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Shellfish Disease Surveillance Programme - Final Report June 2003
NIWA Client Report: AUS2002-018 June 2003 NIWA Project: NAU 03911
(c) All rights reserved. This publication may not be reproduced or copied in any form without the permission of the client. Such permission is to be given only in accordance with the terms of the client's contract with NIWA Australia. This copyright extends to all forms of copying and any storage of material in any kind of information retrieval system.
Shellfish Disease Surveillance Programme - Final Report June 2003 B. K. Diggles
Prepared for
Primary Industries and Resources, South Australia (PIRSA)
NIWA Client Report: AUS2002-018 June 2003 NIWA Project: NAU 03911 NIWA Australia Pty Ltd ABN 87 094 585 994 Level 2, North Tower, Terrace Office Park, 527 Gregory Terrace, Bowen Hills P O Box 359, Wilston, Queensland, Australia 4051 Telephone +61-7-3257 0522, Facsimile +61-7-3257 0566 www.niwa.com.au
D R A F T
10/11/17
Contents Summary ii
Introduction 1
Materials and Methods 1
Results 2
Discussion 7
References 10
Appendices 13
Reviewed by: Approved for release by:
Dr J.G. Cooke Dr J.G. Cooke
Formatting checked
………………………
ii Shellfish Disease Surveillance Programme - Final Report June 2003
Summary
A total of 2238 Pacific oysters (Crassostrea gigas) and 3 cockles (Katelysia sp.) were sampled from
16 sites throughout South Australia (site designations CAI, CB, CO, CW2, DB, DBN, DBS, GB, KIE,
KIW, LN, PJ/BP, SM, SP, ST, WS). The oysters were fixed in 10% formalin then processed for wax
histopathology using standard techniques. Sections taken at two levels in the block for each mollusc
were stained with hematoxylin and eosin and examined under the microscope for pathological lesions,
parasites and disease agents, including those diseases specified by PIRSA and notifiable to the OIE.
The most significant pathological finding was detection of low numbers of microcell-like cells in the
vesicular connective tissue of oysters from 10 sites (CAI, CB, CW2, DB, GB, KIE, KIW, LN, SM and
WS). The cells were between 2 and 4 µm in diameter with a nucleus around 1µm diameter and were
associated with focal or diffuse haemocytosis in most cases. Overall prevalence at the affected sites
was 16.1% and ranged from 66.4% at site LN down to 3.3% at sites CAI and SM. There are two
described microcell genera, namely Bonamia and Mikrocytos. Bonamia spp. are primarily parasites of
flat oysters, but can also infect crassostreid oysters. Mikrocytos spp. have previously been recorded
from crassostreid oysters, and are found in vesicular connective tissue immediately adjacent to foci of
haemocytosis. Definitive diagnosis for both Bonamia spp. and Mikrocytos spp. to satisfy OIE
requirements is based on transmission electron microscopy (TEM) examination of the microcells.
Molecular probes are available, but these have not been validated for southern hemisphere microcells,
and hence their use for diagnostic purposes in Australia and New Zealand is currently limited.
Vesicular connective tissue cells with abnormal hypertrophied nuclei were also evident in oysters from
all sites, but particularly sites CB (82.7% prevalence) and DB (73.6% prevalence). The presence of
these cells may be suggestive of infection by a virus, and/or exposure of oysters to unfavourable
environmental conditions. Another notable lesion found at sites GB, SM and SP (prevalence 0.7 -
1.4%) resembled viral gametocytic hypertrophy. Other disease agents found included a rickettsia-like
organism (RLO) in the epithelium of, and in the connective tissue between, digestive tubules of
oysters from all sites (0.7 - 6.7% prevalence). Parasites and symbionts included Pseudomyicola-like
copepods in the digestive tubules and encapsulated in host tissues by a host response, Ancistrocoma-
like ciliates in the lumen of digestive tubules, and Trichodina-like ciliates externally in the gills and
mantle. The presence or absence of the mudworm Boccardia knoxi could not be determined from
these samples as this species infects the shell, which was removed prior to fixation.
The microcell-like cells observed should be followed up as all microcell diseases of molluscs are
notifiable to the OIE Molluscan Reference Laboratory. Additional work would be required using
TEM and/or molecular techniques to determine whether the microcell-like cells observed here are
indeed true microcells, and if so to indicate whether they are aligned with Bonamia spp. or Mikrocytos
spp. The low intensity of infection in these oysters may hinder TEM diagnosis and some possible
methods for obtaining heavily infected material for TEM are discussed.
