UNIVERSITY OF THESSALYUNIVERSITY OF THESSALY SCHOOL OF MEDICAL SCIENCESSCHOOL OF MEDICAL SCIENCES

FACULTY OF VETERINARY MEDICINEFACULTY OF VETERINARY MEDICINEKARDITSA

LABORATOTY OF ICHTHYOLOGY , AQUATIC ANIMAL HEALTH LABORATOTY OF ICHTHYOLOGY , AQUATIC ANIMAL HEALTH & & AQUACULTUREAQUACULTURE Prof. Fotini Athanasopoulou DVMProf. Fotini Athanasopoulou DVM, , MScMSc, , PhDPhD, , MRCVSMRCVS

..

DISEASE CONDITIONS IN MEDITERRANEAN FISH AND CURRENT RESEARCH IN GREECE

  

  

Viral conditions

VIRUS FISH SPECIES FISH SIZE MAIN CLINICAL SIGNS

NODA VIRUS Dicentrarchus labrax (sea bass)- MAcipenser spp –FWMugil cephalus-FW

Young mainly >150gAll sizes

Nervous signs, apathy, bending of spine, eye lesions, brain hemorrhages

LYMPHOCYSTIS Sparus aurata _MSparidae

young Skin granulomata

SPRING VIRAEMIA OF CARP

Cyprinids 1-2 summers External and internal hemorrhages

VIRUSES- MARINE FISH

LYMPHOCYSTIS

• Sea bream & other marine species•Summer outbreaks•Granulomata on skin & gills•Μacroscopic diagnosis•Horizontal transmission

VIRUSES - MARINE FISH

Εικόνα 3: Ιοyενής εyκεφαλοπάθεια και αμφιβληστροειδοπάθεια σε ιχθύδια λαβρακίου

NODA VIRUS

•Many marine species, sea bass, •also in FW •summer•Nervous signs, distended swim bladder, •Histology, ELISA, IFAT, PCR•Horizontal transmission•High mortality

EPITHYLIOCYSTIS

(Chlamydia sp) RICKETSSIAS / RLO’s

•Many marine spp •Spring and summer•Lethargy, respiratory problems•Μacroscopic examination•Horizontal transmission? μετάδοση ?•High mortality in sea bream

•Many marine spp, sea bass in Greece•Winter•Νervous signs no systemic disease •Histology•Horizontal transmission?• 20% mortality

Bacteria

 

Yersinia ruckeri (ERM)

Salmonidae Adult and young fish

Red mouth

Αeromonas sppA. hydrophila

Α. salmonicida  

Α. salmonicida ssp.nova  

 

SalmonidaeFW   

Cyprinids FW

All   All

Hemorrhagic septicemia

Cytophaga –like*Flexibacter, Cytophaga, All

 young and adults

Skin lesions mainly

FW fish

Bacteria

 

Vibrio (Listonella) anguillarum

All marine fish

All septicemia

 

 

All marinefish All

   

Pasteurella (Photobacterium piscicida)

Septicemia, GranulomataHigh mortality

ΒACTERIA – FW FISH

Yersinia ruckeri

Α. salmonicida ssp.nova

Α. salmonicida Furunculosis

Βacteria

Bacterium 

Fish species

Fish size Clinical signs

Mycobacterium spp.

All Older fish

Internal & external swellings, granulomata, ulcers. Ascites, exophthalus

Nocardia sp. All Older fish,broodstock

Internal & external swellings, granulomata, ulcers. Ascites, exophthalus

Streptococcus sp.Gram+

All mainly trout

All septicemia

Renebacterium sp.Gram+

 Salmonidae

 Adults Abscesses in kidneys, swellings

 

BACTERIA- FW FISH

Renebacterium salmoninarum

Myxobacteria

  

Agent Fish species

Fish size Clinical signs

Saprolegnia sp. ALL ALL Skin lesions

Branchiomyces CYPRINIDS

ALL Gill lesions

Ichthyophonus hoferi

SalmonidaeMarine fish

young Systematic disease

Exophthiala sp Salmonidae ALL Lesiona in kidneys, spleen

Αphanomyces astaci

Decapoda, Astacus spp.

