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PARASITES IN FISHERY PRODUCTS

PARASITES IN FISHERY PRODUCTS. David Di Cave University of Rome Tor Vergata.

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PARASITES IN FISHERY PRODUCTS

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  1. PARASITES IN FISHERY PRODUCTS David Di Cave University of Rome Tor Vergata

  2. Anisakids are ascaridoid nematodes dependent upon aquatic hosts for the completion of their life history, which generally involves an array of invertebrates and fish as intermediate or paratenic hosts, and marine mammals or fish-eating birds, reptiles and fishes as definitive hosts.

  3. Taxonomy • Eukaryota • Metazoa • Nematoda • Secernentea • Ascaridida • Ascaridoidea • Anisakidae • The most frequently recovered anisakid species in fish • belong to the following genera: • Anisakis, Pseudoterranova, Contracaecum, Phocascaris

  4. Adult forms of Anisakis spp. are usually found in cetaceans (whales and dolphins). Pseudoterranovadecipiens matures mainly in pinnipeds (phocids and otariids). Some members of the genus Contracaecum reach maturity in pinnipeds, others in fish-eating birds (e.g cormorants, pelecans and herons). Phocascaris species become adults in pinnipeds of the northern hemisphere.

  5. Anisakid larvae can be identified at genus level by light microscopy, mainly on the basis of the morphology of digestive tract and of excretory system. A-C Pseudoterranova decipiens; D-F Anisakis sp.; G-I Hysterothylacium aduncum; J-L Contracaecum rudolphii. (From Anderson, 2000)

  6. The taxonomy of anisakids is mainly based on the morphology of male adult specimens. The most significant structural characters for species identification are the distribution and pattern of caudal papillae, the spiculae and the morphology of cephalic end (Fagerholm, 1991). From Abollo et al., 1998.

  7. Third stage larvae of anisakids are commonly found in the flesh and the body cavity of a large number of fishes as well as in cephalopods that serve as paratenic hosts. From aquatic.unizar.es/n3/art1401/anisakis.htm

  8. Anisakis sp. larva in the flesh of blue whiting (Micromesistius poutassou) after light candling (from www.usc.es/banim/doc/tppanisa.htm)

  9. Histopathology Histopathological studies in fish revealed damages mostly at the level of stomach wall, liver, gonads and muscles. These includes mechanical damages, necrosis, tissue compression and castration. Clinical signs in fishes are mainly cellular infiltration, and hemorrhage. From Abollo, 1999.

  10. Histopathological studies in cephalopods revealed damages mostly at the level of stomach wall, mantle muscle, nidamentary glands, testicle and ovary. These includes mechanical damages, necrosis, tissue compression and castration. Cellular infiltration is oftenly found associated to encapsulated larvae. From Abollo et al., 1998.

  11. In the stomach of cetaceans, anisakid adults are often found in clusters of individuals embedded in the mucosa and submucosa. Ulcers of 5x3 cm sized associated to anisakids are found in the fundic portion of the stomach. From Abollo et al., 1998.

  12. Anisakids are of medical and economic significance. Larval forms of anisakid nematodes of the genera Anisakis Dujardin, 1845 and Pseudoterranova Mozgovoi, 1951 are in fact the principal aethiological agents of human anisakidosis. This fish-borne anthropozoonosis occurs when the larvae are taken alive after the consumption of raw, undercooked or improperly processed fish or cephalopods that serve as paratenic hosts in the life cycle of these nematodes.

  13. Gastroendoscopic image showing a Anisakis larva extracted from the gastric tract of a 51 year old woman in Southern Italy (D’Amelio et al., 1999)

  14. Section of Anisakis larva in the ileum of a patient. (From http://www.stanford.edu/class/humbio103/parasitepages/ParaSites/anisakiasis/Diag.html)

  15. Anisakid nematodes tend to be very conserved in morphology and allozyme studies have shown that many presumed monospecific species consists of several cryptic species (Nascetti et al., 1993; Paggi et al., 1991; Orecchia et al., 1994; Mattiucci et al., 1997).

  16. More recently, the systematics and phylogeny of anisakid nematodes has been studied using molecular approaches (PCR-RFLP, DNA sequencing, SSCP).

  17. Survey on the presence of Opisthorchis felineus (Platyhelminthes, Digenea) metacercariae in fish from Bolsena and Bracciano lakes (Central Italy)

  18. Human outbreak in Bolsena and Bracciano Lakes (Central Italy)

  19. Fish • Involved species • Prevalence of infestation • Safe fish • Risk

  20. Materials

  21. Metodi/Microscopia Ricerca metacercarie da muscolo di pesce • preparati per schiacciamento di tessuto muscolare; • digestione manuale (50g): • 50g di muscolo, privilegiando parti in prossimità delle pinne; • digestione in soluzione: 1 L di acqua, 8 ml di HCl e 5 g di pepsina; • temperatura di digestione: 37°C; • tempo di digestione: 1 ora; • materiale filtrato attraverso un setaccio con maglia 0.5 mm; • si fa sedimentare il filtrato 10 minuti e si elimina il surnatante; • lavaggi successivi in soluzione fisiologica finché il surnatante non appare limpido; • dopo l’ultimo lavaggio si elimina il surnatante e si mette il sedimento in Piastra Petri; • si osserva al binoculare.

  22. Methods – Molecular biology L’identificazione delle metacercarie è stata ottenuta in PCR, utilizzando 2 differenti protocolli per l’amplificazione di DNA ribosomale: • OPI: in grado di amplificare una regione dell’Internal Transcriber Spacer 2 (ITS-2) dell’rDNA di tutte le specie appartenenti alla famiglia Opisthorchiidae; • OFI: che amplifica una regione della Cytochrome Oxidase I (COI) specifica per O. felineus. Processate singolarmente 65 metacercarie isolate tramite digestione manuale Alcuni dei prodotti di amplificazione di OPI sono stati successivamente sequenziati

  23. Risultati

  24. Prevalenza (%) CI Total 83.1 72.4-90.7 Digestion 73.0 58.7-87.3 Squash 57.0 44.9-69.0 Bolsena 75.0 59.7-86.8 Bracciano 100 81.8-100 Risultati O. felineus in Tinca Intensity max > 50 metacercarie / 50 g flesh 60 metacercarie (92.3%) amplification OPI 48 metacercarie (73.8%) amplifica ion OFI O. felineus recorded both Bolsena and Bracciano

  25. Preparati per schiacciamento

  26. Isolated metacercaria with digestion

  27. Results Definitive hosts Bolsena: Stools Necroscopie Bracciano: 4 cat stool samples all positive near boats Prevalence in cat: 51.6% (CI=33.1-69.8) Confirmed identification in PCR on eggs

  28. Conclusions • Tinca tinca second intermediate host involved in life cycle • Coregonus lavaretus not involved • Cat is the definitive host • O. felineus species of recent introduction or autoctonous never recorded because of use of cooked fish • High prevalence very radicated cycle • Digestion more sensitive technique to detect metacercariae in fish

  29. Conclusions • Difficult control • Limited therapy • Prophylaxis: no raw or uncooked fish • Monitoring the fish species

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