Shellfish Disease Surveillance Programme - Final Report June 2003
1
D R A F T
10/11/17
Introduction
This is the final report produced for Primary Industries and Resources, South Australia
(PIRSA) by NIWA Australia to communicate the results of a histological survey of
the diseases of South Australian molluscs, mainly Pacific oysters (Crassostrea gigas).
This report summarises the results obtained from 2238 Pacific oysters and 3 cockles
(Katelysia sp.) received for on-processing in 3 batches, which arrived at NIWA on 6
January, 20 March and 30 April 2003. The oysters were obtained from 16 sites
throughout South Australia (site designations CAI, CB, CO, CW2, DB, DBN, DBS,
GB, KIE, KIW, LN, PJ/BP, SM, SP, ST, WS). Samples of 150 oysters were supplied
from most sites.
Materials and Methods
A total of 2238 Pacific oysters and 3 cockles were sampled from 16 sites throughout
South Australia. The oysters were fixed in 10% formalin in filtered seawater and
transported to the pathology laboratory where they were cut into standard transverse
sections 5 mm thick (Howard and Smith 1983) and placed into histopathology
cassettes. The tissues were embedded in paraffin wax, and sections 5 µm thick were
cut at two levels in the block. These two sections were then deparaffinized, hydrated,
stained with hematoxylin and eosin, then dehydrated, cleared and mounted on
microscope slides using standard techniques (Howard and Smith 1983).
The two sections per oyster were then examined with a compound microscope at both
low and high magnification for Bonamia spp., Haplosporidium spp., Marteilia spp.,
Mikrocytos spp., and Perkinsus spp., all notifiable disease agents listed by PIRSA and
the Office International des Epizooties (OIE) (OIE 2002). Any other disease agents or
pathological abnormalities observed were also recorded. A semi quantitative scoring
method (light = 1, moderate = 2, heavy = 3) was used to describe the intensity of
parasitic infections, metabolic processes such as diapedesis and some lesions such as
digestive tubule atrophy.
It should be noted that the level of diagnosis achieved by histological techniques is
generally presumptive. Any requirements for definitive diagnosis past genus level for
any of the disease agents listed above requires more detailed analysis. The presence or
absence of the mudworm Boccardia knoxi could not be determined from these
samples as this species infects the shell, which was removed prior to fixation.
Shellfish Disease Surveillance Programme - Final Report June 2003
2
D R A F T
10/11/17
Results
No parasites or pathological lesions were observed in the 3 cockles examined from site
DBN. The most significant pathological finding in the Pacific oysters examined was
detection of low numbers of microcell-like cells in the vesicular connective tissue of
oysters from 10 sites (CAI, CB, CW2, DB, GB, KIE, KIW, LN, SM and WS) (Tables
1, 2). The cells were between 2 and 4 µm in diameter with a nucleus around 1 µm
diameter (Appendices 1, 2, 5, 6). They were associated with focal or diffuse
haemocytosis (Appendices 3, 4) and were extracellular in most cases, though possibly
intracellular in a very few oysters (Appendix 6). Overall prevalence of the microcell-
like cells at the affected sites was 16.1%, and ranged from 66.4% at site LN down to
3.3% at sites CAI and SM (Tables 1, 2). Infection intensity was low at all sites except
LN, where 15 of 99 infected oysters had infections classed as moderate (Table 3).
Focal or diffuse haemocytosis was recorded at all sites (overall prevalence 32.6%) at
prevalences which ranged between 10.7% (Site CO) and 78.5% (Site LN). Prevalence
of haemocytosis increased with increased prevalence of the microcell-like cells
(Figure 1). Most haemocytosis occurred in the vesicular connective tissue but foci
were also recorded in the mantle, gills, digestive gland, gonad and surrounding the gut
(Table 3).
Vesicular connective tissue cells with abnormal hypertrophied nuclei with marginated
chromatin (Appendices 7, 8), possibly due to infection by a virus, were evident in
oysters from all sites (overall prevalence 34.3%), but particularly CB (82.6%
prevalence) and DB (73.6% prevalence) (listed as putative virus in Table 2). Most of
the oyster with high numbers of abnormal connective tissue nuclei (classed as
moderate to heavily affected) were recorded from sites CB (mean lesion intensity
1.69), WS (mean intensity 1.68), and CAI (mean intensity 1.49) (Table 3). Atrophy of
digestive tubules (Appendix 9) was found at low to moderate prevalences in oysters
from all sites (overall prevalence 22.1%, range 3.4 - 39.3%). Severity of tubule
atrophy (mean intensity >1.5) was greatest at sites GB, PJ/BP and ST (Table 3).
