1. CYTOLOGY AND HISTOLOGY FOR MOLLUSCAN DISEASES
Snježana Zrnčić, PhD, DVM
TCDC/TCCT Consultant No 2 – Diagnostics

2. FAO (production data) 2011 Norway Mussels 1649 t
Pectinids 799 t Clams 8 t
O. edulis 4 t
Great Britain
Mussels t
Pectinids t
Clams t
C. gigas t
Cockle 53 t
O. edulis 711 t
Sweden
Musssels 200 t
Ireland
Mussels t
C. gigas t
Pectinids 903 t
O. edulis 793 t
Cockle 245 t
Netherlands
Mussels t
C. gigas t
Clams t
Pectinids 781 t
O. edulis 75 t
Denmark
Mussels t
O. edulis t
Channel Island
C. gigas 600 t
Pectinids 437 t
Mussels 50
Germany
Mussels t
C. gigas 86 t
Belgium
Pectinids 534 t
France
C. gigas t
Mussels t
Pectinids Clams t
Cockle t
O. edulis t
Bulgaria
Mussels 238 t
Slovenia
Mussels 163 t
Croatia
Mussels t
O. edulis 562 t
Spain
Mussels t
Clams t
O. edulis t
Cockle t
C. gigas t
Pectinids 105 t
Portugal
Clams t Cockle t
C. gigas 651 t
O. edulis 1 t
Italy
Clams t
Mussels t
Greece
Mussels t
O. edulis 49 t

3. 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 important
And 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

4. 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…

5. 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

6. Infection with Bonamia ostreae
Non exotic diseases
Infection with Bonamia ostreae
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
Ifremer ©
Ifremer ©

7. Infection by Marteilia refringens
Non-exotic diseases
Infection by Marteilia refringens
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

8. Distribution of bonamiosis
Europe (UK, Ireland, France, Netherlands, Belgium, Italy Spain, Portugal)
Canada (British Columbia)
U.S.A. (California, Maine, Washington)
Susceptible species
Natural hosts : European flat oyster Ostrea edulis, Crassostrea ariakensis
Susceptible 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
Natural 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

9. DIAGNOSTIC TECNIQUE FOR BONAMIA 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

10. 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.

11. Heart and/or digestive gland imprints for cytological examination
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

12. Imprints examination MARTEILIOSIS
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) 1. 2

13. 1.
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). 2.

14. MOLLUSCS PROCESSING FOR DIAGNOSIS BY HISTOLOGY
Tissue slicing in oysters
Tissue slicing in mussels
Fixation, dehydration, embedding

15. 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

16. Parasite – very small cells of 2-5 μm wide (Fig. 2) 1.
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 ) 2.
Bonamia ostreae in connective tissue Ostrea edulis.
H&E staining (X120 magnification).

17. 1.
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. 2.
Young stages of M.refrigens in epithelia of digestive gland of Mytilus edulis and sporulation in digestive diverticulum
H & E x40