larvi 2013

ghent university, belgium, 2-5 september 2013

6th fish & shellfish larviculture symposium

Larval challenge protocols:the quest for virulence and verification

Øivind Bergh

Experimental challengeThe quest for virulence – and protection

Øivind BerghInstitute of Marine Research, Bergen, Norway

Why challenge?

• Basic questions:– Is ”the bug” virulent?– How does it affect the host?– How does it enter the host?

• Applied:– How can we protect the host– How can we verify that the protection is

effective?

Immuno-protection

Immuno-suppression

Larval ontogenesis (time)

Vaccination possibleProtection induced by vaccination

No protection

Vadstein, Mo and Bergh 2004

Challenge experiments on yolksac larvae

•Rearing of larvae in multiwell dishes•72 independent parallel wells•One egg/larvae per well•Larvae hatches in well, lives untilend of yolk sac period•Protocol developed from variouschallenge experiments during twodecades:

–Bergh et al. 1991 J. Fish Dis.–Sandlund et al. 2010 Dis. Aquat. Org.

Immunohistochemistry

Immunohistochemistry – yolksac larvae

Intestinalephithelium - cod

Epidermis -halibut

• Wild type V. splendiduscauses mortality

• Aerolysin negativemutant could not beseparated fromnegative control

Addition of Roseobacter (Rugeria) 27-4positively affected survival

Note: probiotics in lumen– NOT attached to epithelium

Comparative mortality

5 different Vibrio spp3 x V. anguillarumV. splendidusV. salmonicida

3 different fish larvae: turbot, halibut, cod

Most bacteria associated with larvae areopportunists, not able to cause mortalityaloneV. anguillarum is a ”true” pathogen

Challenges via live feed= oral administration

Challenge with V. logei

Intestine 24 hpost challenge

72 hpost challenge

Gills Intestine

V. splendidus

•Immunohistochemstry: as in fish larvae•Experimental setup: as in fish larvae

•However – 10-20 larvae/well•Mortality: read as absence of motility

Thank you for yourattention!