A viral gametocytic hypertrophy -like lesion in the gonad of oysters from sites GB,
SM and SP (prevalence 0.7% - 1.4 %) was characterised by massive hypertrophy of
the nuclei of female gametes (Appendices 10, 11). The enlarged nuclei contained
bizarre chromatin patterns (Appendix 11). Foci of non specific necrosis (Appendix
12) were evident in oysters from all sites (overall prevalence 2.9%, range 0.7 - 9.3%).
Metaplastic changes of the digestive tubule epithelium (a lesion distinct from tubule
atrophy) were observed in one oyster from site DB, while bacterial infections
(Appendix 13) were observed in oysters from DB, DBN, DBS, and SP (prevalence 0.7
- 1.3%) (Table 2). Diapedesis through the gut epithelium (Appendix 14) was observed
S
hel
lfish
Dis
ease
Su
rvei
llan
ce P
rogr
am
me
- F
ina
l Re
por
t Ju
ne
20
03
3
Ta
ble
1.
Pre
vale
nce
of d
isea
ses
liste
d by
PIR
SA
in 2
238
C.
gig
as f
rom
all
site
s.
NP
= n
ot p
ossi
ble
due
to s
am
plin
g de
sign
.
List
ed D
isea
se a
gent
s C
AI
CB
C
O
CW
2 D
B
DB
N
DB
S
GB
K
IE
KIW
LN
P
J/B
P S
M
SP
S
T
WS
Mic
roce
ll -
like
cells
* 3.
3%
6.7%
0%
16
%
22.9
%
0%
0%
8%
5.3%
21
.1%
66
.4%
0%
3.
3%
0%
0%
5.3%
Bo
na
mia
spp.
*
?%
?%
?%
?%
?%
?%
?%
?%
?%
?%
?%
?%
?%
?%
?%
?%
Ha
plo
spo
ridi
um
spp.
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
Ma
rtei
lia s
pp.
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
Mik
rocy
tos s
pp. *
?%
?%
?%
?%
?%
?%
?%
?%
?%
?%
?%
?%
?%
?%
?%
?%
Per
kin
sus s
pp.
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
Bo
cca
rdia
spp.
N
P
NP
N
P
NP
N
P
NP
N
P
NP
N
P
NP
N
P
NP
N
P
NP
N
P
NP
* C
onfir
ma
tion
of t
he p
rese
nce
of
Bo
na
mia
spp.
and
/or M
ikro
cyto
s spp
. req
uire
s T
EM
or
mol
ecul
ar
ana
lysi
s, a
nd is
bey
ond
the
scop
e of
thi
s st
udy.
In
ligh
t of
the
pres
ence
of
the
mic
roce
ll-lik
e ce
lls t
he p
rese
nce
of e
ith
er o
r bo
th t
hese
mic
roce
ll ge
nera
in S
outh
Aus
tra
lian
C.
gig
as c
ann
ot b
e ru
led
out
at
this
sta
ge.
S
hel
lfish
Dis
ease
Su
rvei
llan
ce P
rogr
am
me
- F
ina
l Re
por
t Ju
ne
20
03
4
Ta
ble
2. P
reva
lenc
e of
pa
rasi
tes
and
lesi
ons
(% in
fect
ed)
fro
m h
isto
path
olog
ica
l scr
eeni
ng o
f P
aci
fic o
yste
rs f
rom
al
l site
s.
Site
and
sam
ple
data
C
AI
CB
C
O
CW
2 D
B
DB
N
DB
S
GB
K
IE
KIW
LN
P
J/B
P S
M
SP
ST
WS
A
ll af
fect
ed
site
s (*
all
site
s)
No.
oys
ters
exa
min
ed
150
150
150
150
144
150
150
150
75
71
149
150
150
149
150
150
2238
Pse
udom
yico
la s
p.
2.7
2.7
6.7
8.7
6.3
5.3
2.7
4 8
1.4
3.4
4.7
5.3
5.4
12.7
2.
7 5.
2
Dig
estiv
e tu
bule
atro
phy
13.3
27
.3
20.7
39
.3
20.1
22
.7
24
22.7
24
11
.3
10.7
29
.3
4 3.
4 38
.7
36.7
22
.1
Dia
pede
sis
2.7
2 19
.3
1.3
2.8
14
10.7
28
10
.7
5.6
6 22
12
4.