- Necrosis

 

Fungal diseases

FUNGHI

Ichthyophonus hoferi

Saprolegnia spp

BACTERIOLOGICAL STUDY OF BACTERIOLOGICAL STUDY OF CULTURED AND WILD FISH IN CULTURED AND WILD FISH IN ARGOLIKOS BAY, GREECEARGOLIKOS BAY, GREECE

Passas Ν.1, Yiagnisi Μ.1,2, Bithava Κ.1, Vagianou Βαγιάνου S.1, Τzironi Ε.1 &

Αthanassopoulou F.1

1 Laboratory of Ichthyology & Aquatic animal Health Faculty of Veterinary Medicine, University of Thessaly

2 Laboratory of Nutrition and Fish Diseases, NCMR. Athens, Greece

AimAimIn this study we investigated the possibility of

finding simultaneously the same bacterial isolates in both cultured and wild fish around

sea cages in Greece

Three seasonal samplings of diseased cultured fish (Sparus aurata, Dicentrarchus

labrax) from 3 farms in Argolikos Bay.

Simultaneoysly, samples of wild fish were examined, near the cages (200-500 m) were

problems were present.

Wild fishWild fish

Είδη άγριων ψαριών Απομονωθέντα βακτηρίδια

Boops boops Ph.damselae ssp.damselae

Trachurus trachurus Ph.damselae ssp.damselae, V.vulnificus

Trachurus mediterranei

Ph.damselae ssp.damselae

Boops boops Ph.damselae ssp.damselae,V.harveyi

SpringSpring

SummerSummer

AutumnAutumn

ResultsResultsPhotobacterium damselae subsp. damselae was widely present in the bay and more often isolated from wild fish. It was isolated in all samplings: spring in wild fish, summer & autumn in cultured & wild fish.

In cultured fish the following bacteria were also isolated: Vibrio alginolyticus, V. harveyi, V. parahaemolyticus, V.

aestuarinus, V. tubiashii και V. splendidus II.

Photobacterium damselae Photobacterium damselae subspsubsp. damselae. damselaePresent in water & can be transmitted through waterPreviously described as a new Vibrio pathogen causing lesions to Chromis punctipinnis (Love et al. 1981).Enters through skin Includes isolates causing skin problems and hemorrhagic septicemia in various marine fish sp.Is a pathogen also of mammals and humans (damselae).

Photobacterium damselae Photobacterium damselae subsp.subsp. damselae damselae

Fish pathogens: Photobacterium damselae subsp. piscicida previously Pasteurella

piscicida.

Only Ph. damselae is pathogen for humans.

ConclusionsConclusions

Wild fish can be carriers of P. damselae subsp. damselae.Vibrio spp. such as V. vulnificus and V. harveyi can be isolated from wild fish

VIBRIOSVIBRIOS

ΓΕΝΙΚΑ ΠΕΡΙ VIBRIO spp

Το γένος Vibrio περιλαμβάνει Gram- , οξειδάση θετικούς (εκτός από δύο είδη) και αναερόβιους βακίλους που ανήκουν στην οικογένεια Vibrionaceae.

Eίναι κοινά βακτήρια του νερού σε θαλάσσια και υφάλμυρα οικοσυστήματα Τουλάχιστον 12 είδη Vibrio spp είναι παθογόνα στον άνθρωπο, 10 από τα

οποία συνδέονται με τροφογενείς λοιμώξεις. (Vibrio parahaemolyticus , Vibrio vulnificus, Vibrio alginolyticus)

Προκαλούν τρία κυρίως σύνδρομα: Γαστοεντερίτιδα, μολύνσεις τραυμάτων και σηψαιμία

Agent:Vibrio anguillarum (Listonella anguillarum). 10 serotypes:

01 serotype commonly in mediterranean fish. 02 in eels03 in larvae in France.

Listonella anguillarum

In both cultured and wild fish –epizootics (1817, 1987 and after).

In many fish species Problem in farms before vaccine use Still a problem in «new» cultured fish (halibut, cod

- Bergh et al. 2001) In mediterranean fish sea bass (Dicentrarchus

labrax) is the most sensitive Related infections in molluscs and bivalves and

crustaceans.

Sensitive fish species:

Temperature and infectionTemperature and infection Autumn and spring ( from 20°C to 15°C & vice versa) Accompanied by high daily changes. Recently in Greece the disease appears also in winter at T< 15°C.

