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

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PARASITES IN FISHERY PRODUCTS

David Di Cave

University of Rome Tor Vergata


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.


Taxonomy 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.

  • Eukaryota

  • Metazoa

  • Nematoda

  • Secernentea

  • Ascaridida

  • Ascaridoidea

  • Anisakidae

  • The most frequently recovered anisakid species in fish

  • belong to the following genera:

  • Anisakis, Pseudoterranova, Contracaecum, Phocascaris


Adult forms of 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. 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.


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)


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.


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


Anisakis flesh and the body cavity of a large number of fishes as well as in cephalopods that serve as paratenic hosts. sp. larva in the flesh of blue whiting (Micromesistius poutassou) after light candling

(from www.usc.es/banim/doc/tppanisa.htm)


Histopathology flesh and the body cavity of a large number of fishes as well as in cephalopods that serve as paratenic hosts.

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.


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.


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.


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.


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


Section of forms of anisakid nematodes of the genera Anisakis larva in the ileum of a patient.

(From http://www.stanford.edu/class/humbio103/parasitepages/ParaSites/anisakiasis/Diag.html)


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).


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


Survey on the presence of nematodes has been studied using molecular approaches (PCR-RFLP, DNA sequencing, SSCP). Opisthorchis felineus (Platyhelminthes, Digenea) metacercariae in fish from Bolsena and Bracciano lakes (Central Italy)



Fish Italy)

  • Involved species

  • Prevalence of infestation

  • Safe fish

  • Risk


Materials Italy)


Ricerca metacercarie da muscolo di pesce

Metodi/Microscopia Italy)

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.


Methods – Molecular biology Italy)

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


Risultati Italy)


Prevalenza (%) Italy)

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




Results Italy)

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


Conclusions Italy)

  • 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


Conclusions Italy)

  • Difficult control

  • Limited therapy

  • Prophylaxis: no raw or uncooked fish

  • Monitoring the fish species


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