7 70
.7
33.3
15
.9
Hae
moc
ytos
is
23.3
26
10
.7
46
68.8
25
.3
34
38.7
16
52
.1
78.5
20
15
.3
17.5
30
.7
22
32.6
Anc
istr
ocom
a -li
ke
cilia
tes
11.3
12
25
.3
17.3
12
.5
29.3
26
.7
11.3
14
.7
14.1
14
.1
22
15.3
22
.8
19.3
7.
3 17
.4
Oth
er c
iliat
es
4.7
3.3
2 2
0.7
1.3
1.3
5.3
1.3
0 2
0 4.
7 8.
7 1.
3 3.
3 3.
1
(*2.
8)
RLO
s 6.
7 5.
3 3.
3 1.
3 0.
7 1.
3 6
4 5.
3 1.
4 1.
3 6.
7 6
2.7
4 3.
3 3.
8
Put
ativ
e
Mic
roce
ll
3.3
6.7
0 16
22
.9
0 0
8 5.
3 21
.1
66.4
0
3.3
0 0
5.3
16.1
(*9.
6)
Put
ativ
e V
irus
54
82.7
8
47.3
73
.6
5.3
32.7
28
62
.7
52.1
54
.4
14
5.3
6 2
45.3
34
.3
Nec
rotic
Foc
i 1.
3 0.
7 1.
3 4
3.5
2.7
3.3
3.3
1.3
2.8
1.3
2.7
9.3
3.4
2.7
1.3
2.9
Bac
teria
l Inf
ectio
n 0
0 0
0 0.
7 0.
7 1.
3 0
0 0
0 0
0 0.
7 0
0 0.
8
(*0.
2)
Vira
l gam
etoc
ytic
hype
rtro
phy
0 0
0 0
0 0
0 0.
7 0
0 0
0 0.
7 1.
4 0
0 0.
9
(*0.
2)
Dig
estiv
e tu
bule
met
apla
sia
0 0
0 0
0.7
0 0
0 0
0 0
0 0
0 0
0 0.
7
(*0.
05)
S
hel
lfish
Dis
ease
Su
rvei
llan
ce P
rogr
am
me
- F
ina
l Re
por
t Ju
ne
20
03
5
Ta
ble
3.
Mea
n in
tens
ity o
f pa
thol
ogic
al
lesi
ons
and
loc
atio
ns
of h
aem
ocyt
osis
in
Pa
cific
oys
ters
fro
m a
ll si
tes.
T
he n
um
ber
s be
low
the
mea
n in
tens
ities
are
raw
cou
nts
of a
ffec
ted
oyst
ers
cla
ssifi
ed u
sing
sem
iqua
ntit
ativ
e sc
orin
g m
etho
ds*
(1 =
Lig
ht,
2 =
mod
era
te,
3 =
hea
vy)
.
Site
and
sa
mpl
e da
ta
Inte
nsity
S
core
C
AI
CB
C
O
CW
2 D
B
DB
N
DB
S
GB
K
IE
KIW
LN
P
J/B
P S
M
SP
ST
WS
Dig
estiv
e
tubu
le a
trop
hy
Mea
n*
1.1
1.24
1.
23
1.32
1.
17
1.12
1.
17
1.56
1.
11
1.25
1.
25
1.52
1
1 1.
57
1.35
1*
18
32
25
40
25
30
30
18
16
6 13
25
6
5 28
38
2*
2 8
5 19
3
4 6
13
2 2
2 15
0
0 27
15
3*
0 1
1 0
1 0
0 3
0 0
1 4
0 0
3 2
Dia
pede
sis
Mea
n*
1 1
1.17
1.
5 1
1.05
1.
13
1.05
1.
13
1 1.
56
1.52
1.
06
1 1.
74
1.08
1*
4 3
24
1 4
20
14
40
7 4
6 16
17
7
40
46
2*
0 0
5 1
0 1
2 2
1 0
1 17
1
0 54
4
3*
0 0
0 0
0 0
0 0
0 0
2 0
0 0
12
0
Put
ativ
e
Mic
roce
ll
Mea
n*
1 1
- 1
1 -
- 1
1 1
1.15
-
1 -
- 1
1*
5 10
24
33
12
4 15
84
5
8
2*
0 0
0
0
0
0 0
15
0
0
3*
0 0
0
0
0
0 0
0
0
0
Put
ativ
e V
irus
Mea
n*
1.49
1.
69
1 1.
32
1.37
1
1 1.
29
1.32
1.
41
1.32
1
1 1
1 1.
