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Shellfish Disease Surveillance Programme - Final Report June 2003

NIWA Client Report: AUS2002-018 June 2003 NIWA Project: NAU 03911

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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.


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.


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

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1.

Pre

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nce

of d

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liste

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PIR

SA

in 2

238

C.

gig

as f

rom

all

site

s.

NP

= n

ot p

ossi

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due

to s

am

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sign

.

List

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s C

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C

O

CW

2 D

B

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N

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S

GB

K

IE

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LN

P

J/B

P S

M

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S

T

WS

Mic

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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.

*

?%

?%

?%

?%

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?%

?%

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?%

?%

?%

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?%

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Ha

plo

spo

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um

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0%

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0%

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0%

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Ma

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0%

Mik

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pp. *

?%

?%

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?%

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?%

?%

?%

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?%

Per

kin

sus s

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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

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nce

of

Bo

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and

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. req

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and

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CB

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N

DB

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GB

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P

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ll af

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s (*

all

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No.

oys

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150

150

150

150

144

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150

75

71

149

150

150

149

150

150

2238

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6.7

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Bac

teria

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0 0.

8

(*0.

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7 0

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9

(*0.

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estiv

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7

(*0.

05)

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Mea

n in

tens

ity o

f pa

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ogic

al

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ons

and

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atio

ns

of h

aem

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osis

in

Pa

cific

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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

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2 =

mod

era

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3 =

hea

vy)

.

Site

and

sa

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e da

ta

Inte

nsity

S

core

C

AI

CB

C

O

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2 D

B

DB

N

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GB

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P

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M

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ST

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Dig

estiv

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hy

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1.1

1.24

1.

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1.32

1.

17

1.12

1.

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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

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sis

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1 1

1.17

1.

5 1

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1.

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1 1.

56

1.52

1.

06

1 1.

74

1.08

1*

4 3

24

1 4

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14

40

7 4

6 16

17

7

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46

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0 0

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2 2

1 0

1 17

1

0 54

4

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0 0

0 0

0 0

0 0

0 0

2 0

0 0

12

0

Put

ativ

e

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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

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irus

Mea

n*

1.49

1.

69

1 1.

32

1.37

1

1 1.

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1.32

1.

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1.32

1

1 1

1 1.

68

1*

45

57

12

50

70

8 49

32

34

23

58

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8

9 3

28

2*

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49

0 19

33

0

0 8

11

13

20

0 0

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34

3*

4 18

0

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0 0

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1 3

0 0

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6

Loca

tion

of

Hae

moc

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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

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Su

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- F

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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.


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

gigas and Ruditapes philippinarum larvae in France. Journal of Fish Diseases 24:

369-376.

http://www.oie.int/eng/normes/fcode/A_summry.htm


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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.