Clinical signsClinical signs : :

Vibriosis is a classical gram negative haemorrhagic septicaemia. The outstanding feature of clinical vibriosis is the level of anaemia. Typical signs are red spots on the ventral and lateral areas of the fish, and swollen and dark skin lesions that ulcerate, releasing a bloody exudate.

Hyperacute: no clinical signs except for anorexia and dark colour.

Acute form: Externally: erythema on skin exophthamos, Sometimes also ulcers. Internally: hemorrhagic septicemia, spleen distension , hyperemic intestine

Furunculosis in sea bass D. labrax

Μ. Μ. yiagnisiyiagnisi 1,21,2, Κ. , Κ. BitchavaBitchava22, , Α, , Αggelidesggelides PP..33, , VatsosVatsos Ι.Ν. Ι.Ν.33 και και ΑΑthanassopoulouthanassopoulou FF22..

1 1 Laboratory of nutrition and Fish Diseases, Inst. Of Aquaculture, NCMR,, Hellinikon, Greece2Laboratory of Ichthyology & Aquatic Animal Health,,Faculty of Veterinary Medicine, University of Thessaly3 Laboratory of Ichthyology and Fish Diseases, School of Veterinary Medicine, University of Thessaloniki

ISOLATION AND IDENTIFICATION ISOLATION AND IDENTIFICATION OF OF Vibrio harveyiVibrio harveyi IN CULTURED MARINE IN CULTURED MARINE FISH IN GREECEFISH IN GREECE

Το Vibrio harveyi είναι Gram-ve bacterium common in the sea . Previously called Achromobacter harveyi.

Pathogen of many invertebrates esp. of the family Penaeidae.

Part of natural enteric fauna of marine organisms. Reported as a common opportunist pathogen in many

marine fish.

Vibrio harveyiVibrio harveyi

Vibrio harveyi isolated from internal organs of diseased fish sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) σε εκτροφές της Ελλάδας . Related to management stress or parasitic infections. Infected sea bream showed anorexia, abnormal swimming, dark colour, scale loss, skin ulcers. Infected sea bass showed skin hemorrhages and exophthalmia.Both fish species in the summer and autumn there was a peak of signs that were related to this bacterium. Sea bass was more sensitive with mortalities hoigher than 50%.

Vibrio harveyi Vibrio harveyi in our researchin our research

Isolation was achieved by biochemical and molecular methods. Sequence was very similar to Vibrio alginolyticus.MicroSeq 500 16S rDNA Bacterial Identification kit (Applied Biosystems) and BLAST data were used.

Isolation & identification Isolation & identification

Από τον Απρίλιο του Από τον Απρίλιο του 20062006, Στην Κτηνιατρική Σχολή του ΑΠΘ, λειτουργεί μια , Στην Κτηνιατρική Σχολή του ΑΠΘ, λειτουργεί μια Τράπεζα αναφοράς παθογόνων βακτηριδίων, απομονωμένα από εκτρεφόμενα ψάρια Τράπεζα αναφοράς παθογόνων βακτηριδίων, απομονωμένα από εκτρεφόμενα ψάρια της Ελλάδας.της Ελλάδας.Στο πρόγραμμα αυτό συμμετέχουν επίσης:Στο πρόγραμμα αυτό συμμετέχουν επίσης:Το εργαστήριο Ιχθυολογίας, του Τμήματος Κτηνιατρικής, του πανεπιστημίου της Το εργαστήριο Ιχθυολογίας, του Τμήματος Κτηνιατρικής, του πανεπιστημίου της ΘεσσαλίαςΘεσσαλίαςΤο Ινστιτούτο Υδατοκαλλιεργειών, του Ελληνικού Κέντρου Θαλασσίων Ερευνών Το Ινστιτούτο Υδατοκαλλιεργειών, του Ελληνικού Κέντρου Θαλασσίων Ερευνών (ΕΛ.ΚΕ.Θ.Ε), της Αθήνας(ΕΛ.ΚΕ.Θ.Ε), της Αθήνας