68
1*
45
57
12
50
70
8 49
32
34
23
58
21
8
9 3
28
2*
32
49
0 19
33
0
0 8
11
13
20
0 0
0 0
34
3*
4 18
0
2 3
0 0
2 2
1 3
0 0
0 0
6
Loca
tion
of
Hae
moc
ytos
is
No.
aff
ecte
d 35
39
16
69
99
38
51
58
12
37
11
7 30
23
26
46
33
Con
nect
. Tis
s.
22
22
8 47
75
21
30
47
8
26
82
11
17
18
16
29
Gut
4
13
3 3
10
14
18
5 1
8 31
10
3
6 22
3
Gill
s 1
1 0
6 1
0 2
1 2
0 0
1 0
1 4
0
Dig
estiv
e G
l. 0
0 0
0 0
0 0
0 0
0 0
1 0
0 2
0
Man
tle
3 3
1 7
13
0 1
4 1
3 4
6 1
0 2
1
Gon
ad
5 0
4 6
0 3
0 1
0 0
0 1
2 1
0 0
S
hel
lfish
Dis
ease
Su
rvei
llan
ce P
rogr
am
me
- F
ina
l Re
por
t Ju
ne
20
03
6
0102030405060708090
SM
(3.3
%)
CA
I(3
.3%
)W
S(5
.3%
)K
IE(5
.3%
)C
B(6
.7%
)G
B
(8%
)C
W2
(16%
)K
IW(2
1.1%
)D
B(2
2.9%
)L
N(6
6.4%
)
Pre
vale
nce
of
mic
roce
ll-lik
e ce
lls a
t ea
ch s
ite
Prevalence of haemocytosis
Fig
ure
1. D
iagr
am
sho
win
g tr
end
of in
crea
sing
ha
emoc
ytos
is
with
incr
ease
d pr
eva
lenc
e of
mic
roce
ll-lik
e ce
lls a
t a
ffect
ed s
ites.
Shellfish Disease Surveillance Programme - Final Report June 2003
7
D R A F T
10/11/17
in oysters from all sites (overall prevalence 15.9%, range 1.3 - 70.7%), but occurred at
particularly high prevalence (70.7%) and intensity (mean intensity 1.74) in oysters
from site ST (Tables 2, 3).
Parasites and symbionts found included Pseudomyicola-like copepods (Appendix 15)
in the digestive tubules and encapsulated in host tissues (overall prevalence 5.2%,
range 1.4 - 12.7%), Ancistrocoma-like ciliates (Appendix 16) in the lumen of digestive
tubules (overall prevalence 17.4%, range 7.3 - 29.3%), and Trichodina-like ciliates
(Appendix 17) externally in the gills and folds of the mantle (prevalence 0 - 8.7%)
(Table 2). Rickettsia-like organisms (RLO's) (Appendix 18) was also present at low
prevalence (overall prevalence 3.8%, range 0.7 - 6.7%) in and around the epithelium
of the digestive tubules of oysters from all sites (Table 2). Larger RLO inclusions
were very occasionally observed in the gills (Appendix 19).
Discussion
The detection of microcell-like cells associated with areas of haemocytosis in oysters
from 10 of the 16 sites sampled is significant as all microcell infections in molluscs
are notifiable to the OIE Molluscan Reference Laboratory (OIE 2002). There are two
described microcell genera, Bonamia and Mikrocytos. Bonamia spp. are primarily
parasites of flat oysters, but have also been recorded in crassostreid oysters
(Cochennec et al. 1998, Cochennec-Laureau et al. 2003). Mikrocytos spp. are
parasites of crassostreid oysters and have been recorded from Pacific oysters (M.
mackini, Farley et al. 1988) and Sydney Rock Oysters (Saccostrea glomerulata), (as
M. roughleyi, Farley et al. 1988, but recently reclassified as Bonamia roughleyi
(Cochennec-Laureau et al. 2003)). Mikrocytos mackini is commonly found in
vesicular connective tissue immediately adjacent to abcesses (foci of haemocytosis),
while B. roughleyi and Bonamia spp. are most commonly intracellular within
haemocytes (Hine and Wesney 1994, OIE 2000, B. Diggles, personal observation).
On this basis the microcell-like cells found in the Pacific oysters from these samples
appeared to possess characteristics more like those of Mikrocytos mackini rather than
Bonamia spp., because the vast majority were extracellular.
