cycle, which means that
only the larval stage is susceptible to AGD as grow
out and spawning take place in fresh water and
N. perurans is a marine species.
Acknowledgements
This work formed part of a project of Aquafin CRC
and was supported by the Australian GovernmentÕs
CRC programme, the Fisheries Research and
Development Corporation and other CRC
participants.
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Received: 8 July 2009
Accepted: 12 October 2009