Συντονιστής προγράμματος Συντονιστής προγράμματος ::Παναγιώτης ΑγγελίδηςΠαναγιώτης Αγγελίδης, , Επίκουρος καθηγητής Κτηνιατρικής Σχολής ΑΠΘ,Επίκουρος καθηγητής Κτηνιατρικής Σχολής ΑΠΘ,Email: Email: panangel@vet.auth.grpanangel@vet.auth.gr

Το πρόγραμμα χρηματοδοτείται από το Υπουργείο Αγροτικής Ανάπτυξης & Το πρόγραμμα χρηματοδοτείται από το Υπουργείο Αγροτικής Ανάπτυξης & ΤροφίμωνΤροφίμωνΓεν. Δ/ση ΑλιείαςΓεν. Δ/ση Αλιείας, , Δ/ση Αλιευτικών Εφαρμογών , ΕΠΑΛ Δ/ση Αλιευτικών Εφαρμογών , ΕΠΑΛ 2000 – 2006, 2000 – 2006, Μέτρο Μέτρο 4.64.6

Greek Greek Aquaculture Aquaculture Bacteria Bacteria CollectionCollection

Purpose of this program:Purpose of this program:Isolation and identification of bacterial pathogens of cultured fish in Greece with microbiological, biochemical and molecular methods. Pathogenicity tests of isolates.Hisopathological study of diseased fish.Preservation of isolates for future use & research (vaccines, auto vaccines).Information to anybody involved. Cooperation with other data bases abroad.

Στάδια Συλλογή ψαριών, απομόνωση και

ταυτοποίηση βακτηριδιακών στελεχών Ιστολογική εξέταση Μοριακή ταυτοποίηση Συλλογή των απομονωθέντων βακτηριδίων

στους -80οC

This data bas follows the directions of the World Federation for Culture Collection

Macroscopic lesions and bacteria isolated from cultured fish

Fish species Isolated bacteriaα Macroscopic lesionsDicentrarchus labrax Listonella anguillarum (12) Αιμορραγίες γύρω από τα βραγχιακά

επικαλύμματα, τα πτερύγια, την έδρα, ασκίτης, συμφόρηση εσωτερικών οργάνων

Vibrio alginolyticus (2) Αιμορραγίες γύρω από την έδρα, πληγές, σπληνομεγαλία

Sparus aurata L. anguillarum (1) Αιμορραγίες γύρω από την έδρα, ασκίτης, αιμορραγίες στο ήπαρ

V. alginolyticus (5) Αιμορραγίες γύρω από τα βραγχιακά επικαλύμματα και τα πτερύγια

Photobacterium damsela ssp piscicida (2) Συμφόρηση εσωτερικών οργάνων, όζοι πάνω στα εσωτερικά όργανα, κυρίως στον σπλήνα

L. metsscnikovii (1) Δεν παρατηρήθηκαν βλάβεςUnidentified coccus (1) Αιμορραγίες στο λίπος γύρω από τα

εσωτερικά όργανα Anguilla anguilla Aeromonas spp. (3) Αιμορραγίες στα βραγχιακά επικαλύμματα ,

δερματικά έλκη Oncorhynchus mykiss Aeromonas spp. (1) Αιμορραγίες στα βραγχιακά επικαλύμματα,

τα πτερύγια, σπληνομεγαλία, ηπατομεγαλίαPagrus pagrus L. anguillarum (1) Αιμορραγίες στα βραγχιακά επικαλύμματαCarassius auratus A. hydrophila (1) Αιμορραγίες γύρω από τους οφθαλμούς και

την βάση των πτερυγίων, ασκίτης, σπληνομεγαλία, διόγκωση χοληδόχου κύστης

Cyprinus carpio Aeromonas spp. (not A. salmonicida) (1) Ασκίτης, σπληνομεγαλία, ηπατομεγαλίαPagellus erythrinus -** Ασκίτης , καχεξίαCarassius auratus gibelio A. sombria (1) Αιμορραγίες γύρω από τα βραγχιακά , στη

βάση των πτερυγίων και το στόμα, σπληνομεγαλία, διόγκωση χοληδόχου κύστης λευκόφαιο ήπαρ

Solea solea V. alginolyticus (1) Ασκίτης

Number of examined fish andtheir isolatesNumber of examined fish andtheir isolatesFish species No of cases Total number of

examined fishIsolated bacteria*

Dicentrarchus labrax 16 129 Listonella anguillarum (12)Vibrio alginolyticus (2)