The microcell-like cells found in these samples were slightly larger (2-4 µm) than
recorded for B. roughleyi, (1-4 µm, see Farley et al. 1988), but were very similar in
size and appearance to Bonamia exitiosus when the latter are found in lightly infected
New Zealand dredge oysters (Appendix 5). In any case, due to the very small size of
these cells (which at 1-4 µm are approaching the limit of resolution for light
microscopy), any comparison at the light microscope level is probably meaningless at
this stage. Definitive diagnosis for both Bonamia spp. and Mikrocytos spp. to satisfy
OIE requirements is currently based on transmission electron microscopy (TEM)
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examination of the microcells. Molecular probes are available (Adlard and Lester
1995, Cochennec et al. 2000, Carnegie et al. 2003, Diggles et al. 2003), but these have
not been validated for southern hemisphere microcells and hence their use for
diagnostic purposes is currently limited. This suggests that additional sampling is
required to collect more material, preferentially from site LN, specifically for TEM for
initial attempts at a definitive diagnosis. At the same time, however, it would be
advisable to collect samples for molecular analysis so these could be analysed at a
later date.
One potential problem with collecting additional samples for TEM is the low intensity
of infection in the oysters examined in this sample. Even at the site with the highest
prevalence of microcell-like cells (site LN), of the 99 oysters which were recorded as
infected only 15 (15%) had infections which were classified as moderate. Infected
oysters from all other sites were classified as lightly infected. This is not surprising as
these sites were surveyed in the absence of clinical disease, suggesting that the oysters
with microcell-like cells are perhaps reservoir hosts or carriers of these cells, as
suggested for C. gigas by Bower et al. (1994). It is considered unlikely that microcells
could be visualised by TEM in lightly infected oysters, and the chances of detecting
microcells by TEM in moderately infected oysters may be only slightly higher. This is
because microcells are usually detected by TEM only in diseased, heavily infected
oysters (M. Hine, personal communication). This suggests that before additional
samples are taken for TEM, preferably from site LN, the oysters should be stressed in
a quarantine facility to try and increase infection intensity, perhaps by overcrowding
and/or increase in water temperature (which can promote the course of disease in the
case of Bonamia exitiosus (see Hine et al. 2002)), and /or a decrease in water
temperature, which promotes disease in the case of Mikrocytos mackini (see Hervio et
al. 1996) and B. roughleyi (see Farley et al. 1988). Alternatively, some published
techniques for isolating and purifying microcells (Mialhe et al. 1988, Hervio et al.
1996, Joly et al. 2001) could be utilised to attempt to obtain material for ultrastructural
and/or molecular analysis.
The hypertrophied nuclei with marginated chromatin observed in vesicular connective
tissue cells of oysters from all sites were almost identical to lesions described by
previous workers in C. gigas infected by herpes-like viruses (Hine et al. 1992, Renault
et al. 2000, 2001). Additional work would be required using TEM and/or molecular
techniques to determine whether the cell abnormalities in these oysters were caused by
a viral infection. Many of the oysters with these nuclear abnormalities were in poor
condition with marked digestive tubule atrophy, suggesting they may have been
diseased. However, high numbers of unusual cell nuclei in poorly conditioned oysters
may not necessarily be due to infection by a viral disease agent. It is also possible
these lesions could be associated with unfavourable environmental conditions such as
lack of food and/or very high water temperatures. However, because of the apparent
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association between the nuclear lesions and poor condition, the possibility of a viral
agent must be ruled out before the lesion can be attributed to an environmental cause.
The copepod and ciliate parasites found here are common symbionts of healthy
oysters (McGladdery et al. 1993) and are of little pathological significance. The RLOs
were present at low prevalence (0.7 - 6.7%) and intensity in oysters from all sites.
RLOs and other related intracytoplasmic bacteria are probably ubiquitous in marine
bivalves (Hine and Diggles 2002). Usually they occur at low intensities, as in the
present study, and are not associated with disease. However, if the host becomes
stressed, due to factors which may include unfavourable environmental conditions or
metabolic imbalances post-spawning, the RLOs can proliferate and may cause disease
(Hine and Diggles 2002).
Diapedesis (migration of haemocytes across epithelia), was commonly observed in the
gut epithelium. Diapedesis is a normal metabolic process in oysters, and is used to
remove harmful elements or metabolic by products, as well as parasites such as
Bonamia (see McGladdery et al 1993). However, diapedesis also occurs in healthy
oysters, and hence increased prevalence and intensity of diapedesis at some sites may
not necessarily be related to the presence of disease agents or pollutants. Focal areas
of cell necrosis were also encountered at all sites and appeared to occur in the absence
of obvious disease agents in most cases (non-specific necrosis). In very rare cases
bacterial infections were associated with areas of necrosis and haemocytosis,
particularly in small, poorly conditioned oysters. Due to their low prevalence and
intensity of the bacterial infections observed, it is assumed they resulted from the poor
condition of these oysters (i.e. secondary infections), rather than the alternative of
them playing a direct causative role in a disease process.
