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

FAO (production data) 2011 Norway Mussels 1649 t Pectinids 799 t Clams 8 t O. edulis 4 t Great Britain Mussels 34 247 t Pectinids 23 974 t Clams 11 324 t C. gigas 1 381 t Cockle 53 t O. edulis 711 t Sweden Musssels 200 t Ireland Mussels 33 323 t C. gigas 6 515 t Pectinids 903 t O. edulis 793 t Cockle 245 t Netherlands Mussels 31 300 t C. gigas 3 120 t Clams 1 873 t Pectinids 781 t O. edulis 75 t Denmark Mussels 34423 t O. edulis 804 t Channel Island C. gigas 600 t Pectinids 437 t Mussels 50 Germany Mussels 3 670 t C. gigas 86 t Belgium Pectinids 534 t France C. gigas 116 180 t Mussels 67 525 t Pectinids 26 876 Clams 10 540 t Cockle 1 680 t O. edulis 1 112 t Bulgaria Mussels 238 t Slovenia Mussels 163 t Croatia Mussels 3515 t O. edulis 562 t Spain Mussels 228 837 t Clams 6 851 t O. edulis 3 256 t Cockle 1 411 t C. gigas 1 264 t Pectinids 105 t Portugal Clams 4 816 t Cockle 4 489 t C. gigas 651 t O. edulis 1 t Italy Clams 84 453 t Mussels 61 928 t Greece Mussels 28 522 t O. edulis 49 t

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

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 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 ©

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

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

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

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.

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

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

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.

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

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).

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