CYTOLOGY AND HISTOLOGY FOR MOLLUSCAN DISEASES

Snježana Zrnčić, PhD, DVMTCDC/TCCT Consultant No 2 – Diagnostics

zrncic@irb.hr

Ireland Mussels 33 323 t

C. gigas 6 515 tPectinids 903 t O. edulis 793 t

Cockle 245 t

Great BritainMussels 34 247 tPectinids 23 974 t Clams 11 324 tC. gigas 1 381 t Cockle 53 tO. edulis 711 t

DenmarkMussels 34423 tO. edulis 804 t

NetherlandsMussels 31 300 tC. gigas 3 120 tClams 1 873 tPectinids 781 tO. edulis 75 t

GermanyMussels 3 670 t C. gigas 86 t

France C. gigas 116 180 tMussels 67 525 tPectinids 26 876

Clams 10 540 tCockle 1 680 t

O. edulis 1 112 tSpain

Mussels 228 837 tClams 6 851 t

O. edulis 3 256 tCockle 1 411 t

C. gigas 1 264 tPectinids 105 t

ItalyClams 84 453 tMussels 61 928 t

GreeceMussels 28 522 t O. edulis 49 t

NorwayMussels 1649 tPectinids 799 t Clams 8 tO. edulis 4 t

PortugalClams 4 816 t Cockle 4 489 tC. gigas 651 tO. edulis 1 t

SloveniaMussels 163 t

SwedenMusssels 200 t

BelgiumPectinids 534 t

CroatiaMussels 3515 tO. edulis 562 t

Channel IslandC. gigas 600 tPectinids 437 tMussels 50

Bulgaria Mussels 238 t

FAO (production data) 2011

Transfers and introduction into new area – prerequisite for diseases spreading

Transfers of spat or other stages for cultivation

Transfers for placing on market (re-immersion)

In the global context of molluscs cultivation, transfers and introdution into new area are extremelly importantAnd for the moment recognizhed as mail cause of epizooties and

outbreaks of mass mortalitie There are many others sources of infection and prerequisite

of diseases spreading Balast waters Ship traffic Ornamentaln aquariums

Molluscan diseases control There is no vaccines, drugs...

Molluscans have no capability to produce antibodies

Treatment would have adverse influence to the aquatic environment

In disease free are it is crucial to avoid any introduction of infected populations

International, regional and national standards, guides and recommendation

Effort for improvment of diagnostic methods for molluscan diseases are continuos

Transfers are not single source of infection and danger (for instance: balast waters/global changes…)

In the infected area...The main goal is to minimize disease influence, better

knowledge and understanding og the disease, it is necessary to study relationship of host and pathogen

Development of resistant strains of molluscans…

Listed diseases Annex IV, Part II Exotic diseases

Infection with Bonamia exitiosa Ostrea angasi, O. chilensis

Infection with Perkinsus marinus Crassostrea gigas, C. virginica

Infection with Mikrocytos mackini Crassostrea gigas, C. virginica, Ostrea conchaphila and O. edulis

Non exotic diseases

Infection with Bonamia ostreae Ostrea edulis, O. chilensis, O. conchaphila, O. denselammelosa, O. puelchana

Infection with Marteilia refringens Ostrea edulis, O. chilensis, O. angasi, O. puelchana, Mytilus edulis and M. galloprovincialis

Infection with Bonamia ostreae

Ifremer © Ifremer ©

Non exotic diseases

- lethal infection of flat oyster (Ostrea edulis) haemocytes

- haplosporidians Bonamia spp.- yellow discoloration and extensive

lesions on gills and mantle but most infected oyster appears normally

- lesions in connective tissue of gills, mantle and digestive gland

- intrahaemocytic protistans quickly become systemic leading to the death of oysters

- experimetal infection-cohabiatation of inoculation

- highest prevalence –late winter, early spring

- B. ostreae and B. exitiosa in O. edulis

- different species throughout the world

Infection by Marteilia refringens

Non-exotic diseases

- Marteiliosis – disease caused by Marteilia refringens in flat oyster (Ostrea edulis) european blue mussel (Mytilus edulis) and mediterranean mussel (M.galloprovincialis) - «Aber disease»

- parasite sporulate in digestive gland epithelia causing poor condition index, emaciation comsumption of its reserves of energy (glycogena), discoloration of digestive gland, cessation of growth and mortalities

- Mortalities appeard to be related to the sporulation of the parasita

- presporulation stages occur in epithelia of palps, stomach, digestive ducts and gills

- Infection confined in spring and summer, at temperatures of the sea under 17°C

- high salinity repress sporulation

Distribution of bonamiosisEurope (UK, Ireland, France, Netherlands, Belgium, Italy Spain, Portugal)Canada (British Columbia)U.S.A. (California, Maine, Washington)