Viral gametocytic hypertrophy (VGH) due to a Papillomavirus-like papovirus has
been recorded in both the male and female gonads of Crassostrea virginica (see
McGladdery et al. 1993) and C. gigas (see Farley 1985). In C. virginica the virus
causes hypertrophy of gametes and germinal epithelium , but intensity of infection is
usually low and the infection is not associated with mortality or reduced fecundity
(McGladdery et al. 1993). The nuclear changes observed in C. gigas in the present
study were very suggestive of viral infection, however viral involvement would again
require confirmation by TEM. In any case, given the viruses involved with VGH are
apparently ubiquitous, and that they appear to have minimal adverse effects on oyster
health, prevention of spread of such viruses by restriction of oyster movements
appears unnecessary (McGladdery et al. 1993).
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References
Adlard, RD and Lester RJG (1995). Development of a diagnostic test for Mikrocytos
roughleyi, the aetiological agent of Australian winter mortality of the commercial rock
oyster, Saccostrea commercialis (Iredale & Roughley). Journal of Fish Diseases 18:
609-614.
Bower SM, Hervio D and McGladdery SE (1994). Potential for Pacific oyster
Crassostrea gigas, to serve as a reservoir host and carrier of oyster pathogens. ICES
Council Meeting Papers, ICES, Copenhagen Denmark, 1994. 5 p.
Carnegie RB, Meyer GR, Blackbourn J, Cochennec-Laureau N, Berthe F and Bower S
(2003). Molecular detection of the oyster parasite Mikrocytos mackini, and a
preliminary phylogenetic analysis. Diseases of Aquatic Organisms 54: 219-227.
Cochennec N, Renault T, Boudry P, Chollet B, and Gerard A (1998). Bonamia-like
parasite found in the Suminoe oyster Crassostrea rivularis reared in France. Diseases
of Aquatic Organisms 34: 193-197.
Cochennec N, LeRoux F, Berthe F and Gerard A (2000). Detection of Bonamia
ostreae based on small sub unit ribosomal probe. Journal of Invertebrate Pathology
76: 26-32.
Cochennec-Laureau N, Reece K, Berthe F and Hine PM (2003). Mikrocytos roughleyi
taxonomic affiliation leads to the genus Bonamia (Haplosporidia). Diseases of
Aquatic Organisms 54: 209-217.
Diggles BK, Cochennec-Laureau N and Hine PM (2003). Comparison of diagnostic
techniques for Bonamia exitiosus from flat oysters Ostrea chilensis in New Zealand.
Aquaculture 220: 145-156.
Farley CA (1985). Viral gametocyte hypertrophy in oysters. ICES Identification
Leaflets for Diseases and Parasites of Fish and Shellfish, C.J. Sindermann (ed). No.
25: 5 p.
Farley CA, Wolf PH and Elston RA (1988). A long term study of "microcell" disease in
oysters with a description of a new genus, Mikrocytos (g.n.), and two new species,
Mikrocytos mackini (sp. n) and Mikrocytos roughleyi (sp. n). Fishery Bulletin 86(3): 581-
593.
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Hervio D, Bower S, Meyer GR (1996). Detection, isolation and experimental
transmission of Mikrocytos mackini, a microcell parasite of Pacific oysters
Crassostrea gigas (Thunberg). Journal of Invertebrate Pathology 67: 72-79.
Hine PM and Wesney B (1994). Interaction of phagocytosed Bonamia sp.
(Haplosporidia) with haemocytes of oysters (Tiostrea chilensis). Diseases of Aquatic
Organisms 20: 219–229.
Hine PM and Diggles BK (2002). Prokaryote infections in the New Zealand scallops
Pecten novaezelandiae and Chlamys delicatula. Diseases of Aquatic Organisms 50:
137-144.
Hine PM, Wesney B and Hay BE (1992). Herpesviruses associated with mortalities
among hatchery-reared larval Pacific oysters Crassostrea gigas. Diseases of Aquatic
Organisms 12: 135-142.
Hine PM, Diggles BK, Parsons M, Pringle A and Bull B (2002). The effects of
stressors on the dynamics of Bonamia exitiosus Hine , Cochennec-Laureau & Berthe ,
infections in flat oysters Ostrea chilensis (Philippi). Journal of Fish Diseases 25:
545-554.
Howard DW and Smith CS (1983). Histological techniques for marine bivalve
molluscs. NOAA technical Memorandum NMFS-F/NEC-25. US Dept. Commerce.
