larval challenge protocols: the quest for virulence and ... 5 - thursday... · –sandlund et al....
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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!