Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 87
Amoebic gill infections of rainbow trout in
freshwater ponds
Buchmann, K.*, Nielsen, T.**, Sigh, J.*, Bresciani, J.*
Royal Veterinary and Agricultural University, Frederiksberg C., Denmark*
Aquasearch Vet. DK-7300 Jelling, Denmark**
Abstract
Gill disease of fry and fingerlings associated with extensive mortality in rainbow trout freshwater farms in Denmark have been observed on an increasing number of occasions in the
latest years. Clinical symptoms of hyperventilation indicate respiratory distress. Gross-pathological observations of gills show pale nodules and patches on gill filaments. Histological
and scanning electron microscopic investigations were conducted on gills from trout with
these symptoms. It was found that extensive hyperplasia of gill filaments, clubbing and fusion of filaments and lamellae occurred at high frequency. Heavy colonization of affected
gills with amoeba-like protozoans was observed. The protozoans were approximately 10-20
micrometers in diameter and the density on gill tissue varied but could reach 50 cells per mm
surface section. The gill infection resembled nodular gill disease previously described from
Canada, a disease connected to concomitant infections with bacteria and amoebae. However,
in the present study no colonization with filamentous bacteria could be detected. The amoebae could not be ascribed to the known freshwater amoeba genera recorded in rainbow trout.
The amoeboid organisms co-occurred with a few specimens of Trichodina. Formaldehyde treatments of the affected tank were shown to alleviate the acute situation.
Introduction
Parasite infections in freshwater trout farms
in Denmark have been relatively well studied (Buchmann & Bresciani 1997). Both metazoan and protozoan organisms are known to
occur regularly. Thus, various flagellates and
ciliates are common findings on rainbow trout
gills during fish examinations. However, in
the latest years gill disease problems associated with mortalities have been registered. All
cases have shown hyperplasia of gill fila-
Materials and methods
Rainbow trout fingerlings (body length 10-15
cm) from two freshwater trout farms in Jutland (western part of Denmark) were sampled
in May 2003. Fish were clinically affected and
showed respiratory distress associated with
high mortality. Gills were dissected and fixed
in neutral formaldehyde and brought to the
laboratory for further investigation.
Whole mount preparations
ments, clubbing and lamellar fusion. In order
to elucidate the disease problem the present
Formalin fixed gill filaments were rinsed in
distilled water and stained with haematoxy-
investigation was undertaken on affected gills
from two trout farms in Denmark where out-
lin for 5 min and subsequently embedded in
glycerine-gelatine.
breaks occurred in May 2003.
Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 88
Scanning electron microscopy
Scanning electron microscopy
Formalin fixed gills were post-fixed in 2.5 %
neutral glutaraldehyde (24 h), washed in dis-
The extensive hyperplasia of gill filaments
caused clubbing and fusion of gill lamellae
tilled water, immersed in 2 % tannic acid, dehydrated, critically point-dried, sputtered
(Fig. 1). Amoeba-like organisms were firmly
attached to the gill surface. In certain areas
with gold and studied in a JEOL JSM 840 scanning electron microscope.
these protozoans produced a carpet-like covering of the epithelial substrate (Fig. 2). The
Histology
shape of individual cells was roundish to
ovoid (10-15 micrometers) with the
Formalin fixed gill filaments were dehydrated, embedded in paraffin wax , sectioned
(5 micrometer sections), stained with Giemsa,
haematoxylin/eosin or haematoxylin/alcian
blue and subsequently mounted on slides
with Depex as embedding medium.
ectoplasma forming blebs (Fig. 3), folds or
ridges (Fig. 4). A few Trichodina parasites were
also found on the gills (Fig. 4). No extensive
aggregations of bacteria were detected on the
surface of affected areas.