97 p.
Joly JP, Bower SM, Meyer GR (2001). A simple technique to concentrate the
protozoan Mikrocytos mackini, causative agent of Denman Island disease in oysters.
The Journal of Parasitology 87: 423-434.
McGladdery SE, Drinnan RE and Stephenson MF (1993). A manual of parasites,
pests and diseases of Canadian Atlantic bivalves. Canadian Technical Report of
Fisheries and Aquatic Sciences 1931. 121 p.
Mialhe E, Bachere E, Chagot D, Grixel H (1988). Isolation and purification of the
protozoan Bonamia ostreae (Pichot et al. 1980), a parasite affecting the flat oyster
Ostrea edulis L. Aquaculture 71: 293-299.
Office International des Epizooties (2000). Diagnostic Manual for Aquatic Animal
Diseases. 3rd Edition. OIE, Paris. 237 p.
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Office International des Epizooties (2002). International Aquatic Animal Health
Code. 5th Edition. http://www.oie.int/eng/normes/fcode/A_summary.htm.
Renault T, LeDuff RM, Chollet B, Cochennec N, Gerard A (2000). Concomitant
herpes-like virus infections in hatchery reared larvae and nursery cultured spat
Crassostrea gigas and Ostrea edulis. Diseases of Aquatic Organisms 42: 173-183.
Renault T, Lipart C and Arzul I (2001). A herpes-like virus infecting Crassostrea
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Appendices
Appendix 1. Microcell-like cell (arrow) in vesicular connective tissue surrounded by
host haemocytes in an oyster from site LN. 1500x magnification.
Appendix 2. Two extracellular microcell-like cells (arrows) adjacent to a focal area of
haemocytosis in an oyster from site LN. 1500x magnification.
Lesions observed in Pacific oysters (C. gigas) from South Australia.
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Appendix 3. Multiple foci of haemocytosis (arrows) in the vesicular connective tissue
of an oyster from site LN. 75x magnification.
Appendix 4. Diffuse haemocytosis in the vesicular connective tissue of an oyster from
site LN. 75x magnification.
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Appendix 5. Comparison of a single Bonamia exitiosus (arrow, main picture) in a
New Zealand dredge oyster (Ostrea chilensis) with microcell-like cells from Pacific
oysters from (clockwise from top) sites CW2, LN, DB, GB, WS and SM. Photos of B.
exitiosus, CW2, LN and DB taken at 1500x magnification. Photos for GB, WS and
SM taken at 1000x magnification.
Appendix 6. A microcell-like cell (arrow) apparently inside the cytoplasm of a
haemocyte in the connective tissue of an oyster from site DB. 1500x magnification.
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Appendix 7. Hypertrophied nuclei with marginated chromatin (arrows) in the
connective tissues of an oyster from site DBS. 1000x magnification.
Appendix 8. Digestive tissue atrophy (arrows) in an oyster from site PJ/BP with
numerous hypertrophied cell nuclei (arrowheads). 400x magnification.
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Appendix 9. Severe digestive tubule atrophy (arrows) in a poorly conditioned oyster
from site PJ/BP. The arrowheads indicate necrotic epithelial cells. 100x magnification.
Appendix 10. Normal female gametes from an oyster from site SP. 1000x
magnification.
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Appendix 11. Suspected viral gametocytic hypertrophy in the gonad of an oyster from
site SP. Note bizarre chromatin patterns in the massively hypertrophied nucleus.
1000x magnification.
Appendix 12. A necrotic digestive tubule (arrows) in an oyster from site ST. 200x
magnification.
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Appendix 13. Bacterial colony (arrow) surrounded by haemocytes in an oyster from
site SP. 1000x magnification.
Appendix 14. Heavy diapedesis in the epithelium of the gut of an oyster from site ST.
200x magnification.
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Appendix 15. Pseudomyicola-like copepod encapsulated in the connective tissue of
an oyster from site SM. 100x magnification.
Appendix 16. Ancistrocoma-like ciliate in the digestive tubule of an oyster from site
ST. 1000x magnification.
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Appendix 17. Trichodina sp. from the gills of an oyster from site CW. 800x
magnification.
Appendix 18. Rickettsia-like organisms (RLOs) (arrows and arrowheads) in the
digestive gland epithelium of an oyster from site PJ/BP. 400x magnification.
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Appendix 19. A large rickettsial inclusion (arrowheads) surrounded by infiltrating
haemocytes in the gills of an oyster from site DBS. 100x magnification.