Susceptible speciesNatural hosts : European flat oyster Ostrea edulis, Crassostrea ariakensisSusceptible species without identification of parasite : Ostrea angasi, O. chilensis, O. puelchana

Distribution of marteiliosis- Atlantic Europe, from north Brittany to Portugala; notified case in oyster in Netherlands, in mussels in England- Mediterranean part of Europe from Spain to France, Italy, Croatia, case in u Marocco

Susceptible speciesNatural hosts : Ostrea edulis, Mytilus galloprovincialis and M. edulis

Susceptible species without identification of parasite : Ostrea chilensis, O. angasi, O. puelchana, O. densellamellosa, Crassostrea virginica, C. gigas (only young plasmodia in stomach without sporulation), Cardium edule

DIAGNOSTIC TECNIQUE FORBONAMIA OSTREA AND MARTEILIA

REFRINGENS IN OYSTERS/MUSSELS

• Screening diagnosis of parasite histological or cytological analysis of samples

• Confirmatory dijagnosis – molecular techniques (PCR, in situ hibridisation)

• Study of parasite morphology – electron microscopy

Soft tissue of european flat oyster, Ostrea edulis, visible after removal of the shell

Legend: AM – aductor muscle; G - gills; GO – gonads; L - ligament; M - mantle and U – umbo – part of the shell which connect them. The inhalantn i exhalant siphon of mantle cavity marked as IC and EC.

tissue imprints of small piece of heart/digestive gland should be prepared on the clean, degreased slide

Imprints should be air dried and fixed by methanol

Imprints are stained by commercial kit for blood smears according to the manufacturer instructions (Merck Hemacolor staining kit)-modified Giemsa staining

After staining, slides are washed under tap water, air dried and cover with cover slip

microscopy

Heart and/or digestive gland imprints for cytological examination

Imprints examinationMARTEILIOSIS Slides with tissue imprints

(digestive gland or digestive tract) should be first observed by 10X or 20X dry objective to find area with epithelial cells of digestive gland or digestive tract

early stages of Marteilia refringens occur within epithelia probavnog trakta, a spore u epithelia of digestive gland (Fig. 1)

Parasite is 5-8 μm in size in early stages and 40 μm during sporulation

Cytoplasm of the cells is staining basophilic, nucleus is eosinophilic (blue and red after Hemacolor® kit staining)

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BONAMIOSIS Slides with tissue imprints

(heart, gills, oyster larvae) must be first observed by 10X ili 20X dry objectives: look for areas with 200 haemocyts per eye field at 20X objective.

Bonamia ostreae cells occur within of outside haemocyte (Fig 1)

They appear as very small cells (2-5 μm size) with basophilc cytoplasms and eosinophilic nuclei

As they spread through slide they may appear wider then in histological slide

Multinucleated cells may appear ( Fig. 2).

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MOLLUSCS PROCESSING FOR DIAGNOSIS BY HISTOLOGY

Tissue slicing in oysters Tissue slicing in mussels

Fixation, dehydration, embedding

PROCESSING FOR HISTOLOGY Prepare Davidson’s fixative, formalin or Carson’s Slice the tissue Put into cassette, immerse it in the fixative and leave it

there for at least 24 hours Once samples are fixed they must be dehydrated and

infiltrated by paraffine Embeddin is a process of place tissue in the paraffin

block to enable cutting Removing cassette, cooling of the block, trimming and

cutting tissue into water bath Placing on the glass slide Staining and covering with cover glass, examination

Bonamia spp. within hemocyte or feely in the connective tissue or synuses of gills, mantle and often associated with infllamatory reaction (Fig. 1).

Parasite – very small cells of 2-5 μm wide (Fig. 2)

B. exitiosa usually appears larger than B. Ostreae with a more centred nucleus.

To avoid any doubt the parasite has to be observed inside the haemocyte for a positive diagnosis. This is to avoid false positive results.

Hemocytic infiltration in connective tissue of digestive gland. H&E staining (X20 )

Bonamia ostreae in connective tissue Ostrea edulis. H&E staining (X120 magnification).

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• Young stages of Marteilia refringens are present in epithelia of stomach, intestine and digestive tracts (Fig. 1) and sometimes in digestive tubules. Sporulating stages can occur in epithelia of digestive tubuls (Fig. 2). Free sporangia can occur in intestine lumen

Marteilia refringens sporulationg stages in the epithelia of digestive tubule Ostrea edulis. H & E staining x80.

Young stages of M.refrigens in epithelia of digestive gland of Mytilus edulis and sporulation in digestive diverticulum H & E x40

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