Histology
Results
Farm observations
Fish in the two freshwater farms were showing signs of respiratory distress, lethargy and
Extensive hyperplasia of gill epithelia and
mucous cells was found. The gill structure was
highly disorganized. The epithelial surface
surface swimming associated with mortality.
was colonized by a carpet-like covering of
densely packed amoeba-like organisms of
Gross pathology
sizes between 10 and 24 micrometers (length)
(Figs. 5 and 6). In certain areas at least two
The gill tissue appeared swollen and irregularly structured with whitish or pale patches
spread on the gill filaments.
Whole mounts
When studied at magnification 100 x , 200 x
and 400 x the extensive hyperplasia of gill filaments was evident. The surface of the gills
was in certain areas covered with uni-cellular nucleated amoeboid organisms. A few
specimens of Trichodina sp. were detected at
low densities (approximately one parasite per
gill filament). The size of the amoeba-like organisms varied from 10-20 micrometer. Bacterial aggregations were not detected.
layers of these parasites were found over gill
cells. Mucus associated with aggregations of
amoebae was observed between gill filaments.
The colonizing cells contained a nucleus and
showed highly varying shapes in cross sections.
Discussion
The present investigation was based on formalin-fixed material and it should be stressed
that further studies on fresh preparations and
cultures should be conducted. Likewise, specific diagnosis needs additional transmission
electron microscopy studies. However, the
work suggested strongly that the causative
agent of the gill disease in the Danish trout
farms is an amoeba-like parasite. A disease
Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 89
Fig. 1. Scanning electron microscopy of rainbow
trout gills showing extensive hyperplasia and
clubbing of gill filaments and fusion of gill filaments
(arrow) associated with colonization of numerous
amoeba-like protozoans. Scale bar 1mm.
Fig. 3. Scanning electron microscopy of amoebae
on gill epithelium showing pseudopodia and
membrane blebs. Scale bar 10 micrometers.
(Arrows?)
Fig. 5. Histological section of amoeba-infected gill
filament. Giemsa stain. Scale bar 100 micrometers.
Fig. 2. Scanning electron microscopy of rainbow
trout gill filament heavily colonized with amoebae
forming a carpet-like covering (arrow). Scale bar
100 micrometers.
Fig. 4. Scanning electron microscopy of amoebae
associated with Trichodina sp. on the gills of rainbow
trout. Scale bar 10 micrometers.
Fig. 6. Histological section of amoeba-infected tips
of gill filament. Haematoxylin stain. Scale bar 100
micrometers.
Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 90
with similar clinical findings was previously
et al. 1998, 2000). Gross pathology changes re-
detected in rainbow trout and described as
nodular gill disease (NGD) by Daoust &
ported for NGD have many similarities to the
findings in the present investigation. It is not
Ferguson (1985). NGD has been recorded in a
number of other hosts including charr
clear to what extent other associated parasites
play a role in the amoebic proliferative gill
Salvelinus alpinus (Speare 1999). However,
NGD is normally caused by amoebae in asso-
disease. Trichodinids in association with
amoebae as found in the present study were
ciation with heavy colonization by
filamentous bacteria. Such bacteria were not
also mentioned by Chatton (1909, 1910) and
Sawyer et al. (1974) but the importance, if any,
found on the gills of Danish trout in these
cases, although previously, bacterial coloni-
needs further investigation. At least the density of trichodinids on the gill tissue was very
zation in connection with these particular disease symptoms has been recorded in Danish
low compared to the density of amoebae. Although the clinical and histological work
trout farms (own observations). It should be
noted that the fixation method used in the
presents similarities to NGD and Thecamoeba,
specific diagnosis of the organism found in
study reported herein, could have obscured
the presence of bacteria. The presence of pale
the Danish rainbow trout farms await further
characterization by transmission electron
nodules in the gill material due to extensive
hyperplasia and the presence of clubbed gill
microscopy and in vitro cultivation studies
followed by molecular diagnostic work.
filaments and fused gill lamellae presents a
picture much like NGD. Certain areas of infected gills were colonized by dense layers of
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