Sparus aurata 14 176 L. anguillarum (1)V. alginolyticus (5)

Photobacterium damsela ssp piscicida (2)L. metsscnikovii (1)

Αταυτοποίητος κόκκος (1)

Anguilla anguilla 4 13 Aeromonas spp. (3)Oncorhynchus mykiss 1 1 Aeromonas spp. (1)

Pagrus pagrus 1 4 L. anguillarum (1)Carassius auratus 2 6 A. hydrophila (1)Cyprinus carpio 1 4 Aeromonas spp. (όχι A. salmonicida) (1)Pagellus erythrinus 1 3 -**Carassius auratus gibelio 1 5 A. sombria (1)

Solea solea 1 10 V. alginolyticus (1)Diplodus puntazzo 1 1 V. alginolyticus (1)

* () no of cases from which pathogen was isolated **Σημείωση: no bacterial isolated

Αscites- V. alginolyticus.

Spleen smears Giemsa X300

ΝΝecrotic lesion and granuloma ecrotic lesion and granuloma formation in sea bream kidneys, formation in sea bream kidneys, H&EH&E

Enteritis in sea bass swelling of Enteritis in sea bass swelling of entericenteric. . H&EH&E..

Endocarditis lesion in sea bassEndocarditis lesion in sea bass..H&E. H&E.

ΝΝecrotic lesions and granulomata ecrotic lesions and granulomata formation in sea bream spleen.H&E. formation in sea bream spleen.H&E.

PARASITES PARASITES

ICH CRYPTOCARYON sp

•All FW & tropical fish•Any age •T 3-20οC•Skin lesions, dyspnoia•Direct observation -2 stages in gills and tanks

•All Marine fish•Any age•T >20οC•Skin and gill lesions •Direct observation -2 stages in gills and tanks

METACERCAREA NEMATODA

Anguillicola crassus

Diplostomum spathaceum

•Many FW spp,• esp. Salminidae \•T 3-20οC•Cataract, blindness•Direct observation •Indirect life cycle

•In Swim bladder of eels Also in Salmonidae•Immunosupression, abnormal swimming •Direct observation•Indirect life cycle

TREMATODA ARTHROPODA, COPEPODA

Microcotyle sp.

Lernathropus kroyeri.

Ceratothoa oestroides

Microcotyle sp.

PARASITES- PROTOZOAPARASITES- PROTOZOA

TRICHODINA

CHILLODONELLA sp.•Stress related•Any species, any age

•Direct diagnosis in skin & gill smears

OODINIUM sp. •Marine spp•>20C•Skin lesions•Direct diagnosis in skin & gill smears•High mortality

•All FW fish esp. mullets•Any age at high temperature•Skin lesions•Direct diagnosis in skin & gill smears•Low mortality

Enteromyxum (Myxidium) leei

•Internal organs esp gall bladder •Microscopic direct diagnosis

•Low mortalityCeratomyxa sp.

ΜΥΧΟSPORIDA

•Diplodus puntazzo, sea bream, Pagrus major, P. pagrus•T >20οC

•Internal organs. Cachexia, serious histological lesions in intestine •Direct life cycle•High mortality

Polysporoplasma sparis

Kudoa sp.

Μιcrosporida

•In renal tubules•Any age

•Direct diagnosis in smears of kidneys•Low mortality

In musculature μύς of marine fish •Indirect life cycle???

Polysporoplasma sparis

CONDITIOΝ FISH SPECIES

FISH SIZE CLINICAL SIGNS

Gill disease ALL ALL Respiratory

Gas babble disease

ALL ALL Bubbles in skin, blood vessels

Fat degeneration/necrosis

ALL / (TROUT)

ALL Discolourization of liver

Systemic granulomatosis 

Marine sea bream, P.puntazzo

 

cagesSwelling of kidneys, int. organs /granulomata

Winter syndrom Sea bream

Cages Ascites, gill necrosis, hyperenia of intestine

   

Non infectious diseases

Fat degeneration of liver

Dietory Cataract

Gas bubble diseaseWinter syndromeSystematic granulomatosis